www.nature.com/nature Vol 464 | Issue no.

7286 | 11 March 2010

Climate of fear
The integrity of climate research has taken a very public battering in recent months. Scientists must now
emphasize the science, while acknowledging that they are in a street fight.

limate scientists are on the defensive, knocked off balance Senator James Inhofe (Republican, Oklahoma), the leading climate

C by a re-energized community of global-warming deniers who, by sceptic in the US Congress, who labelled several respected climate
dominating the media agenda, are sowing doubts about the fun- scientists as potential criminals — nonsense that was hardly a surprise
damental science. Most researchers find themselves completely out of considering the source. Some scientists have responded by calling for
their league in this kind of battle because it’s only superficially about the a unified public rebuttal to Inhofe, and they have a point. As a mem-
science. The real goal is to stoke the angry fires of talk radio, cable news, ber of the minority party, Inhofe is powerless for now, but that may
the blogosphere and the like, all of which feed off of contrarian story one day change. In the meantime, Inhofe’s report is only as effective
lines and seldom make the time to assess facts and weigh evidence. as the attention it receives, which is why scientists need to be careful
Civility, honesty, fact and perspective are irrelevant. about how they engage such critics.
Worse, the onslaught seems to be working: some “Scientists must The core science supporting anthropogenic global
polls in the United States and abroad suggest that it is not be so naive as to warming has not changed. This needs to be stated again
eroding public confidence in climate science at a time assume that the data and again, in as many contexts as possible. Scientists
when the fundamental understanding of the climate speak for themselves.” must not be so naive as to assume that the data speak
system, although far from complete, is stronger than for themselves. Nor should governments. Scientific
ever. Ecologist Paul Ehrlich at Stanford University in California says agencies in the United States, Europe and beyond have been oddly
that his climate colleagues are at a loss about how to counter the attacks. silent over the recent controversies. In testimony on Capitol Hill last
“Everyone is scared shitless, but they don’t know what to do,” he says. month, the head of the US Environmental Protection Agency, Lisa
Researchers should not despair. For all the public’s confusion about Jackson, offered at best a weak defence of the science while seeming to
climate science, polls consistently show that people trust scientists distance her agency’s deliberations from a tarnished IPCC. Officials
more than almost anybody else to give honest advice. Yes, scientists’ of her stature should be ready to defend scientists where necessary,
reputations have taken a hit thanks to headlines about the leaked and at all times give a credible explanation of the science.
climate e-mails at the University of East Anglia (UEA), UK, and an These challenges are not new, and they won’t go away any time soon.
acknowledged mistake about the retreat of Himalayan glaciers in a Even before the present controversies, climate legislation had hit a
recent report from the Intergovernmental Panel on Climate Change wall in the US Senate, where the poorly informed public debate often
(IPCC). But these wounds are not necessarily fatal. leaves one wondering whether science has any role at all. The IPCC’s
To make sure they are not, scientists must acknowledge that they fourth assessment report had huge influence leading up to the climate
are in a street fight, and that their relationship with the media really conference in Copenhagen last year, but it was always clear that policy-
matters. Anything strategic that can be done on that front would be makers were reluctant to commit to serious reductions in greenhouse-
useful, be it media training for scientists or building links with cred- gas emissions. Scientists can’t do much about that, but they can and
ible public-relations firms. In this light, there are lessons to be learned must continue to inform policy-makers about the underlying science
from the current spate of controversies. For example, the IPCC error and the potential consequences of policy decisions — while making
was originally caught by scientists, not sceptics. Had it been promptly sure they are not bested in the court of public opinion. ■
corrected and openly explained to the media, in full context with the
underlying science, the story would have lasted days, not weeks. The
IPCC must establish a formal process for rapidly investigating and,
when necessary, correcting such errors.
The unguarded exchanges in the UEA e-mails speak for them-
Scientific glasnost
selves. Although the scientific process seems to have worked as it Russia’s scientific reputation will continue to
should have in the end, the e-mails do raise concerns about scientific dwindle unless it embraces international research.
behaviour and must be fully investigated. Public trust in scientists
is based not just on their competence, but also on their perceived ver since the Soviet Union fell apart in 1991, Russian leaders
objectivity and openness. Researchers would be wise to remember
this at all times, even when casually e-mailing colleagues. E have been vowing to transform their old-line, industrial society
into a modern, knowledge-based economy driven by innova-
US scientists recently learned this lesson yet again when a private tive science and technology. The current Russian president, Dmitry
e-mail discussion between leading climate researchers on how to deal Medvedev, has repeated that ambition frequently — not least as a
with sceptics went live on conservative websites, leading to charges way to overcome Russia’s dependence on oil and gas exports. Unfor-
that the scientific elite was conspiring to silence climate sceptics (see tunately, that transformation continues to be hobbled by outdated
page 149). The discussion was spurred by a report last month from attitudes at the top of Russia’s academic hierarchy.
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© 2010 Macmillan Publishers Limited. All rights reserved
EDITORIALS NATURE|Vol 464|11 March 2010

A small, but telling example came to light last month when the in the interview, English, not Russian, is the international language
popular online newspaper gazeta.ru published an interview with Yuri of science.
Osipov, president of the Russian Academy of Sciences in Moscow. Russian science is already lagging behind that of other nations.
Pressed by the reporter about the very low citation rate for articles According to an analysis published in January by Thomson Reuters,
published in Russian-language science journals, Osipov dismissed Russia produced just 2.6% of the research papers published between
the relevance of citation indices, questioned the need for Russian 2004 and 2008 and indexed by the firm — fewer than China (8.4%)
scientists to publish in foreign journals and said that any top-level and India (2.9%) and only slightly more than the Netherlands
specialist “will also study Russian and read papers in Russian”. (2.5%). Moreover, Russia’s publication output has remained almost
From anyone else, such a response might be dismissed as an off- flat since 1981, even as the output of nations such as India, Brazil
hand comment, perhaps reflecting a bit of stung national pride. But and China was exploding. The situation is so bleak that in October
Osipov is head of the largest and most powerful research organiza- last year, 185 Russian expatriate scientists signed an open letter
tion in Russia, the employer of around 50,000 scientists in more to Medvedev and Prime Minister Vladimir Putin warning of an
than 400 research institutes, and the publisher of some 150 Russian- imminent collapse of Russian science unless something was done
language research journals. What he says and thinks has a big effect to improve the inadequate funding, strategic planning and teach-
on Russian science. Moreover, the undercurrent of scientific nation- ing of science.
alism in his remarks is widely shared by other senior members of the Self-imposed scientific isolationism can only make matters worse
academic establishment — many of whom are products of Soviet — and accelerate the already large emigration of Russian scientists
times, when Russian science was pretty much an all-Russian affair seeking better opportunities in the West. And those who remain
(see Nature 449, 524–527, 528–529; 2007). in Russia are also starting to recognize the danger. Many young
Such parochialism is hopelessly at odds with any dreams of a researchers now eagerly collaborate with Western groups. And many
knowledge-based economy. The knowledge in question flows from older Russian professors continue to produce excellent science under
basic research and technological innovation, which have long since often difficult conditions. They know very well what a grave dis-
moved beyond being just national endeavours. If nothing else, inter- service they would do to their students by asking them to publish
national scrutiny and feedback are essential for winnowing the good in low-profile journals for the supposed sake of national pride. The
ideas from the dead ends. And, as Osipov himself acknowledged answer isn’t to close Russia in, but to open it up. ■

These targets are worthy indeed. But then, they were worthy when
Europe’s research future they were outlined in the Lisbon Strategy, the ten-year economic
plan that was adopted by the EU in 2000. Today that strategy is
The region’s member states must follow through on widely regarded as a failure, largely because the EU member states
their political and scientific commitments. seemed content to bask in the warmth of their good ideas rather than
implementing them.
t is hard to believe on a first reading of the European Commission’s The European Council, which comprises the 27 EU mem-

I 3 March proposal for ‘Europe 2020’ — the new ten-year economic

strategy for the European Union (EU) — that it is important for
scientists. In bland-yet-grandiose prose that invites the eye to slide
ber states, will meet on 25–26 March to start turning the draft
proposal into detailed policy. Only at this point will it become
clear exactly to what extent the council
right off the page, it defines a strategy for Europe to survive the members — whose loyalty to the inter- “Politics is a dicey
current financial crisis and “emerge stronger”. It pompously pledges ests of their own country can sometimes business and EU
to reform research and development and innovation systems “to conflict with their allegiances to the EU research is highly
foster excellence and smart specialisation”. — will allow themselves to be pinned political.”
But this draft is indeed important for scientists. It endorses down.
research as the basis of an economically and socially strong The commission will then have to write a second draft proposal
Europe. It maintains the EU’s goal of raising research expenditure that reflects those details. This final document must be approved by
to 3% of its gross domestic product. It recommits EU member the European Council in June. The consequences will then begin
states to the concept of the European Research Area, which seeks to unfold for researchers on the ground — for example, through
to remove legal hindrances to the free movement of researchers the design of the Eighth Framework Programme for Research and
across the region. It endorses the concept of a single, European- Technological Development, a multibillion-euro grant scheme that
wide patent system, which is badly needed as an alternative to is due to launch in 2014.
the current costly system that requires patents to be registered in Politics is a dicey business and EU research is highly political.
individual countries. And it explicitly reiterates the EU commit- There are still many ways in which things could go wrong for
ment to achieve a significant reduction in greenhouse-gas emis- research as a consequence of Europe 2020 strategy. But this time,
sions, while promoting the development of clean and efficient at least, this draft makes it possible to imagine that things could go
energy sources. right. ■

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© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March 2010

RESEARCH HIGHLIGHTS
The long and the short

M. STEIN
Zool. J. Linn. Soc. 158, 477–500 (2010)
The fossils of many arthropods — invertebrates with hard, external skeletons
and segmented bodies — from 540 million to 250 million years ago have a large
pair of ‘great appendages’ that they probably used to grasp and handle food.
Euarthropods had shorter versions of the appendage, whereas many of the
now-extinct anomalocaridids had longer, leg-like structures.
The position of these animals in the arthropod family tree is
controversial. Martin Stein, now at the University of
Kansas in Lawrence, reports a new arthropod
species that bridges a gap between the
two groups.
The new species, Kiisortoqia
soperi (reconstruction pictured),
discovered in Greenland, was clearly
a euarthropod but had the longer type of appendage.
Stein says this supports a shared ancestry of the long and short
‘great appendages’ and suggests that both evolved from the limbs
of a particular segment of the arthropod body.

METABOLISM adding water to a mixture of acid catalyst and Chapel Hill and her team engineered mice in
ionic liquid as it attacked untreated cellulosic which they could either stimulate or suppress
Warm milk biomass. The process avoids the hazards of calcium signalling. They showed that neither
Cell Metab. 11, 206–212 (2010) working with concentrated acids, and in a activating nor deactivating the calcium rise in
Mother’s milk fires up a heat-generating few hours produces sugar yields of 70–90%, astrocytes affected neurotransmission in the
metabolic pathway in newborn mice. which enzymes take days to achieve. brain’s hippocampus, suggesting that other
Newborns have to rapidly adjust to the However, scaling this up for commercial mechanisms underlie astrocyte signalling.
comparatively chilly environment outside the use could be problematic because of the need
womb. Francesc Villarroya of the University to recycle ionic liquids, which are expensive BIOMATERIALS
of Barcelona in Spain and his colleagues relative to other solvents.
found that expression of a metabolic regulator Squishy particles
gene called Fgf21 increases immediately after NEUROSCIENCE Angew. Chem. Int. Edn doi:10.1002/anie.200906606
birth. However, this increase was seen only (2010)
when pups were allowed to suckle or were Nerve cell talk Researchers have created tiny microgel
given a lipid-rich emulsion to drink. Pups fed Science 327, 1250–1254 (2010) particles that can squeeze through pores just
a glucose solution — a diet similar to what Neuroscientists had long believed that neural one-tenth of their size. This makes them
they would have received in utero — did not cells called astrocytes (pictured below) potentially useful as a biomaterial for tasks
activate Fgf21. provide structural support and nutrients to such as drug delivery — squishy nanoparticles
When the FGF21 protein was injected the neurons they surround. But a debate has can be easily filtered out by the kidneys, so
into newborns that were not allowed to erupted over whether these cells also release don’t need to be degradable by the body.
nurse, genes associated with heat generation signalling molecules that affect neuronal Grant Hendrickson and Andrew Lyon
in brown fat, a tissue specialized in heat communication — and, if they do, how. at the Georgia Institute of Technology in
production, were activated in the pups. It is thought that an increase in astrocyte Atlanta found that 116-nanometre-wide
calcium-ion concentration might trigger the microgel particles, when subject to pressure,
CHEMISTRY release of these signalling molecules. Cendra can pass through a material with pores of
Agulhon at the University of North Carolina at 10 nanometres — similar in diameter to those
Cellulose busters of the kidneys.
A. TOUSSON/PHOTOLIBRARY

Proc. Natl Acad. Sci. USA doi:10.1073/

pnas.0912073107 (2010) CANCER BIOLOGY
Finding a safe, low-cost and high-yield
way to break down cellulose — a major Arsenic activation
component of plant-fuel sources such as Cancer Res. 70, 1981–1988 (2010)
maize stalks — into fermentable sugars Arsenic, a carcinogen found at unsafe
is a challenge in biofuel development. A levels in drinking water in many parts
mild mixture of acid and ionic solvent of the world, may cause cancer by
may prove quicker and cheaper than increasing the activity of the Hedgehog
the enzymes commonly used. signalling pathway, which is known to
Ronald Raines and Joseph Binder at promote cell proliferation.
the University of Wisconsin, Madison, David Robbins, now at the
improved an existing recipe by slowly University of Miami, Florida, and his
144
© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March
NATURE|Vol 464|11
2010
March 2010 RESEARCH HIGHLIGHTS

JOURNAL CLUB
Markus Reichstein
Max Planck Institute for
Biogeochemistry, Jena, Germany
team have shown that arsenic destabilizes NANOTECHNOLOGY A biogeochemist looks at where
a protein that regulates Hedgehog activity,
which may allow the pathway to boost cell Harvesting heat all the emitted carbon dioxide is
going.
growth. Mice exposed to arsenic-laced water Nano Lett. doi:10.1021/nl903267n (2010)
had higher Hedgehog activity. Waste heat from vehicle exhaust pipes Humanity is currently performing
The researchers also analysed 265 human and industrial waste streams could offer a huge global experiment, emitting
bladder cancer samples along with the a sustainable energy source, but current increasing amounts of CO2 into the
arsenic levels in the patients’ tap water. technologies for harvesting thermal energy atmosphere by burning fossil fuels.
Higher arsenic levels correlated with are costly and inefficient. I find it astonishing that although
increased tumour expression of a key player Ray Baughman at the University of we scientifically explore other
in the Hedgehog pathway. Texas at Dallas and his colleagues have planets, we still don’t understand
created a ‘thermocell’ that can be wrapped Earth’s important carbon cycle.
PHYSICS around pipes (pictured below). Made of Corinne Le Quéré at the
carbon nanotube electrodes, the device University of East Anglia in Norwich,
Photon storage for telecoms is three times as efficient as conventional UK, and her team have put together
Phys. Rev. Lett. 104, 080502 (2010) platinum-based thermocells. The difference the pieces of the contemporary
Photons have a quantum mechanical spin, in temperature between the two electrodes global carbon cycle. They analysed
which can be ‘up’, ‘down’ or both. Storing a creates an electrochemical potential observations and modelling results
photon using conventional methods alters this difference, which the thermocell uses to on fossil-fuel emissions and the
state, destroying its quantum information. generate electricity. terrestrial and ocean carbon cycle,
Björn Lauritzen and his colleagues at One of the team’s prototypes can be which are the major contributors
the University of Geneva in Switzerland attached to hot nuclear-reactor pipes. to the atmospheric carbon budget
have found a way to store infrared photons (C. Le Quéré et al. Nature Geosci. 2,
without changing them. The team shone 831–836; 2009).
AM. CHEM. SOC.

a weak infrared laser pulse at a crystal The bottom line is that humans
containing erbium atoms, which had are emitting more CO2 than
previously been excited with a different light projected in the pessimistic
pulse. As an infrared photon was absorbed scenarios outlined by the United
by the crystal, its quantum state spread Nations Intergovernmental
across many erbium atoms. Panel on Climate Change.
Using an electric-field gradient, the group The researchers find that only
triggered the crystal’s re-emission of a photon 40–45% of this CO2 remains in the
encoding the same information as the atmosphere; the rest is ‘cleaned
incident photon a few hundred nanoseconds up’ by the ocean and land — the
later. The efficiency was well below 1%, but ‘carbon sink’. It would be interesting
the technique could prove useful in quantum- EVOLUTION to know whether the fraction taken
communication devices, the authors say. up by oceans and land remains
Creating cooperation constant, because any alterations
CANCER GENOMICS Evolution doi:10.1111/j.1558-5646.2010.00959.x (2010) will change the global climate–
How cooperation evolves between species is carbon-cycle feedback.
Melanoma’s mutations much debated. William Harcombe, currently The study also indicates that
Genome Res. doi:10.1101/gr.103697.109 (2010) at Harvard University in Cambridge, we are moving towards saturation
Some cancer-associated genetic changes are Massachusetts, used bacteria to observe this of the carbon sink, but the
not easily detected with standard technologies. evolution in the lab. uncertainties are large. Many
Researchers have now found mutations linked He plated out Petri dishes with an carbon pools and processes,
to melanoma using RNA sequencing. Escherichia coli mutant unable to produce an particularly those below ground in
Levi Garraway of the Dana-Farber Cancer essential amino acid, and a Salmonella species the soil, are not well understood
Institute in Boston, Massachusetts, and his that consumes waste from E. coli and excretes and are hardly accounted for in
colleagues used a high-speed sequencing small amounts of the amino acid. In two out carbon-cycle models (P. Ciais
technology to sequence RNA from ten of ten dishes, Salmonella mutants arose that Nature 462, 393; 2009).
patients’ melanoma samples. They identified made large amounts of the amino acid. The message from Le Quéré
11 abnormal RNAs resulting from genes that When grown with E. coli and normal et al. is that more observations
had fused in the genome — the first reported Salmonella, the cooperative mutants rapidly are needed, that data should be
gene fusions for melanoma. They also found increased from 1% of the Salmonella fully integrated with models, and
12 instances in which two separate genes were population to more than 80%. When the that these efforts must be more
transcribed, or ‘read’, together to produce bacteria were grown on different media such targeted and coordinated if we are
a mutated RNA, seven of which occurred that the Salmonella no longer relied on the to understand what is going on
in more than one sample. In addition, the E. coli for food, cooperative mutant numbers with the Earth system in our huge
researchers confirmed previous findings that crashed. This also happened when the bacteria experiment.
melanoma has a higher mutation rate than were grown in flasks, suggesting that spatial
other cancers, reflecting DNA damage caused structure of the bacterial colonies is needed for Discuss this paper at http://blogs.
by exposure to ultraviolet light. cooperation to evolve. nature.com/nature/journalclub
145
© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March 2010

NEWS BRIEFING
● POLICY

ESO/S.BRUNIER
European vision: In a new
decadal economic agenda,
the European Union (EU) has
said it hopes to raise spending
on research and development
(R&D) to 3% of the region’s gross
domestic product by 2020. That
measure was supposed to have
been hit this year, but spending
never rose above 2%. The Europe
2020 strategy, released on
3 March, suggests streamlining
bureaucracy to boost private
R&D spending, a single EU-wide
patent system and completion
of the European Research
Area, which would enable the
free movement of researchers
and funding across national
borders. Europe also needs
to refocus its research agenda
onto societal challenges such as
climate change, the plan says. See
Editorial, page 142.
GIANT EYE IN CHILE
Polio drive: More than 85 million The €800-million (US$1.1-billion) European Extremely Large Telescope will probably be built at Cerro
children under the age of five Armazones, in Chile’s Atacama Desert. The European Southern Observatory (ESO) announced on 4 March
will be immunized against polio that its advisory committee had recommended the site (pictured) above Spain’s La Palma and three other
in 19 countries across Africa. Chilean competitors. The Atacama Desert has the best overall sky quality and would allow the 42-metre-
The effort, part of the Global diameter telescope to operate in sync with ESO’s existing Paranal Observatory, the committee said. A final
Polio Eradication Initiative, was decision will be made no later than June.
announced by the World Health
Organization on 4 March. It is
an attempt to stop an ongoing Canada budget hope: Canada’s OCI, which investigates crimes
epidemic of the disease. The 2010–11 federal budget features such as the sale of counterfeit
initiative tried to prevent polio modest increases for major drugs, increased by 73% (after
spreading in 2009, but not enough science funding agencies, but adjustment for inflation) to
children received vaccines. also includes plans to review all $41 million in the decade that
federal support of research and ended in 2008. But the 230-plus-
Swiss animal rights: In development. See page 153 for person office runs with little
a 7 March referendum, more. accountability to senior agency
Switzerland’s voters rejected managers, the report says. The
a proposal for animals to be UK facilities funding: A FDA largely agreed with the
represented by state-funded
lawyers in court proceedings
structural shake-up may aid
Britain’s troubled Science and
SOUND findings. Its commissioner,
Margaret Hamburg, says it is
involving animal rights. The
Zurich canton already provides
Technology Facilities Council,
which distributes most of the
BITES taking action.

lawyers for animals in cases country’s physics and astronomy “My feeling is that
of alleged animal cruelty, but grants but has suffered huge it is going to be very ● BUSINESS
campaigners hoped to extend budget holes since it was created difficult to get a Pharma cuts: AstraZeneca has
the provision across the country, in 2007. See page 155 for more. treaty.” announced details of how it
which already has some of the plans to reshape its research and
world’s toughest animal-rights Lax drug oversight: The US development programme. The
laws. The referendum did pass a Food and Drug Administration Connie Hedegaard, European London-based pharmaceutical
separate proposal to amend the (FDA) has been lax in commissioner for climate company will slash several
change, is pessimistic over
constitution in order to preserve overseeing its Office of Criminal whether the world will draw up early-stage projects in topics
human dignity in biomedical Investigations (OCI), a a new treaty on climate change ranging from neuroscience
research; more detailed legislation report from the Government in 2010. (schizophrenia, depression,
is expected to follow on stem-cell Accountability Office concluded bipolar disorder and anxiety) to
Source: Financial Times
research and gene therapy. last week. The budget for the infectious disease (hepatitis C
146
© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March
NATURE|Vol 464|11
2010
March 2010 NEWS BRIEFING

and most vaccine projects).

NUMBER of confidence” in protection
Research sites in Lund, Sweden,
and in Charnwood and
CRUNCH
of workers and the public at
biocontainment laboratories in THE WEEK
$1bn–6bn
Cambridge, UK, will close. The Fort Detrick, Maryland, despite
company is holding to its earlier
estimate of a net reduction of
finding numerous deficiencies in
the way environmental hazards
AHEAD
1,800 people affiliated with The liability that at the site were modelled. The lab 13–25 MARCH
research and development. (where anthrax researcher David Conservation deals, including
experts polled by
Norwood is pictured, below) was a ban on international trade
Solar support: Germany’s
financial analysts at the source of the anthrax spores in bluefin tuna and greater
government is to reduce state
Swiss bank UBS think that killed five people in 2001. protection for the African
incentives for solar power from GlaxoSmithKline faces The US Army Medical Research elephant, are on the table at a
1 July. Solar feed-in tariffs — the as a result of lawsuits Institute of Infectious Diseases meeting of the Convention on
price an electricity utility must filed over its diabetes is expanding its facilities there International Trade in Endangered
pay to generators of solar energy drug Avandia. under a congressional mandate, Species in Doha, Qatar.
— have been dropping by 8–10% Source: The Guardian
but citizens living nearby had ➧ www.cites.org
a year since they were introduced complained of risks including
in 2000; now they will be cut an inadequate environmental 15–19 MARCH
by a further 16% for roof-top impact statement. The council’s The American Physical Society
panels and abandoned for panels report concluded that it “would hosts its spring meeting in
installed in converted farmland. not be useful” to redo the analysis Portland, Oregon.
Those installing and generating because construction has already ➧ go.nature.com/Y8YXiL
US ARMY/AP

solar power will see lower begun on the project.

returns — meaning that solar 16–19 MARCH
panel manufacturers will have Miami, Florida, hosts a ‘State of
to drop their prices. But as last the Arctic’ conference, which will
year’s plummeting price for solar discuss current understanding
modules has not yet been fully of the rapidly changing Arctic —
passed on to installers, Germany and what can be done about it.
should end up installing about ➧ http://soa.arcus.org
the same solar capacity as in
2009, according to analysts
Bloomberg New Energy Finance Energy call: The US
in London. Department of Energy last
Palace in space: China plans to week announced $100 million
Alzheimer’s disappointment: launch an unmanned orbiting for a third round of research
A closely watched Alzheimer’s a biopharmaceutical company space module in 2011, the programmes at its fledgling
treatment has failed to show in San Francisco, California. But first component of a proposed Advanced Research Projects
an effect on the disease in two in a trial involving 598 patients permanent space station. Agency-Energy (ARPA-E).
late-stage clinical trials, the it performed no better than Tiangong-1, or ‘Heavenly Palace’, The agency is geared towards
pharmaceutical company Pfizer, placebo. Medivation’s share price will first be used for orbital energy research that might not
based in New York, announced plunged after the announcement. docking practice with three be funded through traditional
on 3 March. Latrepirdine spacecraft; after that, crewed science grants. Research
(dimebon) — already used ships will visit the module. proposals are requested in
as an antihistamine in Russia ● RESEARCH Aerospace official Qi Faren told energy storage, electrical
— had seemed promising Pathogen lab confidence: A state media on 3 March that power delivery and energy-
enough for Pfizer to enter into a US National Research Council “technical reasons” had pushed efficient cooling technology.
$225-million deal in 2008 with report published on 4 March back an original launch target of The announcement came at the
the drug’s developer, Medivation, pronounced “a high degree late 2010. inaugural ARPA-E summit.

MARKET WATCH
SOURCE: PNAS

CONSUMER-BASED
More than one-fifth of the carbon dioxide California, published their findings this week CARBON ACCOUNTING
produced by China in 2004 was emitted to (S. J. Davis and K. Caldeira Proc. Natl Acad. Sci Because of exports, China produces
more CO2 than expected from
provide goods and services for non-Chinese USA doi:10.1073/pnas.0906974107; 2010).
domestic consumption.
consumers, mainly in western Europe, the The most comprehensive of its kind, the 5.8
United
6.5
United States and Japan (see chart). The analysis takes trade figures from 113 countries States
CO2 emissions
statistic comes from the latest study to look at across 57 industry sectors and finds that 23% produced
an alternative style of carbon accounting — one of global CO2 emissions (or 6.2 billion tonnes
CO2 emissions
that assigns CO2 emissions to the consumers of CO2) was traded internationally in 2004.
consumed 4.1 Europe 5.1
responsible for them. By contrast, inventories In some wealthy countries, including Britain
such as those reported under the United Nations and France, more than 30% of consumption-
Framework Convention on Climate Change based emissions are imported; in the United 5 billion
tonnes
(UNFCCC) simply tally the amount of gas each States, the figure is 11%. Caldeira thinks that the
5.1 China 4.0
country produces. UNFCCC’s production-based inventories should
Ken Caldeira and Steve Davis, at the Carnegie be supplemented with figures that track where Figures for 2004
Institution for Science’s lab in Stanford, the carbon is ultimately being consumed.
147
© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March 2010

NEWS

Outcry over scientists’ dismissal

Following years of acrimony, two high-profile researchers in Mexico have been expelled from their institute.

R. ROMERO/NEWSCOM
To the scores of scientific luminaries winners of the Nobel Prize in
who support them, the Terrones Chemistry. They unsuccessfully
brothers are two of the brightest stars lobbied for a compromise with
of Mexican science and have raised Mexican President Felipe Calderón
the nation’s profile in nanotechnology. Hinojosa and other top officials.
Yet the federal Institute for Scien- Kroto and some 70 other scientists
tific and Technological Research are again petitioning in support of
of San Luis Potosí (IPICYT) fired the brothers (see Correspondence
Humberto and Mauricio Terrones from H. W. Kroto et al., page 160).
Maldonado in December, and their The appeals by researchers out-
future in their home country is now side Mexico have not calmed the
looking dim. situation. In a written response to
International science leaders say enquiries from Nature, Rosaura Ruiz
that the case serves as an example of Gutiérrez, president of the Mexican
how entrenched scientific bureauc- National Academy of Science, said
racies in developing nations can that the letter sent to President Cal-
drive away promising researchers, derón in 2008 “created a great deal
especially those who have been of unease in the Mexican scientific
trained abroad. community, since its arguments lack
“This is a major loss for Mexican sufficient knowledge of the regula-
science,” says Mildred Dresselhaus, tions in Mexican institutions”. Ruiz
a nanotechnology researcher at the said the case has divided the Mexi-
Massachusetts Institute of Tech- can scientific community and that
nology (MIT) in Cambridge and a “proper channels exist for resolving
former president of the American this disagreement”.
Association for the Advancement Some are pessimistic about the
of Science, who has advocated on chances for resolving the dispute.
behalf of the Terrones. Ljubisa Radovic is a Spanish-speak-
David Ríos Jara, IPICYT’s direc- ing materials scientist at Pennsyl-
tor, rejects these arguments. “The vania State University in University
foreign scientists’ perception that Park who visited San Luis Potosí in
Mexican science is in imminent an unsuccessful attempt to broker a
peril is a flat misconception,” he says, compromise. He blames the impasse
adding that “there are about 20,000 Mauricio Terrones is an established nanotech researcher. on the IPICYT administration. “If
researchers in the country carrying you have stars like the Terrones,” he
out high-quality research in many areas”. the brothers were expelled from their lab in says, “you take care of them.”
Ríos says that he was forced to terminate December. Dresselhaus got a personal view of the
the employment of the brothers because they “I won’t work in a developing nation again,” escalating conflict in mid-December, when she
had violated institute rules and Mexican laws. says Mauricio. “Other Mexican universities are visited IPICYT to participate in the doctoral
Scientists outside Mexico have heard only one afraid to hire us,” adds Humberto. defence of Jessica Campos-Delgado, one of the
side of the story, he says. Terrones’ students. An unusual number of uni-
After receiving their doctorates in the United Centre of attention formed guards were present, apparently to head
Kingdom and doing postdoctoral work abroad, In an e-mail to Nature on 25 February, Ríos off a disturbance by students supporting the Ter-
the Terrones returned to Mexico and estab- accuses the brothers of several “irregularities”: rones, according to the brothers and Campos.
lished the country’s first nanotech lab, located failing to include IPICYT in four technol- The Terrones were intermittently called away
at IPICYT, about a decade ago. They secured ogy patent applications, not securing proper for discussions with their lawyers and adminis-
several large grants, collaborated with top authority to travel extensively last year and trators. Around that time, their e-mail accounts
researchers abroad and published well-cited improperly working for a private university. were shut down, and by the end of December,
papers on carbon nanotubes and buckyballs The Terrones deny any impropriety and blame their pay cheques stopped coming. “The whole
in high-impact journals. professional jealousy for their firing, a charge event was bizarre,” says Dresselhaus.
But several years ago, disagreements arose that Ríos rejects. Kroto, who oversaw Mauricio’s doctorate at
between the Terrones and the administration In 2008, the Terrones brothers’ situation the University of Sussex in Brighton, UK, says
at IPICYT over the operation of their lab (see attracted the attention of prominent scien- that he worries about the Terrones’ remaining
Nature 454, 143; 2008). Tensions between the tists, including British researcher Harold students. In an e-mail addressed to the “Inter-
two sides came to a head late last year, and Kroto and Mexico’s Mario Molina, both national Community” in late January, several
148
© 2010 Macmillan Publishers Limited. All rights reserved
Vol 464|11 March
NATURE|Vol 464|11
2010
March 2010 NEWS

A HIDING PLACE FOR HIV

S. KAULITZKI/ISTOCKPHOTO
AIDS virus escapes
treatment inside progenitor
blood cells.
go.nature.com/ZGu9wW

Climate e-mail rerun

students and staff in the Terrones’ nanotech
group said “we remain extremely worried that
we might be fired on a whim” and that they had
been pressured to retract their criticisms of the
administration. Arriving at work on 5 March, Stanford urging colleagues to calm down and stick
Ríos says that the IPICYT administration University ecologist Paul Ehrlich found to the science. And he hopes that the Inhofe
has offered its full support to the remaining a rambling and highly profane voice report — which says the scientists “violated
students. The current students declined inter- message from someone identifying himself fundamental ethical principles governing
view requests from Nature. as John Q Public. In one of his more lucid taxpayer-funded research and, in some
Campos, who received her degree, says that moments, the caller labelled Ehrlich cases, may have violated federal laws” —
she remains shaken by the Terrones’ firing. She and his colleagues in the climate-science will spark a backlash. “If we don’t have civil
will soon leave Mexico and head for a postdoc- community as “progressive communists discourse, where reality rather than spin is
toral fellowship in Brazil. “I don’t know if I can attempting to destroy America”. the basis of decisions, how are we going to
or will return, if I have to deal with the people Once again, a private e-mail function as a society?” Schneider asks.
who did this,” she says. conversation about global warming had Yet Schneider himself compares Inhofe
That will be a loss for Mexico. During her gone public, inflaming critics and giving to the infamous Senator Joe McCarthy,
doctoral training, Campos spent six months climate scientists yet another lesson in the who led the discredited campaign
in Dresselhaus’s lab at MIT, providing a key challenge of responding effectively. against communists during the 1950s.
contribution to a method to enhance graphene Several days earlier, Ehrlich had taken Inhofe spokesman Matt Dempsey calls
nanoribbons for mass production as semicon- part in an informal discussion with leading the comparison “flat out false” and says
ductors (X. Jia et al. Science 323, 1701–1705; climate researchers on a National Academy the report merely lists a number of laws
2009). MIT, in conjunction with IPICYT, of Sciences e-mail list. They — including the Freedom of
applied for a US patent application on the were exploring how climate “Progressive Information Act and the False
technology, and MIT now is seeking licensing scientists should respond communists Claims Act — that the scientists
agreements. to a recent US Senate report might have violated. Inhofe has
on the ‘Climategate’ e-mails attempting to asked the inspectors general
Helpful discussions leaked late last year from the destroy America.” of a number of federal science
Two of the patents that Ríos says the Terrones University of East Anglia in agencies to determine whether
failed to disclose involve Mexico’s largest juice the United Kingdom. In the 23 February violations actually occurred.
producer, Grupo Jumex in Tulpetlac. The report, Senator James Inhofe (Republican, As a member of the minority party, Inhofe
other two are owned by institutions in Japan: Oklahoma), who has long been the leading can’t do much more than issue statements
the National Institute for Materials Science sceptic in Congress, accused at least and reports. But Schneider says that if the
(NIMS) in Tsukuba and Shinshu University 17 climate scientists of unethical, even Republicans later regain a majority in the
in Wakasato. potentially criminal, behaviour in their Senate, Inhofe could take more concrete
The science for the patents was “partly or handling of climate data. steps, such as forcing climate scientists to
fully developed” at IPICYT, Ríos wrote, and The researchers’ private — and quite testify before Congress and pursuing his
“it goes without saying how serious stealing spirited — discussion was soon disclosed claims in congressional hearings.
intellectual property is”. in conservative media outlets. The initial Scientific societies, including the
Gerd Reiband, a head engineer at Jumex, story, which ran in The Washington Times, American Association for the Advancement
says that the company filed the two patent quoted Ehrlich as saying that climate of Science (AAAS) and the American
applications on its own because the work was scientists must recognize they are in a Geophysical Union, should respond
conducted there, not at IPICYT. There was no “street fight against well-funded, merciless to Inhofe’s report with a declaration of
agreement to financially reward the Terrones enemies who play by entirely different support, says Michael Oppenheimer, a
and their names were included as a courtesy for rules”. Other participants went so far as climate scientist at Princeton University
their helpful discussions. Scientists at the two to suggest taking out advertisements to in New Jersey who is also on Inhofe’s list.
Japanese institutions say similar conditions counter the sceptics, perhaps through a “Defending the scientific community and
applied to their patents. new non-profit organization. scientists from attacks ought to be a central
Ajayan Vinu, a materials scientist at NIMS, Ehrlich stands by his statement, but says part of their mission.”
worked with Mauricio when the Mexican sci- the affair has made it “crystal clear” that The AAAS and other scientific
entist was on a fellowship in Japan. Vinu says there is no such thing as a private e-mail. organizations have repeatedly affirmed the
that he was astounded to hear that advice that “I’m not putting anything in an e-mail that fundamental science of global warming,
Mauricio had offered had played a part in his I don’t want to appear on the front page of but some officials are wary of responding
dismissal. The Washington Times or Fox News,” he directly to Inhofe. Alan Leshner, AAAS
“Tell them they are crazy,” says Vinu, who says, adding that the sceptics “are trying chief executive, says he is focused on trying
was not contacted for details by IPICYT offi- to keep the scientists busy and to keep the to ensure that policy-makers distinguish
cials. “This is dangerous for science.” scientists from doing their job, and they between the controversies and the science.
The brothers remain in San Luis Potosí and are doing extremely well”. “What I don’t want to see is that this set of
are considering legal action to fight the termi- Stephen Schneider, a climatologist at incidents is used as an excuse to deny the
nation of their employment. ■ Stanford University in California who scientific findings.” ■
Rex Dalton is on Inhofe’s list and participated in the Jeff Tollefson
See Correspondence, page 160. National Academy discussion, says he is See Editorial, page 141.

149
© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March
NATURE|Vol 464|11
2010
March 2010 NEWS

JUSTUS SUSTERMANS (1636)

GALILEO BACKED
COPERNICUS DESPITE
DATA
Early telescopes suggested
that Earth stood still.
go.nature.com/OV49Sv

Apollo 15
Biology thinks
big to stay cuts
A grain of the
mineral apatite
shows off
rainbow hues
under a
microscope.
Biologists with grandiose research

F. MCCUBBIN, CARNEGIE INST. WASHINGTON

Locked inside are
hydroxyl ions proposals often turn to the risk-tolerant
(orange, inset) Human Frontier Science Program (HFSP)
revealing the to get themselves started. However, the
presence of
300 μm 30 μm world’s only intercontinental research
water.
funding agency is now working hard to
convince its members — 13 individual
nations plus representation from the
European Union — that frontier research is
a wise investment in tough economic times.
The 20-year-old HFSP distributes about
US$60 million a year to support research
into complex biological systems. Last
week, the programme’s secretary-general,
geneticist Ernst-Ludwig Winnacker, met
with an elite group of about two dozen
scientists to discuss where the frontiers of
biology might lie.
Participants agreed that genetic
technologies will continue to be central
to frontier biology; that fields such as
microscopy will continue to drive it; and that
new developments in chemistry will feed it.

NASA
Apollo 14 Tom Henzinger of the Institute of Science
Apatite grains in a basalt and Technology in Vienna also convinced
sample contain about as much participants that pure mathematics could
water as has been found in become an increasingly useful tool in
some volcanic rocks on Earth.
understanding biological processes.
“We remain open to any approach — the
three sole criteria remain excellence, risk
and biology,” says Winnacker, who has
for its formation, and that the heavy comet chlorine in volcanic rocks, which can pro- previously served as secretary-general of the
MOON: LICK OBSERVATORY

water mixed into the Moon’s magma ocean. vide some information about ancient water European Research Council and president
The comets would have struck Earth too, but because of the way chlorine would have of the German research agency the DFG.
because the young planet had a significantly bound to it to form other compounds. He The general messages from the
larger supply of water, the heavy water depos- says that the estimated water concentrations brainstorming session will feed into the
ited on Earth was greatly diluted. reported last week are at the upper limit of the organization’s first formal strategy report,
Alternatively, perhaps the heat of the range allowed by his chlorine results. which will be crucial when the HFSP’s
impact evaporated the lighter water from the Sorting out the debate will require new members meet in May. Winnacker is already
Moon, leaving it enriched in heavier water. samples. To Clive Neal at the University of bracing himself for trouble: Japan, which
Or maybe the impactor itself contained large Notre Dame in Indiana and chair of a NASA normally contributes around half of the
amounts of heavy water. lunar advisory group, that means returning budget, has this year cut its contribution
According to Elkins-Tanton, “this may all to the Moon. But before then, researchers will by 5%, and the global financial crisis is
lead to a reconsideration of the giant-impac- be revisiting the rocks collected by Apollo squeezing all members. Winnacker hopes
tor theory,” especially in how it relates to the astronauts. that the report will help to secure continued
origin of lunar water. At the meeting last week, Taylor drew an funding for this unique programme, whose
But not yet. The heavy-water results could index card out of his wallet that contained unbureaucratic support of big ideas is
reflect nothing more than a specific spot of the five-digit codes for three more samples popular with scientists. “It is a paradox
enrichment — the impact site of an ancient he was going to retrieve from the vaults at the that science is international, but funding
comet, for example. And some researchers Johnson Space Center, on the other side of is regional or national,” Winnacker notes.
aren’t convinced that the Moon is a wet as the Houston from the conference. He has made “The HFSP is the only agency with none
new results suggest. the trip many times before. “Being an old of the political borders that make joint
Chip Shearer at the University of New fart,” he says, “I know the ropes.” ■ funding so complicated.” ■
Mexico in Albuquerque has been analysing Eric Hand Alison Abbott
151
© 2010 Macmillan Publishers Limited. All rights reserved
NEWS Vol 464|11 March 2010
NATURE|Vol

Plant biologists fear for cress project

Is enthusiasm withering for funding studies into Arabidopsis thaliana?
The brilliant career of a diminutive weed may INSIDE ARABIDOPSIS was this idea that if we march through every

CUSTOM LIFE SCIENCE IMAGES/ALAMY

have hit a snag. Arabidopsis thaliana has been The Arabidopsis 2010 project has linked gene and look at its phenotype with a knockout
the darling of plant biologists for some 30 years thousands of the plant's genes and proteins mutant, that would give us great insight into the
because of its small genome and rapid growth, to their biological function. functional identity of every gene,” says Philip
and in 2000 it became the first plant to have its Benfey, a plant biologist at Duke University in
genome sequenced. To capitalize on this, the Durham, North Carolina. “The reality was
US National Science Foundation (NSF) Identified more than
much more complicated.”
soon afterwards dedicated US$200 mil- Studied microRNAs
350 genes with an
and the regulatory
lion towards determining the function of networks that control
essential role in seed Systems approach
every Arabidopsis gene by 2010. development In 2008, the project team drafted a proposed
flower development
Now time is nearly up, although the Arabidopsis 2020 programme that would
work is far from done. And with the NSF focus on systems biology, an approach that
unlikely to extend the project, Arabidopsis Analysed the would use the large data sets generated in the
researchers fear that the plant’s popularity with expression of 2010 programme to develop models of plant
27,103 specific
funders is on the wane. sections of DNA
function.
The appeal of Arabidopsis is as a stand-in That programme, however, is unlikely to be
for unwieldy food crops that grow funded, according to Parag Chitnis, deputy
slowly, take up lots of space and have Identified more than 4,000 gene director of the NSF’s Division of Molecular
large genomes. By studying thousands of mutations that cause defects in Studied transcription and Cellular Biosciences. “Now we are
photosynthesis, chloroplast factors involved in
plants in a single greenhouse, scientists responses to infection
thinking beyond just one model organism,”
can conduct experiments in a fraction of metabolism and development he says.
the time and space required for crop spe- The NSF’s Plant Genome Research Pro-
cies. But sequencing technology is improv- gram and the Department of Agriculture’s com-
ing so quickly that genome size is no longer petitive funding programme have increasingly
the barrier it once was. As more crop genomes favoured grants for work on agricultural species
are sequenced, including maize (corn) and rice, in recent years. The NSF has also begun phas-
researchers can increasingly study crop species ing out funding for TAIR, which is partially
directly, rather than relying on a model plant. funded by 2010 project funds (see Nature 462,
“There’s obviously a drive back to increased 258–259; 2009).
funding of crop plant research,” says Mark Stitt “People seem to feel, ‘Oh, Arabidopsis is
at the Max Planck Institute of Molecular Plant Mapped gene done. Let’s just move to the real stuff,’” says
Physiology in Potsdam, Germany. “This is expression in Natasha Raikhel at the University of California,
fair and good, but there is a quite serious risk developing roots Riverside. “Well, it’s not done.” Raikhel adds
that some of the advances made in Arabidop- that researchers have only just begun to tackle
sis research in the past ten years may not be some key biological questions in Arabidopsis,
sustained.” such as how cell walls form. Researchers devel-
Data from the NSF’s 2010 project (see oping ways to turn cellulose into ethanol need
‘Inside Arabidopsis’ for a few highlights) have to know how plants build their cell walls, but it
been used to tackle fundamental questions Washington in Stanford, California. However, is often more difficult to characterize the func-
about development, pathogen resistance and Huala estimates that direct experimental data tion of genes in biofuel crops.
hormone signalling. Annotation of the Arabi- about function are available for only a third of And although Benfey welcomes additional
dopsis genome — the linking of biological data all Arabidopsis genes. funding for crop research, he notes that the
to sequence information — is Money was one hurdle. extensive data already available for Arabidopsis
now considered among the “People seem to feel, Although the project was make it the best species to advance plant systems
highest quality of all sequenced ‘Oh, Arabidopsis is designed for an annual budget of biology. It would be difficult to build general
genomes. $100 million, it actually received models of flowering, for example, using data
But the project has fallen done. Let’s just move about a fifth of that sum. Then collected from many different plants, he says.
short of its original goal of to the real stuff.’ Well, there were biological obstacles. Even crop researchers have a good word
determining the function of it’s not done.” Arabidopsis has many large for the little weed. “It’s very important for
every gene. Most Arabidopsis gene families whose constitu- the entire plant and agricultural community
genes have been characterized in some fashion, ent genes have overlapping functions — a com- that Arabidopsis research should continue
for instance by observing their expression in a mon phenomenon in plants. Knocking out one to be funded.” says Edward Buckler, a maize
high-throughput assay, says Eva Huala, direc- gene in the family often did not affect the plant, geneticist at Cornell University in Ithaca,
tor of the Arabidopsis Information Resource because other genes were able to compensate. New York. ■
(TAIR), a database at the Carnegie Institution of “When the programme was designed, there Heidi Ledford
154
© 2010 Macmillan Publishers Limited. All rights reserved
Vol
NATURE|Vol
464|11 March
464|11
2010
March 2010 NEWS

WOODY SHRUBS DON’T

C. TAYLOR
SUCK UP WATER
Clearing savannahs might
make drought worse.
go.nature.com/VlScpq

GRAPHIC DETAIL

*SCIENCE & ENGINEERING INDICATORS: 2010 (NATL SCI. BD);

INTERNATIONAL COMPARATIVE PERFORMANCE OF THE UK RESEARCH BASE (EVIDENCE LTD, 2009)
60 an influential 2005 report by
the US National Academies
Securing 50
Share of global funding
for research (2007 data)*
that advocated increased funding
to maintain America’s pre-eminent
UK science 40
Share of world's most
highly cited papers
position in science. The
British politicians have given no (1998–2008)†
Royal Society argues along
assurances that science will be Percentage similar lines: “Our scientific
protected from the deep cuts in 30 leadership, which has taken
public spending that are predicted decades to build, can quickly
to follow this year’s general 20 be lost,” it warns.
election (see Nature 463, 410–411; The report asks the UK
2010). But Britain’s Royal Society 10 government to outline plans
is trying to ward off the expected for increased research funding
blow. In a report entitled The over a 15-year period (2011–26).
0
Scientific Century: Securing Our United Japan China Germany France United It also recommends expanding
Future Prosperity, released on States Kingdom a tax credit for research
9 March, the venerable society and development to stimulate
argues that slashing support bigger proportion of the most rising research funding in India, investment by businesses, and
for research would be a false highly cited research papers than China and Brazil, along with outlines the need for policies
economy, and would harm the its share of global funding would significant investment for science that encourage researchers
nation’s long-term economic suggest (see graphic). But seen in the stimulus packages from overseas to work in the
prospects. this impressive track record is in the United States, France and country. ■

†
The United Kingdom has “fragile” and brings a “risk of Germany. Richard Van Noorden
traditionally punched above its complacency”, the report The dossier models itself on ➧ http://royalsociety.org/
weight in science, garnering a says, noting competition from Rising Above the Gathering Storm, the-scientific-century

A rescue plan for UK physics funding

Britain’s most troubled research council drastic cuts. But this year it has again as the European Southern Observatory,
is about to undergo radical surgery. On come up short, by £40 million. “That was headquartered in Garching, Germany, and
4 March, UK science minister Paul Drayson leading to damage on the grants end of its CERN, Europe’s particle-physics laboratory
unveiled his plan to reform the Science responsibilities,” says Drayson. near Geneva, Switzerland, in order to
and Technology Facilities Council (STFC), Drayson set up a ministerial commission minimize the impact of the pound’s poor
the main research council that provides to look into what he describes as the exchange rate. Third, the UK subscription
funding for particle physics, space science, “tough problem” of balancing the STFC’s to the European Space Agency (ESA),
nuclear physics and astronomy. commitments and grants, resulting in a the STFC’s most costly international
The plan, which promises to end years proposed three-part reform. First, the commitment, will be moved to a new
of financial turbulence for the council, has council will keep the running costs for British space agency that is scheduled to be
gained tentative support from the physics its large facilities separate from grant launched later this month.
community, which had previously been funding and budget them over a six-year The plan “is just good business sense”,
incensed over repeated budget shortfalls period. Second, it will enlist the Bank says Kirby-Harris. By making sure that
that forced the STFC to slash grants and cut of England to help it manage the cost of the STFC’s fixed costs are appropriately
operations (see Nature 462, 396; 2009). overseas subscriptions to institutions such budgeted, the council should be able
“We’re very pleased,” says Robert Kirby- to better serve the physicists and
DIAMOND LIGHT SOURCE

Harris, chief executive of the London-based astronomers who depend on it for grants,
Institute of Physics, which represents the he says. Moving the ESA subscription
country’s physicists. But missing from the to the new space agency makes sense,
plan is more money for research funding. adds Andrew Fabian, president of the
Grants will still be cut back, and Britain still Royal Astronomical Society in London,
plans to withdraw from the multinational although the implications of this move for
Gemini telescope project in 2012. astronomers are not yet clear.
The STFC was formed in 2007 by The proposed changes should happen
merging two councils: one that managed after the government’s next spending
large facilities and another that disbursed review, which is currently scheduled to
physics and astronomy grants. Almost follow a national election that is expected
immediately it became clear that the to be called in May. Any change of
organization faced an £80-million New proposals could safeguard funding for big government could yet upend the plan. ■
(US$121-million) shortfall, leading to UK facilities such as the Diamond Light Source. Geoff Brumfiel
155
© 2010 Macmillan Publishers Limited. All rights reserved
 NEWS FEATURE Vol 464|11 March 2010
NATURE|Vol

Welcome to the Atomic

Weapons Establishment
With the launch of a powerful laser facility, Britain’s most secretive
lab is opening up to academics. Geoff Brumfiel secures a preview.

A
ldermaston is a picturesque English target chamber — and the fact that the AWE is have started to change over the past decade,
village with red-brick houses, tidy sharing it at all. says Steven Rose, a physicist at Imperial Col-
gardens and an inviting local pub. The motivations for collaboration are not lege London who headed the AWE’s plasma-
But just to the south lies a much entirely selfless. The defence establishment physics division from 2001 to 2004. “The old
more forbidding set of buildings. Behind dou- wants to provide scientists inside the security argument that secrecy was paramount I think
ble-fencing and guarded gates is the sprawling cordon with the sort of intellectual stimulation perhaps holds less sway these days,” he says.
campus of the Atomic Weapons Establishment needed to keep them on their toes; it also has “They’ve realized that you can do some things
(AWE), Britain’s most hush-hush research lab- an incentive to nurture the wider community in collaboration with the outside world.”
oratory. The 304-hectare facility, a converted of physicists from which it draws its staff. “We Enter Orion, the £183-million (US$274-
Second World War airfield, is home to the want to demonstrate that we maintain high million) laser on which construction began in
roughly 4,000 scientists, engineers and tech- standards for our science,” says 2005. The machine has ten con-
nicians who are responsible for maintaining Daryl Landeg, the AWE’s chief “The defence ventional lasers (see graphic),
Britain’s nuclear warheads. scientist. And academics far ministry is each of which can deliver 500
The Ministry of Defence, which oversees the beyond Aldermaston are keen joules of energy in about a nano-
AWE, is notoriously tight-lipped about the lab’s to cross the fence. At present, notoriously tight- second (a billionth of a second).
activities, and scientists who work there gen- Europe has only a handful of lipped about the It also has two short-pulse lasers,
erally try to keep a low profile. But within the comparable laser facilities, says lab’s activities.” which deliver the same amount
next few months, the AWE’s most ambitious François Amiranoff, director of of energy in just half a picosec-
and expensive scientific project is due to be the Laboratory for the Use of Intense Lasers ond (a trillionth of a second).
completed, and it is prompting the lab to open at the École Polytechnique near Paris. “A facil- Inside Orion’s main building, workers in
up its doors — at least a chink. ity like Orion is very, very interesting for the Teflon suits and hairnets are busily scrambling
The new Orion facility will be home to scientific community,” he says. around gigantic white scaffolding. The struc-
12 high-powered laser beams capable of heat- ture will soon hold the mirrors, amplifiers and
ing and compressing material to millions of Secrecy rules lenses needed to boost and focus the 12 beams
degrees Celsius in less than a nanosecond. Britain established its atomic-weapons pro- onto their target, which lies in a separate cham-
Housed in a gleaming building the size of gramme at the current AWE site in 1950, after ber behind 1.5 metres of solid concrete, a shield
a soccer pitch, the laser system will pro- close involvement in the Manhattan Project. The necessary to contain the radiation generated
vide physicists at Aldermaston with crucial campus, which also houses facilities for manu- when the laser beams hit. The exceptional
data about how components of their ageing facturing and storing sensitive nuclear materi- cleanliness in the laser halls and target area (and
nuclear weapons behave. Under current plans, als, is shielded by the nation’s strict secrecy laws, expected of visitors) is about more than aesthet-
around 15% of Orion’s time will be offered to and has historically shunned visitors. Things ics: a stray hair in the path of the intense beam
academics wanting to study con- could cause irregularities and crack
CROWN COPYRIGHT

ditions on stars or inside giant ORION LASER an expensive mirror or grating.

Ten long-pulse and two short-pulse lasers allow precise control of temperature
planets. And in that open spirit, and pressure in target materials.
Orion’s main mission, like that of
researchers there invited a Nature 4-metre-diameter target chamber the NIF, is to explore how nuclear
reporter in for a look around. Radiation shielding weapons work, particularly as they
In most respects, Orion is get older. In 1998, Britain ratified
the smaller cousin of the US the Comprehensive Nuclear-Test-
National Ignition Facility (NIF) Ban Treaty, an international agree-
in Livermore, California, which is ment prohibiting tests of nuclear
already running academic experi- weapons. Scientists in Britain and
ments. When operating at full steam, worldwide have therefore been
the NIF will use 192 lasers to create busy developing computer models
around 4 million joules of energy, to simulate nuclear warheads and
some 100 times more powerful than work out whether the weapons
Orion. What makes the AWE’s laser Two short-pulse will still detonate after decades
beamlines
notable is the exquisite precision in storage, and what type of deto-
that it will give researchers in con- Laser-pulse nation will result. What is missing,
Ten long-pulse compressors
trolling the heat and compression however, are actual data.
beamlines
exerted on the materials placed in its US scientists hope that the more
156
© 2010 Macmillan Publishers Limited. All rights reserved
 Vol 464|11 March
NATURE|Vol 464|11
2010
March 2010 NEWS FEATURE

laser — and the academics are eager to try out its

capabilities. “It will have significant characteris-
tics that no other laser in the United Kingdom or
indeed Europe has,” says Mike Dunne, director
of the Central Laser Facility at the Rutherford
Appleton Laboratory near Didcot, UK. Dunne
says that Orion should relieve the strain on the
facility’s Vulcan laser, Orion’s massively oversub-
scribed civilian counterpart.

Extreme conditions
Physicists studying astronomical objects find
themselves in a situation not dissimilar to
that of nuclear-weapons scientists: unable to
recreate the extreme conditions inside a star,
for example, and largely dependent on com-
plex computer models to show how they work.
Orion will not reach stellar temperatures and
pressures, but it will be able to create flows of
hot ionized gas with the sorts of high magnetic
fields and temperatures that mimic parts of
stars and other astronomical objects. By scaling
up the data from Orion, astrophysicists should
be able to improve their models, Dunne says.
The laser facility can also inform more
abstruse, fundamental work in atomic physics.
Current theory does well at describing normal
matter and extremely hot matter that has been
stripped of electrons. But it cannot describe
situations in which atoms are subjected to high
levels of radiation without losing all their elec-
trons. By heating a test material, then probing it
with its short-pulse beams, Orion can provide
data that can extend existing theories into this
middle region, says Amiranoff. This warm,
dense matter may exist at the heart of gas giants
Target chamber: Orion’s twelve lasers will explore conditions inside ageing thermonuclear weapons. such as Jupiter, or even within Earth’s core.
Scientific collaborations are not slated to
powerful NIF will contribute some of those case, the two stages that make up a weapon. begin on Orion until the second half of 2012,
A. COX/AWE CROWN COPYRIAGHT 2010

data, by generating temperatures and pressures The first stage, or primary, is a few kilograms of and proposals to use the facility will be sub-
so high that they will spark nuclear fusion in plutonium that are compressed by conventional mitted through the system being used for
small quantities of two hydrogen isotopes, deu- explosives until they begin a runaway nuclear Vulcan and other UK lasers, rather than being
terium and tritium. This fusion process would reaction. The radiation from that reaction is determined by weapons scientists. This open
resemble conditions inside the most powerful then focused onto the ‘secondary’, the stage in peer-review of proposals “is absolutely critical
stage of a modern thermonuclear weapon. which hydrogen isotopes create a much larger to gaining the confidence of the community”,
If the NIF is a thermonuclear hammer, then blast using nuclear fusion. Researchers want to Dunne says. “If some committee inside of
Orion is a scalpel. The smaller facility will never know what the opacity is and how it changes the wire assessed relative merits, there would
achieve full-scale fusion, but it will be able to with age so that they can model radiation’s flow always be the suspicion that they were picking
carefully control conditions inside test materials from the primary to the secondary and verify topics that helped their programme rather than
such as uranium. Pressure and temperature usu- whether the warheads will still work. The other just because they were good science.”
ally go hand-in-hand, explains Peter Roberts, parameter — the equation of state — describes Back on the AWE’s campus, the weapons
head of the AWE’s plasma-physics department. how a material behaves at enormous pressures scientists are eagerly preparing for their new
“You pump up a tyre with a bicycle pump and it and temperatures. By generating data on these guests. Tom Bett, who helps to manage con-
gets hot,” he says. But Orion can get around this. and other crucial parameters, Orion will give struction, shows off a data-analysis room pur-
It can compress a material with its long, nano- nuclear-weapons scientists the information they pose-built for unclassified visitors and lined
second pulses then suddenly heat it with its very need to ensure that their models are correct. with sleek computer terminals. (The weap-
short, half-picosecond pulses. The result is ‘iso- “You can’t look this stuff up,” Rose says. ons work will be done in a separate room, he
choric heating’, an unusual condition in which a The researchers running the NIF often explains, and the data will be stored on servers
material is heated so quickly that it doesn’t have emphasize the giant laser’s applications in locked inside vaults.)
time to expand. This capability allows Orion to energy production and fundamental science On the ground level, floor-to-ceiling
probe materials at wide-ranging combinations over its military role; it could, for example, lead windows illuminate a bright reception area —
of temperatures and pressures. to new reactors that produce electricity using the first thing that visiting researchers will see
In particular, researchers will use Orion tiny fusion implosions. Orion’s scientists are as they are welcomed to the new laser facility.
to explore two key parameters for materials much less circumspect. “We’re working on “Those windows,” Bett adds proudly, “are all
used in nuclear weapons: their opacity and weapons physics fundamentally,” Roberts says. bullet-proof.” ■
their equation of state. The first describes how Nevertheless, he and others at the lab are eager to Geoff Brumfiel is a senior reporter for Nature
radiation travels through a material — in this give civilian scientists an opportunity to use the based in London.
157
© 2010 Macmillan Publishers Limited. All rights reserved
 NEWS FEATURE Vol 464|11 March 2010
NATURE|Vol

What to make with DNA origami

Chemists looking to create complex self-assembling nanostructures are turning
to DNA. Katharine Sanderson looks at the science beneath the fold.

D
NA is the kind of polymer that chem- itself. So in 2004 and 2005 he spent months, he precision that many people have been looking

H. DIETZ/TECHNICAL UNIV. MUNICH

ists dream about. Because its comple- says, programming in his underpants, trying for. Origami scaffolds, sheets or bricks of folded
mentary sequences can bind to one to work out a way to bend a 7,000-base-pair DNA, are packed with known sequences that
another, individual molecules of the viral genome to his will. In his design he visu- could be used to position DNA-binding mol-
right sequence will assemble all by themselves alized how the genome could be folded into ecules just a few nanometres apart. And the
into intricate shapes and structures at the nano- a predetermined, two-dimensional shape. new, larger structures can contain upwards of
scale. DNA can weave together and bind other Knowing the sequence of the virus at every 200 sites for affixing such molecules, compared
molecules, allowing it to serve as a scaffold for twist and turn, he was able to write comple- with only a handful on pre-origami structures.
complex nanomachinery. mentary DNA sequences, about 16-base-pairs This type of precision engineering could be a
DNA nanoengineering is dreamy, but dif- long, that would essentially staple boon to nanoengineers wanting to position
ficult. Researchers have been putting together the folds in place. He ordered components on nanoelectronic circuits or for
carefully chosen segments of DNA to form the ‘staples’ from a DNA-syn- bioengineers looking to place proteins in
P. W. K. ROTHEMUND

sheets, tubes, even simple machines such as thesis company, mixed them close, accurate proximity to one another.
tweezers since the early 1980s. But back then, with his virus in a buffer Now the challenge is to go beyond the
designing these structures could take months that stabilized the DNA and novelty of Rothemund’s smileys and
to years. And because researchers were focused then heated and cooled the a dozen or so other demonstration
on designing them from scratch, they could mixture, allowing the single patterns and build structures
use only the short segments, no more than stranded viral DNA to bind with a practical purpose. Here’s
150-base-pairs long, that DNA synthesizers with the staples (see graphic, what several researchers are
could manufacture. This in turn constrained opposite). The result, viewed using dreaming of doing.
the size and complexity of the designs. “The atomic-force microscopy, was the smiley
problem is that we don’t just want to make face and several other shapes, created by Make a ruler
small stuff, we want to make complicated small what he called DNA origami1. Rothemund’s technique was a door
stuff, cheaply and easily,” says Paul Rothemund, The ease of DNA origami was a break- opener for Friedrich Simmel, a biophys-
a computational bioengineer at the California through, dispensing with the intricacies of pre- icist at the Technical University of Munich in
Institute of Technology in Pasadena. cise DNA engineering and other metamaterials Germany. Suddenly, Simmel says, he was able
Rothemund wondered whether he could development. “It’s like being able to bake a cake to have even “rather sloppy” physics students
create the complicated stuff using a longer, and not pay attention to the ingredient ratios,” making DNA structures with ease. Simmel has
naturally occurring piece of DNA, such as says Rothemund. But with the right ingredients used DNA origami to make a ruler to meas-
the genome of a virus, and folding it over on complex structures can be built with the kind of ure distances between single molecules and
158
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol
Vol 464|11 March
464|11
2010
March 2010 NEWS FEATURE

STAPLING A SMILEY Stapled DNA

With about 250 specific ‘staple’ strands of DNA,
Paul Rothemund folded a 7,000-base-pair viral genome
into a 10-nanometre ‘disk with three holes’.

Viral genome

Staple strands

calibrate super-high resolution microscopes. control all the electron-transfer sites then we that were complementary to loose ends on the
Simmel designed a DNA origami rectangle can improve the efficiency,” says Yan. DNA triangles5. The work is still quite crude,
measuring 100 nm by 70 nm and included says Rothemund. They haven’t got an active
some staple strands labelled with fluorescent Put a drug in a box device yet. “Much of the next five years will
dye molecules. When the DNA folds, two Last year, Kurt Gothelf, director of the Centre be spent perfecting techniques to place DNA
labelled staples sit at opposite ends of the rec- for DNA Nanotechnology at Aarhus Univer- origami on surfaces where we want them and
tangle in precise locations. This ruler can be sity in Denmark, and his colleagues, reported making this technique widely available to other
used to calibrate high-tech microscopes that that they had made a box from DNA origami3. scientists,” he says. Importantly, they’ve tacked
can resolve objects smaller than the diffraction One strand of DNA holds the lid shut; a sepa- down the shapes rather than having them
limit of light — roughly 200 nm (ref. 2). The rate DNA ‘key’ springs it open. The invention “bobbing around like jellyfish in solution”, says
kinds of molecules generally used for calibrat- prompted many to wonder what could be put Rothemund. “I didn’t think anyone would solve
ing such scopes, such as loose pieces of DNA or into the box and subsequently released. that problem in ten years. Now we’ve got DNA
filamentous proteins, are not ideal because they William Shih, a DNA nanotechnologist at origami lined up like little ducks in a row.”
are flexible and their dimensions can change. Harvard Medical School in Boston, Massachu-
Simmel wants to use the ruler and related setts, is keen to exploit this kind of vessel for Pushing past smileys
structures to help track the movement of drug delivery, but the challenges are significant. The grand schemes go on. Researchers imagine
molecular motors. Because fluorescent tags Getting the box to pass through a cell membrane an artificial ribosome capable of building cus-
allow them to use light microscopy, as opposed is going to be difficult, he says. He proposes cov- tom enzymes, a matrix for supporting artificial
to atomic-force microscopy or electron micros- ering the box with a membrane similar to those organs, or a DNA origami network designed to
copy, researchers will be able to view molecular sported by some enveloped viruses. support a neuronal network connected to elec-
processes as they happen. That’s important, These viruses also have special proteins to trical circuitry. Researchers are attempting all of
Rothemund says, once scientists begin cou- facilitate entry. Shih says that he can take viral these, but they will face some serious hurdles.
pling proteins to DNA origami. Rulers like proteins or related proteins and fix them to Buffer solutions must be finely tuned, otherwise
Simmel’s will be useful to watch how those the outside of his DNA cages, but structures fall apart. And large,
proteins behave. he faces a long list of challenges.
“Now we’ve got complicated structures can take
“There’s a lot of stuff,” Shih says. DNA origami up to a week to fold completely.
Build an artificial leaf “At this point it’s just a concept.” lined up like little Change is needed to the
DNA origami could allow researchers to put basic, almost slapdash synthesis
biomolecules together according to speci- Go for gold
ducks in a row.” approach of using unpurified
fication. A grail of sorts for many engineers Many have talked about using DNA origami as DNA that isn’t adequately sequenced, says Shih,
working at the nanoscale is photosystem II, a substrate for nanoelectronic circuitry, such “just because we got away with it so far doesn’t
a complex of more than 20 protein subu- as in plasmonic devices. mean we want to get away with it forever”.
nits and accessories that helps to split water Plasmonic devices couple light waves with But as long as this growing group of research-
into hydrogen ions and oxygen during charges on a metal surface and offer the speed of ers keeps trying to make different shapes, other
photosynthesis. information transfer that light provides, but at applications will appear. Rothemund can’t pre-
Attempts to recreate photosystem II, or even sizes smaller than those to which technologies dict quite how, though. “The problem is that
some of the catalysts involved in electron trans- such as fibre optics are limited. Current litho- you can do all these little cool things but they
port, have been disheartening. DNA origami graphic techniques run into trouble when trying in no way form a whole system. It’s not exactly
could provide the scaffold to hold proteins in to arrange metallic materials such as gold into clear what kinds of systems we’re going to be
place, says Hao Yan, a biochemist at Arizona patterns with features smaller than about 100 nm able to build.”
State University in Tempe. As part of a collabo- in size. Rothemund says that gold spheres might He’s working hard to find out. “In the past six
ration funded by the US Department of Energy be positioned using DNA origami to make struc- months, I’ve mostly been back at home, coding
at the university’s Center for Bio-Inspired Solar tures with better optical qualities. in my underwear again, which hopefully means
Fuel Production, he has been looking to use He has already taken steps, in concert with good things are happening.” ■
DNA origami as the basis for an artificial leaf IBM Almaden in San Jose, California, towards Katharine Sanderson is a reporter for Nature
that makes hydrogen fuel from water. arranging metal nanoparticles. Last year they in London.
Yan plans to use a cage-like DNA structure to managed to arrange DNA triangles on a litho-
position the proteins and to bind manganese, graphically patterned surface4. Jennifer Cha 1. Rothemund, P. W. K. Nature 440, 297–302 (2006).
a crucial component of the water-oxidation from IBM and her colleagues subsequently 2. Steinhauer, C., Jungmann, R., Sobey, T. L., Simmel, F. C. &
process. The goal is to better direct electron showed that they could place gold nanopar- Tinnefeld, P. Angew. Chem. Int. Edn 48, 8870–8873 (2009).
3. Andersen, E. S. et al. Nature 459, 73–76 (2009).
flow in an artificial system that requires pre- ticles smaller than 10 nm onto each origami 4. Kershner, R. J. et al. Nature Nanotech. 4, 557–561 (2009).
cise placement of components. “If we can really structure by affixing strands of DNA to the gold 5. Hung, A. M. et al. Nature Nanotech. 5, 121–126 (2009).
159
© 2010 Macmillan Publishers Limited. All rights reserved
OPINION NATURE|Vol 464|11 March 2010

CORRESPONDENCE
Science and Mexico Mildred S. Dresselhaus Massachusetts 1474; 2009) and the global Another barrier to CCS is
Institute of Technology, USA biodiversity observation system that countries with a weak
are the losers in Morinobu Endo Shinshu University, GEO BON (R. J. Scholes et al. manufacturing capability are
institute politics Japan Science 321, 1044–1045; 2008) not in a strong position to
Alan L. Mackay Birkbeck College, will be valuable tools in this develop lucrative carbon capture
Events at Mexico’s Instituto University of London, UK collaboration. technologies. The Australian
Potosino de Investigación Ljubisa R. Radovic Pennsylvania State Anne Larigauderie DIVERSITAS, 57 government, for example, has
Científica y Tecnológica (IPICYT) University, USA Rue Cuvier–CP 41, 75231 Paris Cedex committed Aus$2.4 billion
have escalated to crisis point (see 05, France (US$2.2 billion) to its CCS
page 148). We the undersigned e-mail: anne@diversitas-international.org Flagships Programme. But two
call on the world’s academic Colour-coded targets Georgina M. Mace Imperial College of the projects rely on capture
community to help reverse the London, Centre for Population Biology, technologies from Japan
damage currently being done would help clarify Silwood Park, Ascot SL5 7PY, UK (Mitsubishi in the ZeroGen
in this research institution, biodiversity priorities Harold A. Mooney Department of Project) and the United States
once a shining example for all Biology, Stanford University, Stanford, (GE in the Wandoan Power
developing nations. In this International Year of California 94306, USA Project). Governments need to
After battling for two years, Biodiversity, we should be setting balance their desire to support
Humberto and Mauricio Terrones ambitious but realistic targets emerging domestic CCS
— acclaimed leaders of IPICYT’s for biodiversity policy over the Barriers to carbon technologies against importing
prestigious nanoscience and next ten years. Those shaped potentially better technologies
nanotechnology group — have at last month’s sixth Trondheim capture and storage from abroad.
been removed from office. This Conference on Biodiversity in may not be obvious CCS solutions are also
flies in the face of the presumed Norway will be refined by the subject to the vested interests
commitment of the Consejo Subsidiary Body on Scientific, There’s more to ‘Buried trouble’ of national politics. In the coal-
Nacional de Ciencia y Tecnología Technical and Technological than whether carbon dioxide heavy economies of Canada,
(CONACYT), Mexico’s highest Advice in May and at the should be injected under urban the United States and Australia,
research authority, to seek a Conference of the Parties of the areas or offshore (Nature 463, for example, governments
“solution that will be a product Convention on Biological Diversity 871–873; 2010). Some barriers to promote CCS in their emissions-
of open negotiations carried out (CBD) in October. carbon-sequestration measures reduction promises, but they have
with tolerance, good will, great As participating scientists in are less immediately noticeable been reluctant to mandate the
objectivity and agreements that the international biodiversity than public opinion. technology.
avoid personal aggression and programme DIVERSITAS, we For example, the technology The real barrier to CCS
radicalization of positions”. This is welcome the draft set of 2020 should be incorporated into is that, even in enthusiastic
the wording of a recommendation targets proposed by the CBD. But developing energy systems. In countries, the focus is on selling
solicited by CONACYT from a the targets continue to mix the most scenarios produced by CCS solutions rather than on
prestigious group of scientists biodiversity we value highly (that the Intergovernmental Panel on mandatory CCS deployment.
formed to help prevent this is, the conservation agenda) and Climate Change (IPCC), much Advocates should commit to a
outcome. the biodiversity we urgently need larger volumes of CO2 will be firm timeline for mandatory CCS
An international group of some to secure the benefits people captured in China and India by on all new and retrofitted large
75 scientists has been working derive from fully functioning 2050 than in developed countries. emitters.
hard with us for two years to broker ecosystems. But the scale and pace of energy- Frances Bowen International Resource
a solution to this sorry affair, to no To resolve competing demands, systems development, and Industries and Sustainability Centre,
avail. Our hope is that President these different priorities should be the necessary carbon capture Haskayne School of Business,
Felipe Calderón will step in and made explicit by categorizing the and storage (CCS) technology University of Calgary, 2500 University
avert further damage. Otherwise, targets according to their primary transfers, are daunting. Drive NW, Calgary, Alberta T2N 1N4,
the prestige of Mexico’s science motivation. We suggest the use People optimistic about CCS Canada, and Smith School of Enterprise
and the prospects for its of red targets to stem urgent technology transfer to developing and the Environment, University of
technological development will deleterious biodiversity loss, countries should remember that Oxford, UK
suffer, as young Mexican scientists green targets for conservation transferring even cost-saving e-mail: fbowen@ucalgary.ca
won’t return after being trained in priorities and blue targets to technologies (transgenic seeds,
research abroad. secure the long-term benefits for example) has been difficult. Contributions to Correspondence
The academic community from functioning ecosystems. We should develop transfer may be submitted to
should join forces to reverse The CBD should work closely incentives by recognizing CCS correspondence@nature.com.
this situation. with the science community investments within the Clean Please see the Guide to Authors at
Harold W. Kroto Florida State to develop these targets for Development Mechanism of the go.nature.com/cMCHno. Science
University, USA changing environments and in the Kyoto Protocol. We must also publishing issues that may be of
e-mail: kroto@chem.fsu.edu light of new scientific discoveries. limit uncertainties surrounding interest to authors are regularly
Pulikel M. Ajayan Rice University, The proposed Intergovernmental CCS investments in developing featured at our blog Nautilus
Texas, USA Platform for Biodiversity and countries, particularly in (http://blogs.nature.com/
Anthony K. Cheetham University of Ecosystem Services (H. Mooney protecting intellectual property in nautilus), where we welcome
Cambridge, UK and G. Mace Science 325, capture technologies. comments and debate.
160
© 2010 Macmillan Publishers Limited. All rights reserved
Vol 464|11 March 2010

OPINION
Accelerating HIV vaccine development
Translational-research programmes supported by flexible, long-term, large-scale grants are needed to turn
advances in basic science into successful vaccines to halt the AIDS epidemic, says Wayne C. Koff.

2008 REPORT ON THE GLOBAL AIDS EPIDEMIC (UNAIDS, 2008)

he world needs a vaccine as soon as

T possible to help halt the inexorable spread

of HIV. But vaccine development has
not kept pace with breakthroughs in basic sci-
IN NEED OF A VACCINE
The number of people living with HIV* continues to grow, and AIDS-
related illnesses remain one of the leading causes of death globally.

ence. However, during the past year, advances

in vaccine research and development (R&D)
have generated great excitement. These include
the first, partial protection in humans by an
HIV vaccine1; identification of a new target
for broadly neutralizing antibodies on HIV’s
surface2; and evidence for significant vaccine-
induced control of simian immunodeficiency
virus (SIV) in non-human primates3.
New leads for vaccine discovery should come
from linking HIV vaccine research with the
broader field of viral immunology, and under- Estimated HIV
standing the early events in HIV pathogenesis prevalence (millions)
— as recommended elsewhere in this issue of
Nature4,5. Translational-research programmes
0 0.02 0.15 1.0 2.5 6.0
need to be expanded to connect this basic sci- No data
ence with existing vaccine-development tools, *2007 estimates

including hypothesis-driven clinical trials to

assess novel immunogen designs. This will (such as for measles, mumps and rubella); This approach identifies infected subjects
probably require solutions to two problems inactivation (for polio); and virus surface protein with broadly neutralizing serum antibody
that have remained unsolved for more than subunits or virus-like particles (for hepatitis B responses, isolates the corresponding broadly
20 years: the design of immunogens to elicit and human papilloma virus). neutralizing monoclonal antibodies (bnMAbs),
broadly neutralizing antibodies to prevent There are, however, three formidable characterizes the interaction of these bnMAbs
HIV infection, and the design of immunogens obstacles for vaccine developers attempting with the virus envelope, and then engineers
to elicit robust cellular immune responses to to raise HIV-specific neutralizing antibodies. immunogens based on this information.
control HIV infection. First, the virus is hypervariable so a vaccine However, in an era of global economic
Most importantly, the field should launch must elicit broadly neutralizing antibodies uncertainty, the multidisciplinary centres and
flexible, large-scale, long-term funding mech- to counter myriad circulating isolates of HIV. consortia likely to be required to adequately
anisms ideally by the beginning of 2011 that Second, the target for broadly neutralizing anti- address this problem are currently too few and
invest in multidisciplinary teams rather than bodies, the envelope spike, is very unstable and in jeopardy of not achieving critical mass or
in projects. Such mechanisms would foster so is difficult to recreate in a form that can act long-term commitment. This is due in large
greater innovation and shorten the timeline to as a vaccine6. Lastly, most highly conserved tar- part to increasingly restrictive funding mech-
a safe and effective HIV vaccine. gets for HIV-neutralizing antibodies are hard anisms such as milestone based, short-term,
to access on the virus spikes7. However, broad project-specific funding.
The neutralizing-antibody problem and potent neutralizing antibodies against
Licensed viral vaccines protect against dis- HIV have been identified from HIV-infected The cellular immune-response problem
ease by priming the immune system before subjects8, demonstrating the feasibility of Several lines of evidence, from human HIV
pathogen exposure. This generates antibody inducing such antibodies. Unfortunately, natural history and simian immunodeficiency
responses that can prevent infection, and experimental attempts to elicit such antibodies virus (SIV) studies, suggest that cell-mediated
cellular responses that target and eliminate have to date universally failed. immune responses are required to control
virus-infected cells. Virus-specific neutraliz- Recent technological advances — in struc- HIV by keeping the viral load very low or
ing antibodies bind to proteins on the surface tural biology, cryoelectron tomography, com- undetectable. Because control of infection is
of viral particles and stop them from infecting putational biology, B-cell immunobiology, and required to prevent disease, and as the best
host cells. Neutralizing antibodies can also bring high-throughput robotic micro-neutralization licensed vaccines against other pathogens
about the destruction of virus-infected cells, via screening assays — have been brought together do not necessarily completely prevent infec-
cellular effector mechanisms. Where natural to try to ‘reverse engineer’ a solution to this tion, a successful HIV vaccine will probably
virus infection elicits robust neutralizing-anti- problem9. These studies may inform the rational also need to elicit cell-mediated immune
body responses, vaccines have been developed design of vaccines against other highly vari- (CMI) responses capable of controlling HIV
by using attenuated versions of the live virus able viruses such as influenza and hepatitis C. infection. This is difficult because the virus
161
© 2010 Macmillan Publishers Limited. All rights reserved
OPINION NATURE|Vol 464|11 March 2010

becomes persistently established in host-cell to the application of B-cell screening and

ADAPTED FROM REF. 12

reservoirs within days of exposure, allowing HIV VACCINE INVESTMENT micro-neutralization technologies central to
In 2008, global research-and-development
only a small window of opportunity for CMI- spend totalled US$868 million. the recent identification of a new target on
based control. HIV for broadly neutralizing antibodies2.
Three HIV vaccine concepts have now Public sector Additional mechanisms to enhance bio-
$731 million
advanced through efficacy trials and none pharmaceutical investment, particularly
Philanthropy
controlled HIV infection as measured by viral $104 million in process development for vaccines, could
load in the blood. In contrast, a small subset of Industry include advanced market commitments,
HIV-infected people can control HIV infection $33 million intellectual-property incentives, and direct
to nearly undetectable levels without antiret- public-sector support for vaccine R&D.
roviral therapy10, and some SIV vaccines can Translational-research teams must be estab-
control pathogenic SIV infections to similarly on. These include the Neutralizing Antibody lished. The Howard Hughes Medical Institute
low viral loads11. To solve this problem, three Consortium (NAC) set up by the Interna- (HHMI) successfully supports internation-
key issues need to be addressed. First, elucidat- tional AIDS Vaccine Initiative (IAVI); the ally recognized basic-research scientists with
ing the mechanisms of CMI-based control of US National Institute of Allergy and Infec- a salary plus a portion of laboratory costs for a
HIV infection in humans and of SIV in SIV- tious Diseases (NIAID) intramural Vaccine minimum of five years, without restriction on
immunized monkeys would provide important Research Center; and the recently established their research direction. This attracts talented
leads. Second, improving functional assays of Ragon Institute in Boston, Massachusetts. scientists and fosters innovation. Few HHMI-
CMI responses would enhance preclinical and Teams associated with these three institutions like mechanisms exist for teams focused on
clinical prioritization of candidate vaccines. have been responsible for many of the most HIV vaccine translational research. Ten per
Third, researchers need to design and screen promising recent advances2,7,10,13–15. cent of the annual investment in HIV vaccine
immunogens capable of outwitting HIV’s ability These institutions have established prac- R&D should be refocused to provide such a
to mutate rapidly and evade CMI responses. tices that emphasize long-term (eight years or mechanism for HIV vaccine development.
Again, such complex challenges require more) institutional commitment. They reward In sum, major funders will bring a successful
multidisciplinary teams, and a different proven scientific leadership and track records HIV vaccine closer by establishing long-term
approach to funding translational research. rather than project-specific proposals; they translational-research programmes, attracting
try to link researchers with those from out- scientific talent and technologies, providing
Enhancing translational research side HIV vaccine R&D; and they limit major greater incentives for industry participation
Total global investment for HIV vaccine R&D project reporting and reviews of laboratories and focusing on rational vaccine design. These
for 2008 was US$868 million (see chart), a to multi-year cycles, increasing time for HIV initiatives should be formulated over the com-
10% decrease from 2007 (ref. 12). However, vaccine design. ing months, debated at the international AIDS
only 5–10% was focused directly on trying to Building on these successes, three new trans- Vaccine 2010 conference starting on 28 Sep-
solve the two major vaccine-design problems lational-research funding mechanisms should tember in Atlanta, Georgia, and implemented
detailed above. Moreover, funding for trans- be instituted. by the beginning of next year. This effort will
lational research has generally been limited Young investigator awards should be set up also provide a framework for the rational
to 3–5 year programmes that primarily sup- by major stakeholders of the Global HIV Vac- design of vaccines against other global infec-
port small academic consortia, and lack many cine Enterprise (www.hivvaccineenterprise.org) tious diseases. ■
of the industrial-style disciplines needed for for scientists under age 35. These awards would Wayne C. Koff is senior vice-president of
vaccine discovery and development. Collec- attract the best new scientists to the challenges research and development, and chief scientific
tively, such projects have a very low probabil- of HIV vaccine R&D by offering 5–7 years of officer at the International AIDS Vaccine
ity of success. In addition, academic scientists salary and flexible funding. Investigators would Initiative, 110 William Street, 27th Floor, New
must split their time between research, getting be free, within broadly defined research pro- York, New York 10038, USA.
grants, teaching and fulfilling administrative grammes, to redirect their funds as new data e-mail: wkoff@iavi.org
and managerial responsibilities, limiting their emerge, in exchange for expending at least 75% 1. Rerks-Ngarm, S. et al. N. Engl. J. Med. 361, 2209–2220
time focused on the scientific problems. of their effort on the two major scientific prob- (2009).
The funding mechanisms from national lems noted above. Developing such grants will 2. Walker, L. M. et al. Science 326, 285–289 (2009).
3. Hansen, S. G. et al. Nature Med. 15, 293–299 (2009).
public-sector research agencies have been require stakeholders, including public-sector 4. Virgin, H. W. & Walker, B. D. Nature 464, 224–231 (2010).
effective in fostering basic research, primarily research agencies and non-profit foundations 5. Haase, A. T. Nature 464, 217–223 (2010).
through investigator-initiated programmes. to invest in young scientists as components of 6. Pancera, M. et al. Proc. Natl Acad. Sci. USA 107, 1166-1171
(2010).
These should continue. However, these mecha- multidisciplinary teams, rather than relying on 7. Chen, L. et al. Science 326, 1123–1127 (2009).
nisms are not optimal for attracting new talent traditional investments in specific projects. 8. Stamatatos, L., Morris, L., Burton, D. R. & Mascola, J. R.
to HIV vaccine research, fostering innovation in Incentives for expanded biopharmaceutical Nature Med. 15, 866–870 (2009).
9. Burton, D. R. et al. Nature Immunol. 5, 233–236 (2004).
vaccine discovery, establishing links with indus- investment need to be set up. Vaccines are 10. Miura, T. et al. J. Virol. 83, 3407–3412 (2009).
try or providing long-term multidisciplinary made in industry, yet industrial involvement 11. Koff, W. C. et al. Nature Immunol. 7, 19–23 (2006).
commitment. It is unfortunate then that phil- in HIV vaccine R&D is minimal because of 12. HIV Vaccines and Microbicides Resource Tracking
anthropic foundations and other donors aiming the scientific challenges, market disincentives Working Group Adapting to Realities: Trends in HIV
Prevention Research Funding (July 2009); available at
to fill the translational science gap have increas- and opportunity costs. Support for industrial http://www.hivresourcetracking.org.
ingly tilted towards the public-sector model. investment — from the Bill & Melinda Gates 13. Simek, M. D. et al. J Virol. 83, 7337–7348 (2009).
There are examples of successful integration Foundation and IAVI — to Theraclone Sci- 14. Hessell, A. J. et al. Nature Med. 15, 951–954 (2009).
15. Košmrlj, A. et al. Nature (in the press).
of basic and translational research practices ences in Seattle, Washington, and Monogram
that should be learned from and expanded Biosciences in San Francisco, California, led See Reviews, page 217, and Perspectives, page 224.

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Vol 464|11 March 2010

BOOKS & ARTS

Evolution of the motor car

A proposed reinvention for urban motoring based on ultra-small electric vehicles does not
address the bigger environmental or social challenges, finds Daniel Sperling.
disruption of today’s models, particularly be integrated with a smart grid and how the

SMART CITIES
Reinventing the Automobile: Personal
Urban Mobility for the 21st Century in cities. Such a proposal might be expected adoption of road pricing might encourage the
by William J. Mitchell, Christopher E. from Mitchell, an architect who directs the transition to small, energy-efficient vehicles.
Borroni-Bird and Lawrence D. Burns smart cities research group at the Media Lab Focusing on large cities, they do not address
MIT Press: 2010. 240 pp. $21.95, £16.95 of the Massachusetts Institute of Technology inter-city or even suburban travel.
in Cambridge, Massachusetts. Yet the promo- It is easy to be sceptical: small, electric,
tion of small vehicles seems surprising com- connected cars in big cities will not solve our
How do we reconcile people’s desire to own ing from Borroni-Bird, a General Motors transportation and energy challenges. Large,
cars with the need to reduce pollution and executive, and Burns, the firm’s former head dense cities house only about 20% of the world’s
dependency on oil? This dilemma has inspired of research and development and strategic cars and light trucks. And there are many chal-
much technological and policy innovation, planning. General Motors is a company with lenges that the authors hardly mention. Will
from hybrid electric vehicles and smart traffic a long commitment to large cars and trucks ultra-small vehicles be economically attractive?
information to congestion charging and car- that, in the past, has vociferously opposed Is there much demand for small cars? Will the
sharing schemes. Yet these developments have regulations on fuel and greenhouse gases pricing of road space be widely accepted? Is
had little impact on the function and perform- aimed at reducing vehicle size. it feasible to build specialized roads for small
ance of our urban transport systems, which Reinventing the Automobile is vehicles in cities?
have barely changed in the past 50 years. provocative, aiming to overturn “Reinventing the Yet they introduce some big
As remarkable consumer products, conven- more than a century of vehicle ideas that could play an impor-
tional cars can last for more than 10 years with and urban design. It proposes car will be harder tant part in building livable cit-
little maintenance and withstand huge forces four main ideas: transform the than putting a ies and a low-carbon world. More
if they crash. But they still require someone to basic principles of car design; man on the Moon connected and automated vehi-
grasp the steering wheel and press the pedals. connect vehicles wirelessly to cles — not just cars but trucks
Because they run using combustion engines and share information on roads and — and much more operating on dedicated high-
petroleum fuels, they have a huge carbon foot- traffic; set up ‘smart’ electric- important.” speed roads — are desirable and
print. And they take up valuable space. They ity grids that manage energy inevitable.
are also an extravagance, sitting unused for 90% supply and demand; and impose real-time Reinventing the Automobile is well written
of the time on average and costing more than pricing and travel-on-demand services that and its ideas are illustrated by many draw-
US$8,000 a year to own and operate. give people greater flexibility and reduce the ings, graphs and tables. Above all the authors
The solution, according to authors cost of transportation systems. deserve credit for taking on a challenging issue.
William Mitchell, Christopher Borroni- The authors focus mainly on upgrading car As they put it, reinventing the car will be harder
Bird and Lawrence Burns, is to “reinvent the design. They also address the practicalities of than putting a man on the Moon — and much
automobile” to accommodate the growing shifting to their proposed system, including more important. ■
demand for cars in the megacities of India, how to convert combustion-engine vehicles Daniel Sperling is professor of engineering
China and the rest of the world. What’s to electric propulsion, how information and and environmental science and policy at the
needed, they propose, is an ultra-small vehi- communication technologies might be used University of California, Davis, California 95616,
cle powered by electricity or hydrogen that to increase vehicle flows without increas- USA, and co-author of Two Billion Cars.
will not cause the environmental and social ing road space, how electric vehicles might e-mail: dsperling@ucdavis.edu
163
© 2010 Macmillan Publishers Limited. All rights reserved
OPINION NATURE|Vol 464|11 March 2010

Space to contemplate
and have tried to limit development of the

ESO/H.H. HEYER
site. Astronomers have generally responded
with respect, by negotiating agreements to
protect the mountain’s unique environment.
The Edge of Physics: A Journey to Earth’s California, where Edwin Hubble measured By contrast, Paranal’s only neighbours in the
Extremes to Unlock the Secrets of the the expansion of the Universe in the 1920s. high Atacama desert are vast copper mines.
Universe/Dispatches from the Frontiers of Ananthaswamy explains how a reverential atti- Chileans travel far to work in this arid land-
Cosmology tude was expected from astronomers working scape, where nothing grows except the lush
by Anil Ananthaswamy there, and how for many years women were vegetation cocooned in the geodesic dome
Houghton Mifflin Harcourt/Gerald banned from the site because they were con- of the observatory’s space-age hotel. The
Duckworth: 2010. 336 pp. $25/£16.99 sidered a distraction. Reflecting this austere gardener who tends this isolated oasis empa-
atmosphere, the astronomers’ basic sleeping thizes with the astronomers: “We are devoted
accommodation is still called The Monastery. to the things we know, and we are a very good
Amazing discoveries tend to follow a satellite Ananthaswamy stays on for a few nights with complement.”
launch or telescope’s first light. Physicists have the neighbouring community of Camaldolese The political agendas behind these experi-
traced galaxies across most of the visible Uni- monks to contemplate the site in solitude and ments are largely skated over. An exception is
verse and have seen the top quark. Yet they lie silence. One of the monks, a distant relative of the negotiations that have led to South Africa
awake at night worrying that they don’t know George Ellery Hale who founded the observa- — together with Australia — becoming a pro-
what makes up 96% of the Universe, what dark tory in 1904, explains how he too seeks “a deep posed site for the Square Kilometre Array, a
energy is or why anything has mass. experience that one can’t express”. giant radio telescope due to be built over the
This gap between theory and observation Ananthaswamy takes a lift some 700 metres next decade to scan for pulsars and the first
concerns science writer Anil Ananthaswamy. below ground into the vast caverns of Soudan stars. This ambitious plan for South African
In The Edge of Physics, he travels the planet to mine in Minnesota to interview scientists astronomy hinges on the enthusiasm and
see for himself the experiments that are chal- working on the Cryogenic Dark Matter Search savvy of Bernie Fanaroff, radio-astronomer-
lenging our view of nature. Along the way he experiment installed there. He flies to Siberia turned-political adviser to the South African
meets the researchers who suffer for their sci- to visit the neutrino-detection experiment in government.
ence in bleak locations: from macho Siberians Lake Baikal’s crystal-clear waters. Scientists The final chapters explore the most
who sleep on the heaving ice of Lake Baikal here must endure sub-zero winter temperatures extreme experiments. Ananthaswamy heads
to catch neutrinos, to technicians who work to check the detectors that hang on strings like to Antarc tica to witness balloon launches
every day deep in a Minnesotan mine to detect jellyfish tentacles deep below the frozen delayed by bad weather and the struggles to
dark matter. Ananthaswamy seeks to capture surface. To bring good luck, the Russian physi- bury the IceCube neutrino detectors beneath
the peculiar motivations that draw people to cists toss some of their vodka onto the ice. the ice. Struck by the complexity and scale of
these remote sites of discovery. Chile’s Paranal observatory and Hawaii’s the Large Hadron Collider at CERN, Europe’s
Confessing that his journey is a pilgrim- Mauna Kea telescopes highlight clashes particle-physics lab near Geneva, Switzer-
age, Ananthaswamy begins and ends his tale between science and locale. Mauna Kea’s land, he pauses to note the tendency of the
at observatories that share peaks with mon- volcanic peak is considered sacred by many researchers to describe the ephemeral par-
asteries. The first site is Mount Wilson in Hawaiians, who leave offerings on its summit ticles they seek in certain terms. Although
164
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 OPINION

Genius who shuns the limelight

California, Berkeley, he vanished for several
Perfect Rigor: A Genius and the
years. None of his colleagues knew it, but he
Mathematical Breakthrough of the Century
was working in solitude on a proof of the
by Masha Gessen
Poincaré conjecture — a famous problem in
Houghton Mifflin Harcourt: 2009.
topology about three-dimensional boundaries
256 pp. $26
of four-dimensional spheres. After reappear-
ing briefly to explain his proof in a whirlwind
Mathematics rarely makes the news. One US tour, he vanished again. His whereabouts
exception was the proof of the century-old today are unknown.
Poincaré conjecture in 2003. When Russian Gessen describes the best and the worst that
mathematician Grigory ‘Grisha’ Perelman Soviet maths had to offer; the best being the
posted his solution on the Internet, people support given to gifted youths such as Perel-
took notice. Experts found that it was correct man through maths clubs and competitions,
and Perelman was awarded the highest the worst being the rampant anti-Semitism
honour in maths, the Fields Medal, by the that pervaded maths departments until
International Mathematical Union in 2006. perestroika in the late 1980s. Had it not been
Yet the laureate refused to attend the award for the strenuous efforts of some professors to
Clear views from Chile’s Paranal Observatory ceremony in Madrid, leaving King Juan Car- support Perelman, even a prodigy of his stature
are protected through agreements to limit los, who was to present the medal, waiting in might have been prevented from pursuing
light pollution by neighbouring mines. vain. Media interest again soared: here was a graduate studies because of his Jewish back-
long-haired weirdo who had flouted the King, ground. Gessen, a self-described ‘maths junkie’
he does not travel into space to visit the thus confirming every prejudice about math- brought up in Moscow, is ideally placed to tell
Planck spacecraft in person, he writes of ematicians. Tales of priority disputes and of discriminatory practices in the Soviet
how its forthcoming results on the cos- plagiarism quickly followed. academic world — her parents are Jewish
mic microwave background will soon Given his supreme problem-solving ability engineers who suffered through such ordeals.
challenge cosmologists’ knowledge of the and eccentric behaviour, Perelman’s personal- The book falls short in one important
early Universe. ity begs analysis. Perfect Rigor fills that need. respect: like others who have tried, Gessen
The Edge of Physics is an accomplished Science writer Masha Gessen has researched was unable to contact Perelman in person.
and timely overview of modern cosmology the reclusive mathematician thoroughly, Putting on a brave face, she writes that she was
and particle astrophysics. Ananthaswamy’s interviewing his teachers, maths coaches and not burdened by any allegiance to Perelman’s
characterizations of the many physicists colleagues in the United States, Russia and narrative. Unfortunately, lacking tangible
he meets are on the mark; yet the actors’ Israel. She also consulted psychologists about facts, she nevertheless speculates on why he
number and brevity on the stage may his behaviour, which, she suggests, has much shunned both the medal and the public. With-
baffle the uninitiated. It is especially gratify- in common with Asperger’s syndrome. out any evidence from supporting interviews,
ing that he spends time with the dedicated Born in St Petersburg (at that time Lenin- Gessen suggests that his great achievement
technicians and support staff, not just the grad), Perelman became a maths prodigy. At made him condescending towards the work
big scientific names. 16 he won a gold medal at the 1982 Interna- of others. This guesswork is unwarranted.
Ananthaswamy conveys that cutting- tional Mathematical Olympiad with a perfect One person who knows the Russian mathe-
edge science is a human endeavour. score. After studies at Leningrad State Univer- matician’s true motives is John Ball, the then-
Ending his journey, and his story, at India’s sity, the Steklov Institute of Mathematics in St president of the International Mathematical
Hanle observatory in the Himalayas, he Petersburg and a postdoc at the University of Union who travelled to St Petersburg in an
again notes the confluence of an observa- attempt to persuade Perelman to accept the
F. ROBERTS/NEWSCOM

tory and a monastery at a remote location. award. Ball reveals only that Perelman was
He urges that these sites must be protected allegedly disappointed by the dishonourable
from environmental threats such as climate behaviour of some unnamed mathematicians.
change and oil pipelines. A more press- Gessen, by proffering gratuitous speculations,
ing brake to grand experimentation is the both misleads the reader and does Perelman
global recession. Yet his interviews show grave injustice.
that understanding ultimately comes from Until 2006, the Poincaré conjecture was one
individual curiosity and motivation, not of the most famous open problems in maths;
expensive equipment. Perhaps in the quieter now it is one more theorem. For Perelman,
economic times ahead, physicists will have proving the conjecture was sufficient reward in
more liberty to contemplate the Universe itself — no prize or recognition was needed.
around them. ■ Perfect Rigor reminds us that it is journalists
Joanne Baker is Nature’s Books & Arts editor, and the public who want more. ■
and an ex-observational cosmologist. She is George Szpiro is a writer based in Jerusalem,
author of 50 Physics Ideas You Really Need to Grigory Perelman disappeared after proving the Israel. His forthcoming book is Numbers Rule.
Know. Poincaré conjecture about the topology of a sphere. e-mail: g.szpiro@nzz.ch
165
© 2010 Macmillan Publishers Limited. All rights reserved
OPINION NATURE|Vol 464|11 March 2010

Q&A: Peter Hessler on urbanization in China

In Country Driving, the final book in his China trilogy, Peter Hessler recounts his 11,000-kilometre drive across China to
see at first hand the effects of rapid industrialization. The New Yorker journalist explains how mass migration to cities
brings out people’s resourcefulness, but also how the speed of social and environmental change leads them to seek
meaning in their lives.

What did you hope to reveal about China separated, geographically and emotionally.

M. LEONG/REDUX/EYEVINE
in your trilogy? There is a sense of emptiness, a sense of
Each book has a different focus: River Town something lacking. I met many ‘searchers’ in
(2001) looks at geography and place, Oracle China, people who are concerned about what
Bones (2006) looks at history and time, and else there is in life beyond material success.
Country Driving looks at development and They want a community and people they
economics. I examine those issues through feel connected to. Many have developed a
the experiences of the average Chinese religious instinct — becoming followers of all
person, in particular people who are making sorts of faiths — but the religious institutions
the transition from the countryside to the in China are poorly developed.
city. They are the driving force of China’s
industrial revolution. A main point is How do people reconcile their sense of
to look at how individuals, families and individual identity with the imposed
communities respond to those changes and collectivism?
new challenges. Individualism is becoming more
characteristic in China. It arises outside
How have improved roads and the the education system. The people who
automobile boom affected the nation’s become migrants or entrepreneurs
rural regions? educate themselves, change their lives
Along my journey I was amazed by the pace and adjust their outlook. And people have
of transition. I saw how quickly villages in learned to take different roles in different
northern China are emptying out because circumstances: a farmer may act sometimes
young people are migrating to cities for Peter Hessler: struck by Chinese adaptability. as a villager, as a city person, an entrepreneur
jobs, leaving young children behind to be or an obedient Party member. This is part
raised by their grandparents. Many rural into factory towns become resourceful of the general flexibility and adaptability of
regions are far from urban centres so there and self-reliant. It is a tough world: when this generation: they are not stuck in a single
are few opportunities for villagers to improve negotiating jobs, they have to learn how to framework.
their livelihood. Roads and infrastructure manipulate people. There are positive aspects
allow them to get out — the young people to this, such as increased individualism and Does China’s political system help
hit the road for the east and the south. capability, but the climate can be a bit brutal or hinder its long-term economic
Building highways is normally the first step and lawless. development?
to urbanization. But in China, people can’t In some ways it has been an asset in the
buy and sell rural land. To develop it, local How is China’s industrial revolution past few decades. This is a society that has
governments often move farmers off their different from those in Europe and the pulled more people out of poverty than any
land, turn it into urban land by building United States? other in history; it has a high literacy rate,
highways and infrastructures, and then sell it One of the main differences is motivation. life expectancy has gone up, the per capita
on at market rates. A shortage of labour in Europe and income has increased. It deserves credit
the United States inspired people to be for that. The question is how it gets to the
You followed the progress of a newly innovative and efficient. China has so many next stage of development. It may reach a
established factory town. What did you workers that there is no need to save labour. point when people become more resistant
notice? This makes it a good place for low-margin and demanding. When that happens, the
I was struck by the adaptability of production, but not so good for innovation system may either collapse or adapt to new
individuals. Spending time in a factory town, or for making high-value-added products. challenges. But we haven’t yet reached that
you realize just how quickly things are being China’s political system, which lacks party point. ■
built and how rapidly urbanization is taking competition, and its educational system, Interview by Jane Qiu, Nature’s retained
place. One thing that impressed me was the which is very controlled and traditional, correspondent in Beijing.
functionality of the development system — also limit the possibility of innovation. e-mail: jane@janeqiu.com
it is flawed, but the flaws are not fatal. It is
very chaotic, the rules may be unfair, there is Has the country’s rapid development Country Driving: A Journey Through China
corruption, people tend to have a short-term affected people’s sense of wellbeing? from Farm to Factory
view, but it works — for now at least. People The pace of change can be overwhelming. by Peter Hessler
know the rules and know how to advance There is a breakdown in values and sense HarperCollins: 2010. 448 pp. $27.99
to improve their situation. Those who move of community. Families become more
166
© 2010 Macmillan Publishers Limited. All rights reserved
Vol 464|11 March 2010

NEWS & VIEWS

STRUCTURAL BIOLOGY

When four become one

Robert Craigie
Every machine is made of parts. But, as the new structure of the HIV integrase enzyme in complex with
viral DNA shows, one could not have predicted from the individual parts just how this machine works.
Retroviruses such as human immunodeficiency
virus type 1 (HIV-1) integrate a DNA copy of
their genome into their host genome as an
obligatory step in their replication cycle. The
DNA cutting-and-pasting reactions that lead to
integration are mediated by the virus’s integrase
enzyme. The structures of this enzyme’s three
domains — the core catalytic domain, the
amino-terminal domain and the carboxy-
terminal domain — have been solved previously.
But the spatial organization of the domains with
viral DNA when in the active nucleoprotein
complex (known as the intasome), which car- Amino-terminal domain Core catalytic domain Carboxy-terminal domain
ries out integration, has remained unknown. In
this issue (page 232), Hare et al.1 describe the Figure 1 | The pieces of the intasome puzzle. An integrase monomer consists of three structural domains:
structure of a retroviral intasome. The struc- the amino-terminal helical bundle, the core catalytic domain and the carboxy-terminal domain. Viral
ture clarifies many poorly understood aspects DNA integration into the host genome is, however, catalysed by a highly stable nucleoprotein complex
of retroviral-DNA integration. What’s more, called the intasome, in which four integrase monomers bridge a pair of viral DNA ends — a structure
it provides a solid platform for understanding that Hare et al.1 have now solved, putting the pieces of the puzzle together. Inhibitors of HIV-1 integrase
bind to the intasome rather than to free integrase, making the new structure particularly valuable.
the mechanism by which the inhibitors of this
essential viral enzyme act (Fig. 1).
Integration of retroviral DNA has been structure of the intasome complex. Hare et al.1 tetramer and DNA, however, is unlike any of
extensively studied at the biochemical level. show that all of the earlier models are wrong. the postulated models.
Such analyses showed that the DNA cutting- But why has the path to this level of under- Extensive surface contacts between integrase
and-pasting steps are mediated by a mecha- standing been so tortuous? Retroviral inte- and viral DNA, as well as protein–protein
nism similar to that used by the transposase grases are notoriously difficult proteins to contacts, are the glue holding the structure
enzymes found in bacteria and more complex work with: even using mutations that improve together. The major contribution of DNA–
organisms. The structures of the catalytic their solubility, the integrase and its DNA protein contacts to the stability of the com-
domain of retroviral integrases revealed2,3 an substrate aggregate under the conditions plex is reminiscent of the complex formation
active site that is common not only to these required for assembly of the active complex. between the Tn5 transposase and DNA7. The
transposase enzymes, but also to members of A more fundamental problem is that, on dimer interface of the catalytic domains,
a larger family of polynucleotidyl transferase initial binding to viral DNA, the integrase which is present in all crystal structures of this
enzymes4. Beyond confirming the mechanism does not show strong sequence specificity, domain, is preserved as expected. Only two of
of catalysis, however, these structures were and stable complexes are formed only after the the four active sites in the tetramer, one from
disappointingly uninformative. For instance, initial binding6. Mixing integrase with DNA each catalytic-domain dimer, associate with
they indicated that the catalytic domain is therefore traps nonspecific aggregates that are viral DNA, whereas the amino- and carboxy-
dimeric, with the two active sites on opposite unsuitable for structural studies. terminal domains of these subunits contribute
sides of the nearly spherical dimer. This spacing Hare et al. took advantage of their finding to stabilizing the tetramer. The other catalytic
is incompatible with the spacing of the catalysis that the integrase enzyme of the prototype domains in each dimer lie far from the DNA
sites on the two strands of host DNA. Clearly, foamy virus (PFV) — a close relative of HIV-1 substrate, and their corresponding amino- and
the functional complex would need at least a — has much more favourable biophysical carboxy-terminal domains are disordered.
pair of such dimers for two active sites to be properties than its HIV-1 counterpart. Using The past few years have seen great progress
close to each other. PFV integrase, they thus successfully crystal- in the development of therapeutic antiviral
In addition to the structures of the individual lized an integrase in complex with viral DNA, drugs that target integrase. These drugs have
amino- and carboxy-terminal domains of retro- a feat that has defied the best efforts of many been developed through large-scale screening
viral integrases, the two-domain structures of groups for the past two decades. efforts. The inhibitor raltegravir — like other
the catalytic domain with either the amino- or As predicted from the biochemical studies, promising integrase inhibitors — binds inte-
the carboxy-terminal domain have been deter- the authors’ intasome structure consists of a grase in complex with DNA rather than bind-
mined5. On the basis of these ‘partial’ struc- tetramer of integrase bridging a pair of viral ing the free enzyme in solution. Knowing little
tures, many models have been proposed for the DNA ends. The organization of the integrase about the integrase structures when in complex
167
© 2010 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS NATURE|Vol 464|11 March 2010

with DNA, therefore, it has not been possible to go without drastic structural rearrangement.
visualize the details of how these drugs interact The stark contrast between reality and the
with the enzymes’ active site, much less to seri- now-redundant previous models cautions
ously consider applying rational design to their against embarking on exuberant model build-
development. ing with few constraints imposed by experi-
50 YEARS AGO Hare et al.1 now change that situation. Their mentation. That said, the integrase enzymes
Strange World of the Moon. An structure succinctly shows how integrase of HIV-1 and PFV are sufficiently similar to
Enquiry into Lunar Physics. By inhibitors displace the 3ʹ end of the viral DNA justify the use of PFV integrase in complex
V. A. Firsoff — Persistent observers from the enzyme’s active site, thus blocking its with both viral DNA and antiviral inhibitors
of the Moon, many of them integration activity. Furthermore, they find for reliably modelling the binding of these
amateurs with small instruments that the active site, which seemed to lie in a inhibitors to the HIV-1 enzyme. ■
and little scientific training, have featureless landscape in the earlier structures of Robert Craigie is in the Laboratory of Molecular
often reported changes of various the catalytic domain alone, is partially buried Biology, National Institute of Diabetes and
kinds in the appearance of the in a rugged surface — features that may afford Digestive and Kidney Diseases, National Institutes
lunar surface; these have tended potential sites for anchoring inhibitors. of Health, Bethesda, Maryland 20892, USA.
to be met with scepticism by Despite the giant leap forward that this e-mail: bobc@helix.nih.gov
most professional astronomers study1 takes our understanding of retroviral
… The present book, which integrases, questions remain. First, four of the 1. Hare, S., Gupta, S. S., Volkov, E., Engelman, A. &
Cherepanov, P. Nature 464, 232–236 (2010).
attempts to interpret changes domains in the integrase tetramer are disor- 2. Dyda, F. et al. Science 266, 1981–1986 (1994).
seen by the author and other dered. Are they involved in the interaction 3. Bujacz, G. et al. J. Mol. Biol. 253, 333–346 (1995).
observers in terms of known with the host DNA, as Hare et al. suggest? 4. Rice, P., Craigie, R. & Davies, D. R. Curr. Opin. Struct. Biol. 6,
physical and chemical laws, is Do they mediate another function, or are 76–83 (1996).
5. Chiu, T. K. & Davies, D. R. Curr. Top. Med. Chem. 4, 965–977
timely … in view of the increased they simply redundant? Also, the target host (2004).
interest in the Moon resulting DNA is not present in the structure, although 6. Li, M. et al. EMBO J. 25, 1295–1304 (2006).
from the new facilities offered there seems to be only one place for it to 7. Davies, D. R. et al. Science 289, 77–85 (2000).
by space rockets … Mr. Firsoff
attributes ray systems to erosion
by running water, fed from large
deposits of ice accumulated ATMOSPHERIC CHEMISTRY
below the spongy surface rocks,
and he suggests that the maria,
also, are filled by impure ice …
Wider role for airborne chlorine
Basically, however, the author’s
arguments only show that his
Roland von Glasow
ideas are barely possible; he has Unexpected chlorine chemistry in the lowest part of the atmosphere can
certainly not shown that they are
at all likely. In particular, the idea
affect the cycling of nitrogen oxides and the production of ozone, and
that water is present on the lunar reduce the lifetime of the greenhouse gas methane.
surface is highly implausible.
From Nature 12 March 1960. When people hear about chlorine in the atmos- and plants, and it acts as a greenhouse gas.
50 & 100 YEARS AGO

phere, many probably think of the chlorine To understand the relevance of Thornton
100 YEARS AGO that comes from man-made chlorofluoro- and colleagues’ discovery, it is necessary to
Survival of Man. A Study in carbons (CFCs) and harms the ozone layer briefly explain the general role of nitrogen
Unrecognised Human Faculty. By in the stratosphere, some 25 kilometres above oxides in the atmosphere. Nitrogen oxides
Sir Oliver Lodge — To most people Earth’s surface. However, on page 271 of this are released from anthropogenic combus-
the question of the survival of issue, Thornton et al.1 report their observa- tion processes (for example, in cars and power
human personality is the greatest tions of large amounts of reactive chlorine plants) or the burning of biomass, but are also
problem of life … At present compounds at Earth’s surface, in the middle produced naturally by lightning. In the tropo-
the chief interest of the subject of the continental United States. sphere, the light-induced reaction of nitrogen
centres round the theory of cross The authors’ measurement site is far from dioxide (NO2) is the only way to form ozone.
correspondences, emanating known natural chlorine sources, such as the Nitrogen dioxide can also react with ozone to
chiefly from the so-called ocean, or strong anthropogenic sources (for produce nitrate radicals (NO3•), which can then
controls that are manifested in example, coal combustion or steel production). react with more NO2 to form dinitrogen pent-
certain well-known and much These findings are important because they oxide (N2O5). In other words, two molecules of
discussed automatic scripts, and reveal a previously unconsidered chemical NO2 are ‘stored’ in one molecule of N2O5.
claim to represent the surviving pathway that generates a large amount of Dinitrogen pentoxide is stable only at low
personalities of Myers, Hodgson, chlorine radicals in the troposphere (the lowest temperatures in the absence of light. If exposed
and others … Similarly, indications 10 kilometres or so of the atmosphere). Such to sunlight, the two molecules of NO2 stored
of known personalities, examples radicals will react with many volatile organic within it are released and can continue to pro-
of typical intellectual activities compounds (VOCs), including the potent green- duce ozone. It has been shown2,3 that a strong
continuing after earthly existence house gas methane (CH4), speeding up their loss process for N2O5 is its reaction on the
has ceased, may accumulate to removal from the atmosphere. Furthermore, surface of aerosol (Fig. 1) — tiny omnipres-
such intensity that no hypothesis this pathway will extend the lifetime of nitro- ent particles in the atmosphere that originate
is so simple or so effective as that gen oxides, the main precursors for ozone pro- from sources such as combustion processes,
which involves the acceptance of duction in the troposphere. Ozone is central to dust and salt from the oceans.
the belief in their manifest survival. atmospheric chemistry, but can also be a pollut- Thornton and colleagues’ atmospheric meas-
From Nature 10 March 1910. ant — in high concentrations it is toxic to animals urements1, taken at night just outside Boulder,
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NATURE|Vol 464|11 March 2010 NEWS & VIEWS

States alone is much larger than the first global

a Night-time b Night-time Daytime estimate of the compound’s production6, and
ClNO2 Sunlight is similar to recent estimates of global ClNO2
production in coastal regions5.
HCl N2O5 HCl N2O5
Clƭ + NO2 If other continental regions also act as
CH4 and sources of ClNO2, then the cycling of nitrogen
Many volatile oxides and chlorine through this compound is
steps organic
compounds
of global relevance. A more thorough investiga-
2NO3– NO3– tion of this reaction pathway is therefore war-
Cl– Cl– ranted, involving many more measurements of
ClNO2 and of background chlorine compounds
Aerosol in general. As a large part of the uncertainties
particle
in Thornton and colleagues’ model relate to the
O3
actual reaction of N2O5 on aerosol particles, this
Deposition Deposition reaction should also be studied in more detail,
in the hope of finding generally applicable
Ground
parameters for the process.
Numerical models of atmospheric halogen
Conventional model New model
chemistry have to include many compounds to
Figure 1 | Schematic of the uptake of N2O5 on aerosol particles. a, Conventional picture of aerosol fully describe the various reaction cycles. Yet
uptake of hydrochloric acid (HCl) and dinitrogen pentoxide (N2O5), leading to chloride (Cl−) and many of these compounds, including certain
nitrate (NO3−) ions, respectively. In the absence of further reactions both are eventually deposited on key intermediates such as hypobromous acid
the ground with the aerosol particle. b, Mechanism supported by the measurements of Thornton (HOBr) and hypochlorous acid (HOCl), have
et al.1: nitryl chloride (ClNO2) is formed at night in the reaction of N2O5 on particles containing so far not been identified in the atmosphere,
chloride. The next day, sunlight breaks ClNO2 into chlorine radicals (Cl•) and nitrogen dioxide
despite the fact that new instruments have pro-
(NO2). The chlorine radicals can then attack the greenhouse gas methane (CH4) and volatile organic
compounds, and the resulting peroxy radicals and NO2 lead to the formation of ozone (O3).
vided much insight into halogen reaction cycles
in recent years1,5. To keep making progress,
scientists working in the laboratory, the field
Colorado, indicate an additional major reaction understanding of where this kind of chemistry and with numerical models must collaborate
pathway for N2O5 (Fig. 1). If it reacts at night on might be most prominent. at the early stages of projects, to find ways of
aerosol particles that contain chloride, then one On this basis, Thornton et al.1 assumed that detecting and quantifying ‘new’ compounds7.
of the two NO2 molecules stored within it can the ingredients for ClNO2 formation are ubiq- Such an approach would be invaluable in
form nitryl chloride (ClNO2). This is released uitous in the atmosphere of the continental answering the questions raised by Thornton and
to the atmosphere, whereas the other NO2 mol- United States and used a computer model of colleagues’ study1. ■
ecule forms a nitrate ion (NO3−). Under most the atmosphere to estimate the continent-wide Roland von Glasow is at the School of
conditions, nitrate ions remain in the particles impact of this reaction. A notable uncertainty Environmental Sciences, University of East
and are eventually deposited on the ground. The in the model is the efficiency of ClNO2 forma- Anglia, Norwich NR4 7TJ, UK.
following morning, ClNO2 is broken down by tion, which depends on the available surface e-mail: r.von-glasow@uea.ac.uk
sunlight into a chlorine radical and NO2. This area of aerosol particles, the particles’ composi-
1. Thornton, J. A. et al. Nature 464, 271–274 (2010).
effectively extends the lifetime of NO2, allowing tion (especially their chloride content) and the 2. Dentener, F. J. & Crutzen, P. J. J. Geophys. Res. 98,
it to produce more ozone and to be transported relative humidity. The authors’ simulations sug- 7149–7163 (1993).
by air currents away from its source. The chlor- gest that 8–22% of all nitrogen oxides emitted 3. Evans, M. J. & Jacob, D. J. Geophys. Res. Lett.
doi:10.1029/2005GL022469 (2005).
ine radical, meanwhile, can react with VOCs, in the United States cycle through the ClNO2 4. Finlayson-Pitts, B. J., Ezell, M. J. & Pitts, J. N. Nature 337,
including CH4. The chain of reactions that fol- pathway, which will therefore affect the lifetime 241–244 (1989).
lows eventually leads to the production of per- and transport of those compounds as described 5. Osthoff, H. D. et al. Nature Geosci. 1, 324–328 (2008).
6. Erickson, D. J., Seuzaret, C., Keene, W. C. & Gong, S. L.
oxy radicals that (along with nitrogen oxides) earlier. The researchers’ estimate of the amount J. Geophys. Res. 104, 8347–8372 (1999).
are key ingredients of ozone formation. of ClNO2 produced over the continental United 7. www.hitt-task.net
This reaction pathway for N2O5 had already
been observed4 in the laboratory in 1989, but
confirmation of its role in the atmosphere
didn’t occur until 2008, when Osthoff et al.5 SUPRAMOLECULAR CHEMISTRY
observed high ClNO2 concentrations in coastal
regions and ship plumes — places where chlor-
ide from sea salt meets pollution. Crucially,
Sticking to sugars
Thornton et al.1 have now observed the same
pathway well away from likely local sources of
Anthony P. Davis
chloride, and provide compelling reasons to If evolution has had trouble making effective carbohydrate receptors,
believe that this chemistry is more widespread
than previously thought. The authors suggest
what hope do humans have of creating synthetic versions? A method for
that the chloride required for the production preparing libraries of such receptors boosts the chances of success.
of ClNO2 could be supplied from a plethora
of man-made and natural sources. Long-term Carbohydrate recognition — the process in receptors for carbohydrates, and are beginning
atmospheric measurements from deposition which receptors bind to specific carbohydrate to succeed. Reporting in Angewandte Chemie,
networks at many locations in the United States molecules — is a difficult task, best left, one Pal et al.1 describe a family of peptide-based
suggest that this might indeed be the case, but might think, to biological molecules that have receptors that incorporates synthetic, carbo-
the sources and amounts of ‘background’ chlor- evolved to do the job. Chemists have neverthe- hydrate-binding amino acids. By screening
ide need to be better quantified to improve our less taken up the challenge of finding synthetic 400 variants of these receptors, the authors
169
© 2010 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS NATURE|Vol 464|11 March 2010

discovered one that selectively binds to a involved two boron-based molecular units11,
tumour-associated carbohydrate that might a OH HO O
selected for their ability to bind diols at neu-
be used as a diagnostic marker for cancer. R1 B + R1 B + 2H2O tral pH. Pal et al. incorporated these units into
The field of supramolecular chemistry OH HO O a short peptide in which two other structural
concerns systems that assemble from mol- Boronic Diol units could be varied (Fig. 1c). By installing
ecules (and/or ions), and therefore involves acid 20 different variants at each of the two adjust-
the design of molecules that interact con- able positions, the authors synthesized a library
trollably with other chemical species. Of the b OH OH OH OH of 400 different receptors. They then screened
various goals in the area, carbohydrate recog- OO O the receptors to see which, if any, bound to
HO OR2 TF antigen
nition in water is among the most challeng- HO HN TF antigen.
ing. A typical carbohydrate, such as glucose, is O Although most of the library showed no sign
festooned with hydroxyl (OH) groups, which of binding, a few members were active. The best
can make strong hydrogen bonds to recep- c O O structure (Fig. 1c) bound to TF antigen rela-
tors in non-polar solvents. But in water, which B OH HO B tively weakly when compared with biological
has hydroxyl groups of its own, carbohydrate receptors, but its performance compared well
hydroxyl groups merely serve as camouflage: with other state-of-the-art synthetic receptors.
although carbohydrate hydroxyls can form NH HN O Moreover, Pal et al. found that carbohydrates
O
O H O H O
hydrogen bonds to receptors in aqueous solu- R3 N N O other than TF antigen did not bind to this
tion, they must displace water molecules in the N N N receptor, unless they were closely related in
H O H O H
process. This creates an extra energetic hurdle structure to TF antigen.
to binding compared with the same process in When the authors made a compound that
non-polar solvents. MeO mimicked the structure of their best receptor,
To create a driving force for carbohydrate but which lacked boron atoms, they observed
recognition in water, substrates must there- Figure 1 | Synthetic carbohydrate receptors.  that the affinity of TF antigen for this com-
fore fit exceptionally well into their receptors. a, Boronic acids react reversibly with diol pound was reduced — as would be expected
Evidence suggests that even biological recep- groups in molecules. B is boron, R1 can be any if the boron atoms are involved in substrate
tors find this requirement difficult to achieve, organic group. b, Diol groups are ubiquitous in binding. Intriguingly, however, the reduction
as the binding of carbohydrates to lectins (the carbohydrates, such as TF antigen, a tumour- in affinity was not as much as would have been
associated disaccharide. One of the diol groups is
main class of carbohydrate-binding proteins) highlighted in blue. R2 can be a lipid or a protein.
anticipated. This implies that substrate binding
is often quite weak2. c, Pal et al.1 have made a library of 400 synthetic involves not only the boron-containing groups,
The complexity of carbohydrate structures carbohydrate receptors. The example shown but also non-covalent interactions with other
increases the problem even further. The build- is the receptor that binds most strongly to TF parts of the receptor.
ing blocks of carbohydrates are sugars known antigen. The boroxole groups (red) react with Screening of Pal and colleagues’ synthetic
as monosaccharides. Unlike proteins or nucleic diols in carbohydrates in a variant of the reaction receptor1 against a wide range of disaccharide
acids, which form only one kind of bond shown in a. The green parts of the molecule are structures is required to show that it is genu-
between their building blocks, there are sev- structural components that are varied to produce inely selective for TF antigen. It may also be
eral different ways in which monosaccharides other library members. R3 is a polymeric group necessary to increase the affinity of this class
that is used to help dissolve the receptor, as
can link together. A small set of these mono- of receptor for the target antigens before they
required for carbohydrate-binding assays.
mers can therefore generate a huge range of can be used in practical applications. Never-
oligosaccharide structures3. Such complexity theless, the success of Pal and colleagues’ strat-
is clearly a headache for chemists trying to reversible reaction between boronic acids egy for discovering carbohydrate receptors is
make synthetic carbohydrate receptors, but it (a class of boron-containing compounds) and encouraging — there is no obvious reason why
is useful in nature — where oligosaccharides diol groups (pairs of OH groups that are ubiqui- receptors for other oligosaccharides could not
provide efficient systems for information stor- tous in carbohydrates), thus making receptors be found in this way, either by screening the
age, and are used extensively as labels for cells that form covalent bonds to their substrates8,9. existing library of receptors or by synthesizing
and proteins4. Carbohydrate recognition thus This reaction (Fig. 1a) proceeds well in water, a different set of compounds based on a modi-
has roles in such diverse processes as fertili- but it often requires high pH to work, a con- fied general design. If this new method lives
zation, infection, cell–cell recognition and dition that isn’t compatible with biological up to its promise, carbohydrate recognition ‘on
protein transport. processes. What’s more, it has proved difficult demand’ may be just around the corner. ■
But synthetic carbohydrate receptors are to develop highly selective receptors based on Anthony P. Davis is in the School of Chemistry,
worth the effort involved in making them, as this process. University of Bristol, Bristol BS8 1TS, UK.
they hold great potential. For example, they In spite of the problems, recent results6 sug- e-mail: anthony.davis@bristol.ac.uk
could be used in diagnostic methods to detect gest that particular carbohydrates can indeed
carbohydrate biomarkers of disease, comple- be targeted by carefully designed synthetic 1. Pal, A., Bérubé, M. & Hall, D. G. Angew. Chem. Int. Edn 49,
1492–1495 (2010).
menting the limited supply of natural lectins. receptors. There remains, however, the issue of 2. Toone, E. J. Curr. Opin. Struct. Biol. 4, 719–728 (1994).
Perhaps more importantly, if receptors can be generality — with so many targets of interest, 3. Laine, R. A. Glycobiology 4, 759–767 (1994).
made that inhibit naturally occurring carbo- we really need a modular receptor system that 4. Gabius, H.-J. (ed.) The Sugar Code: Fundamentals of
hydrate–protein interactions, they would open can adapt to bind to any given carbohydrate. Glycosciences (Wiley, 2009).
5. Davis, A. P. & Wareham, R. S. Angew. Chem. Int. Edn 38,
up new avenues for drug discovery. Pal et al.1 have succeeded in making just such a 2978–2996 (1999).
Supramolecular chemists have therefore system by merging the two main approaches of 6. Kubik, S. Angew. Chem. Int. Edn 48, 1722–1725 (2009).
worked on carbohydrate recognition for about carbohydrate-receptor design, and by using the 7. Ferrand, Y., Crump, M. P. & Davis, A. P. Science 318,
619–622 (2007).
20 years. Broadly speaking, they have split into technique of combinatorial chemistry. Their 8. James, T. D., Sandanayake, K. R. A. S. & Shinkai, S. Angew.
two groups. The first group has devised recep- target substrate is TF antigen — a disaccharide Chem. Int. Edn Engl. 35, 1910–1922 (1996).
tors that rely solely on non-covalent inter- composed of galactose and N-acetylgalactos- 9. James, T. D., Phillips, M. D. & Shinkai, S. Boronic Acids in
actions with their substrates5 — an approach amine sugars that is present in 90% of human Saccharide Recognition (R. Soc. Chem., 2006).
10. Yu, L.-G. Glycoconjugate J. 24, 411–420 (2007).
that, until recently6,7, has had limited success cancers (Fig. 1b)10. 11. Bérubé, M., Dowlut, M. & Hall, D. G. J. Org. Chem. 73,
in water. The second group has exploited a The authors’ general design for receptors1 6471–6479 (2008).

170
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NATURE|Vol 464|11 March 2010 NEWS & VIEWS

SEX DETERMINATION

An avian sexual revolution

Lindsey A. Barske and Blanche Capel
Hormones are not all-powerful in determining whether birds develop with
male or female features. Chickens that are genetic sexual mosaics reveal
that individual cells also have a say in the matter.

Male and female vertebrates have long been heterogametic (ZW). The authors analysed the
thought to owe their distinct sexual charac- sex-chromosome genotype of numerous tissues
teristics — their sexual phenotypes — to the from each side of the body, expecting to find
action of hormones produced by the gonads. evidence of aneuploidy. Instead, all assayed tis-
This basic paradigm maintains that sex deter- sues were mixtures of ZW and ZZ cells. Tissues
mination occurs during embryonic develop- from the ‘male’ side of the body were biased
ment when genetic or environmental factors in favour of ZZ cells, whereas ‘female’ tissues
instruct the bipotential gonads to differentiate had higher proportions of ZW cells. Because all
as ovaries or testes1,2. These organs, in turn, tissues in the body are exposed to an identical
secrete hormones that secondarily instil a hormonal milieu, the authors argue that cells
female or male phenotype in the reproductive must respond to hormones differently depend-
tract and brain — and, in species with external ing on their sex-chromosome constitution — in
sexual dimorphism, in tissues such as muscle other words, they have cell-autonomous sexual
and feathers. According to this view, sex- identity. These results build on an analysis of
chromosome genotype is relevant only in the a zebra finch gynandromorph that was also
gonadal cells where primary sex determination composed of Z- and W-bearing cells6. Like the
occurs, because non-gonadal cells adopt a sex- chicken gynandromorphs, the zebra finch dis-
ual phenotype according to the hormones they played bilaterally asymmetric male and female
sense rather than their own complement of phenotypes, which, in this case, extended to
sex chromosomes1. On page 237 of this issue, neural and behavioural traits. Figure 1 | Funky chicken. Bilateral chicken
however, Zhao et al.3 argue that, in birds, sex To test their sexual-identity hypothesis, gynandromorphs, such as those studied by
determination occurs in cells across the entire Zhao and colleagues transplanted donor Zhao et al.3, are composed of cells of both sexual
body, not just in the gonads. tissue labelled with green fluorescent protein genotypes — ZZ (male, blue) and ZW (female,
Mammals use an XX/XY system to deter- into unlabelled hosts to construct same-sex or pink). These mosaics resolve into male and
mine sex, whereby the Y-chromosome-linked mixed-sex chimaeric embryos. As expected, female patterns of coloration, wattle length and
comb height, despite exposure to an identical
Sry gene activates testis differentiation. If an Sry donor cells integrated into host gonads, but
hormonal environment. This suggests that avian
transgene is expressed in gonadal cells of an XX ZW donor cells did not adopt Sertoli (male) cells adopt a sexual identity on the basis of their
embryo, the embryo will form testes and dif- cell fate and were not incorporated into testis sex-chromosome genotype, not the gonadal
ferentiate as a phenotypic male, even though all cords of ZZ hosts. Similarly, ZZ donors never hormones they sense. (Concept by L. A. Barske.)
of its cells are XX. There is one clear exception: expressed the female marker aromatase in a
marsupial mammals, in which differentiation host ovary, suggesting that their sexual iden-
of a pouch or scrotum occurs before gonad dif- tity was already fixed by the time they arrived development sexual identity is established;
ferentiation and depends on X-chromosome in the host tissue. whether the proportions or level of mixing
dosage4,5. So animals with two X chromosomes These results contrast with mouse chimae- between ZZ and ZW cells determines whether
develop a pouch, and those with one develop ras established at the eight-cell stage, in which a chimaera develops as a gynandromorph or a
a scrotum. XX (female) cells can adopt Sertoli cell fate and simple phenotypic male or female; and whether
Zhao and colleagues’ challenge3 to the classic XY (male) cells are incorporated into ovarian ZW-biased gynandromorph tissues that retain
model is based on an analysis of avian gynan- follicles7,8. Likewise, human patients with a a large ZZ component (or vice versa) retain a
dromorphs. Gynandromorphs are naturally mixed cellular complement of male and female subtle mosaic character or experience a com-
occurring organisms that have both male and genotypes (46,XX/46,XY) display intersex munity effect that permits resolution into a
female structures, with examples cropping up rather than mosaic phenotypes9, suggesting pure female (or male) phenotype.
among crustaceans, birds and insects. Depend- that the dominant influence on mammalian What factors could establish the sexual
ing on the stage of development at which cells is the hormonal environment (although identity of cells across the body? Genes that
gynandromorphy is established, the division there may be exceptions in the brain10). are sex-linked and differentially expressed
between male and female tissues may fall along Interestingly, another study11 involving arti- early in development are good candidates.
the midline, producing a bilateral gynandro- ficial ZZ/ZW chicken chimaeras found not Smith et al.12 recently offered compelling evi-
morph, or occur in a mosaic pattern across the only that these chimaeras were not gynandro- dence that the Z-chromosome gene DMRT1
body. The condition can result from sex-chro- morphic at sexual maturity, but that, in some acts as a dose-dependent master regulator of
mosome aneuploidy (that is, aberrant loss or cases, ZZ donor cells actually induced ZW male sex-determination in the chicken. But
gain of a sex chromosome) or from abnormal hosts to develop as males with functional testes. DMRT1 is expressed at detectable levels only
fertilization events. This discrepancy might be explained by differ- in the gonads13 and is therefore unlikely to
Zhao et al. investigated the cellular basis ences in the developmental stage at which the determine sexual identity in other tissues. In
of this phenomenon in three chicken gynan- transplant was performed, the degree of mix- species such as the fruitfly Drosophila and
dromorphs with bilateral asymmetry (Fig. 1). ing between donor and host cells, or the pro- the nematode Caenorhabditis elegans, sex-
Birds have ZZ/ZW sex chromosomes, where portional representation of the two cell types. chromosome dosage compensation is closely
males are homogametic (ZZ) and females are Future work should determine when during linked to the molecular pathway that controls
171
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NEWS & VIEWS NATURE|Vol 464|11 March 2010

sex determination, such that each cell estab- Department of Cell Biology, Duke University 8. Burgoyne, P. S., Buehr, M. & McLaren, A. Development 104,
683–688 (1988).
lishes its sexual identity at the same time that Medical Center, Durham, North Carolina 27710,
9. Krob, G., Braun, A. & Kuhnle, U. Eur. J. Pediatr. 153, 2–10
X-chromosome dosage is regulated14. However, USA. (1994).
even in Drosophila, a cell’s sexual identity can e-mail: b.capel@cellbio.duke.edu 10. Arnold, A. P. J. Neuroendocrinol. 21, 377–386 (2009).
be overridden by secreted signals15. 11. Kagami, H., Clark, M. E., Verrinder Gibbins, A. M. & Etches,
1. Jost, A. Phil. Trans. R. Soc. Lond. B 259, 119–130 (1970). R. J. Mol. Reprod. Dev. 42, 379–387 (1995).
How should Zhao and colleagues’ results3 be 2. Lillie, F. R. Proc. Natl Acad. Sci. USA 3, 464–470 (1917). 12. Smith, C. A. et al. Nature 461, 267–271 (2009).
reconciled with the substantial body of work 3. Zhao, D. et al. Nature 464, 237–242 (2010). 13. Raymond, C. S., Kettlewell, J. R., Hirsch, B., Bardwell, V. J. &
4. O, W.-S., Short, R. V., Renfree, M. B. & Shaw, G. Nature 331, Zarkower, D. Dev. Biol. 215, 208–220 (1999).
involving hormone-induced sex reversal in 716–717 (1988).
chickens? Seventy-five years of research have 5. Renfree, M. B. et al. Phil. Trans. R. Soc. Lond. B 322, 41–53 (1988). 14. Cline, T. W. & Meyer, B. J. Annu. Rev. Genet. 30, 637–702
6. Agate, R. J. et al. Proc. Natl Acad. Sci. USA 100, 4873–4878 (1996).
definitively demonstrated that oestrogen is 15. DeFalco, T. et al. Dev. Cell 14, 275–286 (2008).
(2003).
necessary and sufficient for female develop- 7. Palmer, S. J. & Burgoyne, P. S. Development 112, 265–268 16. Smith, C. A. & Sinclair, A. H. BioEssays 26, 120–132 (2004).
ment: ZZ (male) embryos exposed to oestro- (1991). 17. Wolff, E. & Wolff, E. J. Exp. Zool. 116, 59–97 (1951).
gen develop as females, whereas ZW (female)
embryos depleted of oestrogen develop as
males16. ZZ ‘females’ revert to a male pheno-
type at puberty if the hormone treatment is dis- COSMOLOGY
continued16, possibly because ZZ cells cannot
produce enough oestrogen to maintain ovarian
structures. By contrast, adult ZW ‘males’ have
Gravity tested on cosmic scales
fairly normal-looking testes, indicating that,
when oestrogen is absent, ZW cells can become
J. Anthony Tyson
Sertoli cells and organize into male patterns. Einstein’s theory of general relativity has been tested — and confirmed
Do these hormone treatments simply override
cell-autonomous sexual identity? Investigation
— on scales far beyond those of our Solar System. But the results don’t
into the adult phenotypes of castrated chick exclude all alternative theories of gravity.
embryos should show whether an underlying
sexual identity does indeed exist in the absence Our understanding of the physics that under- accounted for a wide range of observations1.
of hormones. Although this procedure was pre- lies the dynamical evolution of the Universe But the physics of dark matter and dark energy
viously attempted using irradiation, the animals and the development of cosmic structure is is not understood. Although there are sugges-
did not survive to hatching17. driven by astronomical observations. His- tions from particle physics about the nature
A final question is whether cell-autonomous torically, measurements on galaxy and larger of dark matter, that of dark energy remains a
sexual identity will turn out to be a common cosmological scales conflicted with predic- mystery.
element in the arsenal of sex-determining tions based on a cosmological model that Frustrated by the lack of theoretical candi-
systems in vertebrates, with variable influ- combined Albert Einstein’s theory of gravity dates for dark energy, some researchers have
ence on the outcome of sexual differentiation. (general relativity) and the standard model of instead considered models in which the Uni-
Perhaps it will. These funky chickens, oddities particle physics. Modifications to this model verse’s dynamics, and so gravity itself, devi-
of nature that they are, may well provide new were later introduced, involving dark matter ates from that predicted by general relativity
perspectives on questions of sexual identity and dark energy (a dilute component that has on ‘cosmological’ scales — those larger than
long thought to have been resolved. ■ been proposed to explain the observed recent galaxies and clusters of galaxies. But how can
Lindsey A. Barske and Blanche Capel are in the acceleration of the Universe’s expansion), that one tell the difference between models that

APPLIED ECOLOGY

CAPMC
Grass and the X factor
Symbiosis between plants and which plant diversity is desirable.
microorganisms is widespread, Like many plants, it does not live
but research into its effects on the alone, and has what might be called
composition of a plant community the X factor — symbionts that in the
is in its infancy. Such studies case of tall fescue often include the
require patience. Jennifer Rudgers fungus Neotyphodium coenophialum.
and colleagues have spent six Like the grass, the fungus comes
years investigating the relative in different genotypic forms. For The authors’ salient finding is The significance of this line of
performance of cultivars of tall example, one common form (KY-31) that Georgia-5 plus AR-542 allows research extends well beyond the
fescue grass with different forms produces alkaloids that are toxic to greatest growth of other grasses and specific performance and application
of a symbiont, and now report the certain herbivores; another form herbaceous flowering plants, and of tall fescue. Plant breeders are
results and their recommendations (AR-542) does not. produces fewest inflorescences (and becoming ever-more imaginative in
for the use of this versatile yet By planting experimental plots, so fewer seeds). Those characteristics exploring the intricate options offered
vexatious grass (J. A. Rudgers et al. Rudgers et al. set out to test, among would be appropriate for applications by different genotypic mixes of plants
J. Appl. Ecol. doi:10.1111/j.1365- other things, how two cultivars of in which tall fescue has a job to do but and symbionts. Ecologists have a big
2664.2010.01788.x). the tall fescue Lolium arundinaceum where diversity of vegetation is the task on their hands in checking the
Tall fescue could well be the plant inoculated with KY-31, AR-542 or aim, for example in preventing soil ecosystem consequences — and in
that makes a lawn near you. It is neither, affect the plant species erosion. There would also be reduced taking into account the many other
also grown for forage (pictured). composition of the plots. The two fescue spread from the planting conditions that affect vegetation
It is highly invasive, however, and cultivars chosen were Georgia-5 site. Jesup with either symbiont is growth and invasiveness.
counts as a weed in situations in and Jesup. preferable for monoculture. Tim Lincoln

172
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NATURE|Vol 464|11 March 2010 NEWS & VIEWS

include dark energy and modified dynamics, they used Tegmark

M. BLANTON & SDSS

12 h
models of gravity? In 2007, Zhang and colleagues’ measurement6
et al. 2 developed a method for h of the distribution of galaxy
distinguishing between these two 16 velocities (the redshift distortion
cases that is minimally affected by effect, which is visible in Figure
our lack of knowledge of some key 1 as radial (redshift) smear-
parameters. In this issue (page 256), ing of many of the (red) galax-
Reyes et al.3 apply Zhang and col- ies). They took care to address
leagues’ method to data from the remaining systematic errors.
Sloan Digital Sky Survey (SDSS; And after applying correction

8h
Fig. 1) and find consistency with factors derived from computer
general relativity and one modi- Redshift (z) simulations, they measured this
fied form of it. diagnostic of departure from

0.02

0.04

0.06

0.08

0.10

0.12

0.14
Reyes and colleagues’ measure- general relativity on scales of
20 h

ments are significant not just about 14–70 Mpc to a precision

because they are consistent within of 16%. Although the results are
error with general relativity, but consistent with general relativity
also because they point the way to and with one class of large-scale
future high-precision tests that will deviation from it, a particular
better distinguish between general example of a model of modified
relativity and some variant models. gravity, termed tensor–vector–
This is important because gravity h scalar, which aims to explain
4
is the least tested of the forces in both dark matter and the recent
nature. The predictions of grav- 0h cosmic acceleration, seems less
ity theory have undergone high- Figure 1 | Slices through the SDSS three-dimensional map of galaxies. Earth likely.
precision tests that have yielded is at the centre, and each point represents a galaxy. The radial coordinate is the Zhang et al.2 pointed out that
remarkable agreement4, but these redshift, and the angular coordinate, in units of hours, is the right ascension the next generation of surveys
were performed only on scales of (the celestial equivalent of longitude). The outer circle is at a distance of about will yield per-cent-level preci-
our Solar System or smaller. The 700 megaparsecs. Redder points denote galaxies composed of older stars. sion on this diagnostic. But these
effects of modified general rela- The regions between the slices are not mapped because dust in the Milky Way future surveys will do even more
tivity on scales a hundred billion obscures the view in these directions. The clustering of galaxies (caused by than that. With billions of gal-
times larger may leak down to the gravitational pull of huge unseen masses of dark matter) can be seen. Also axies charted across 80% of the
small-scale effects, and this could evident is the redshift-space distortion, a radial smearing of3 galaxies due to age of the Universe (100 times
their infall velocities near high-density regions. Reyes et al. combine these
be tested by high-precision meas- measures of the effects of gravity in a sample of galaxies from the Sloan Digital the number of galaxies in Reyes
urements of the Earth–Moon dis- Sky Survey (SDSS), which extends to somewhat higher redshift than those seen and colleagues’ study3), other
tance using laser ranging5. here, with another measure of the dark-matter mass in these cosmic structures: probes of dark energy7,8, which
Departures from general rela- the ‘gravitational lens distortion’ of the shapes of yet more distant galaxy images. could more sensitively test the
tivity on cosmological scales will modified general-relativity alter-
cause the growth of dark-matter native, will become possible. By
structure and the cosmic geometry to differ dark-matter fluctuation will cancel out in the combining observations of the cosmic micro-
from that predicted by Einstein’s theory, in ratio of two different methods for measuring wave background (relic radiation from the Big
ways that are in principle distinguishable. How- the dynamics of a large sample of galaxies. The Bang) with galaxy data, unprecedented studies
ever, a full investigation of any such effects, by two methods involve imaging and spectros- will become possible, such as directly observ-
tracing the dynamical history of the Universe copy. For a population of galaxies, the masses ing the homogeneity of the Universe on large
over cosmic time, is currently fraught with of the huge dark-matter haloes in which they scales. The recent acceleration of the Universe,
systematic errors. Perhaps if general relativ- are embedded can be determined in two ways: whether caused by dark energy or a manifes-
ity breaks down on scales of clusters of galax- from the distribution of galaxy velocities (via tation of a modification of gravity on scales
ies, there is sufficient information encoded in spectroscopy) and from the correlation of the a hundred billion times larger than the Solar
the dynamics and the mass of a large sample galaxies with the weak distortion of the shapes System, presages new physics. Experiments
of galaxies to detect this. Under the opposing of background galaxies by the effect of gravita- in the next decade promise even greater
influences of gravity due to dark matter and tional lensing (via imaging). Modified theories insight into the fundamental physics of the
the cosmic expansion, there is a natural rela- of gravity affect these two quantities differently Universe. ■
tionship between the clustering of galaxies, from Einstein’s gravity theory, producing an J. Anthony Tyson is in the Department of Physics,
the mass of the underlying dark matter and the observable change in their ratio compared with University of California, Davis, One Shields
velocity distribution of the galaxies that could the general-relativistic prediction. Avenue, Davis, California 95616, USA.
be used to test general relativity. But there are In a first measurement of this diagnostic, e-mail: tyson@physics.ucdavis.edu
challenges in going from observations of these Reyes et al.3 used imaging and spectroscopy of
properties to a test of any departure from grav- more than 70,000 luminous red galaxies in the 1. Spergel, D. N. et al. Astrophys. J. Suppl. Ser. 170, 377–408
(2007).
ity theory on large scales. SDSS at a mean distance of about 1,700 mega-
2. Zhang, P., Liguori, M., Bean, R. & Dodelson, S. Phys. Rev.
One problem is that galaxies are a biased parsecs. Clustering of these galaxies was meas- Lett. 99, 141302 (2007).
tracer of dark matter, and this bias varies with ured by correlating their relative positions in 3. Reyes, R. et al. Nature 464, 256–258 (2010).
scale. Another is that we do not know to suf- space. To calculate one measure of dynamics, 4. Will, C. M. Space Sci. Rev. 148, 3–13 (2009).
ficient precision how the density of dark matter the authors determined the mass versus scale 5. Murphy, T. W. Jr Space Sci. Rev. 148, 217–223
(2009).
fluctuates across space in the Universe. Zhang for the underlying dark-matter assembly by
6. Tegmark, M. et al. Phys. Rev. D 74, 123507 (2006).
et al.2 recognized that, if a combination of means of weak gravitational lensing, using the 7. Jain, B. & Zhang, P. Phys. Rev. D 78, 063503 (2008).
several types of measurement is used, the bias shapes of 30 million background galaxies, also 8. Song, Y.-S. & Koyama, K. J. Cosmol. Astroparticle Phys.
and other poorly known quantities such as the in the SDSS. For the second measure of the doi:10.1088/1475-7516/2009/01/048 (2009).

173
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www.nature.com/nature Vol 464 | Issue no. 7286 | 11 March 2010

EXOTIC MATTER

G
Cover illustration ases, liquids and solids are the three most REVIEW
Artwork by N. Spencer common types, or phases, of matter that
176 The enigma of supersolidity
we might think about day to day. But
these are not the only phases into which S. Balibar
matter can be classified. Consider, for instance, metals,
antiferromagnets, ferroelectrics, liquid crystals and PERSPECTIVE
Bose–Einstein condensates or the many solid phases
183 Superconductivity gets an
into which liquids can crystallize.
iron boost
There are numerous ways in which a large number
Editor, Nature
of particles can be ordered, whether the particles are I. I. Mazin
Philip Campbell
Publishing
fundamental (such as electrons) or composite (such
Nick Campbell as atoms and molecules). This ordering is determined REVIEW
Claudia Deasy by the quantum nature, mutual interactions and 187 Non-Abelian states of matter
Insights Editor symmetries that govern the particles. Crucially,
Karl Ziemelis A. Stern
the macroscopic properties of matter usually differ
Production Editor substantially from those of its microscopic constituents.
Davina Dadley-Moore PERSPECTIVE
In fact, numerous complex many-body systems show
Senior Art Editor
Martin Harrison
emergent phenomena that are associated with the 194 The birth of topological
Art Editor
‘whole’ but that cannot be understood solely in terms insulators
Nik Spencer of the fundamental laws that govern their microscopic
J. E. Moore
Sponsorship ‘parts’. This complexity is perhaps best encapsulated
Amélie Pequignot in Philip Anderson’s ‘more is different’ idea, from his
Reya Silao often-quoted 1972 Science article, and is part of the REVIEWS
Production challenge and the beauty of condensed-matter physics. 199 Spin liquids in frustrated
Jocelyn Hilton
In this Insight, we expose examples of matter magnets
Marketing
Elena Woodstock
for which ‘more is different’ holds true in striking L. Balents
Emily Elkins ways. We venture into the enigmatic world of
Editorial Assistant supersolids and superconductors, as well as into
Emma Gibson the frustrated landscapes inhabited by spin liquids 209 Electron liquids and solids in
and spin ice. We take a look at the one-dimensional one dimension
confines of nanowires and carbon nanotubes, where V. V. Deshpande, M. Bockrath,
correlated electrons form liquids and solids with L. I. Glazman & A. Yacoby
unique properties that are determined by their low
dimensionality. And we dive deep into the exotic
realms of topological insulators and non-Abelian states
of matter, where quantum effects come with a twist.
We hope that these articles provide a window onto
the world of many-body physics, which controls
myriad aspects of the world around us, and we
thank the authors and reviewers for their invaluable
contributions.
Dan Csontos, Associate Editor

175
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010|doi:10.1038/nature08913

The enigma of supersolidity

Sebastien Balibar1

A ‘supersolid’ is a quantum solid in which a fraction of the mass is superfluid. As a remarkable consequence, it is
rigid, but part of its mass is able to flow owing to quantum physical processes. This paradoxical state of matter
was considered as a theoretical possibility as early as 1969, but its existence was discovered only in 2004, in
4
He. Since then, intense experimental and theoretical efforts have been made to explain the origins of this exotic
state of matter. It now seems that its physical interpretation is more complicated than originally thought.

Quantum properties of matter are paradoxical, especially when they beyond some threshold it may enter as so-called magnetic flux quanta,
show up at the macroscopic level. This is true for superfluidity (Box 1), which are quantized vortices of electrical charges.
and even more so for the newly found supersolidity of solid 4He. A solid In a classical solid, each individual particle resides on one particular
is rigid because its atoms (or molecules) occupy particular positions in site — a lattice site if the solid is a crystal — and is localized such that the
space — they are ‘localized’ — and are ‘distinguishable’, which means crystal is rigid and responds elastically to shear stress. A classical solid
that an atom at one position can be distinguished from another at a dif- inside a box is forced to rotate with the box walls when the box rotates.
ferent position. By contrast, a superfluid moves without friction because However, in a quantum solid, particles fluctuate a lot around their aver-
it is a collective wave of matter in which atoms are indistinguishable and age positions, with the result that atoms may exchange places with their
delocalized. Given this, it is not clear how a system can be ‘supersolid’, neighbours. If this exchange is easy enough, some of the atoms may flow
which means being solid and superfluid at the same time. In a super- through the otherwise rigid network and some of the mass may stay at
solid, part of the mass flows without friction through the rest, which rest while the rest rotates. Eventually, if this flow becomes superfluid,
remains solid, but there is no consensus on how this occurs. As will the solid is said to be supersolid. Some of the mass is delocalized and
become evident, the enigma of supersolidity “continues to defy agreed the remainder is localized. The question to be answered is whether this
theoretical explanation”, as A. J. Leggett said recently1. paradoxical state of matter really exists.
In 2004, Kim and Chan2,3 found some experimental evidence for this Let us start with some early ideas. In 1969, Thouless23 and, independ-
phenomenon in so-called torsional oscillator experiments on solid 4He ently, Andreev and Lifshitz24 considered a quantum crystal in which, at
samples. Since then, several groups have confirmed that anomalies exist low temperatures, there could be ‘vacancies’, that is, empty sites where
not only in the rotation properties4–9 of solid 4He but also in its elastic atoms are missing (Fig. 1). These authors further conjectured that such
properties10–13 and in its specific heat14,15. The whole set of experimental vacancies could behave like mobile quantum particles because they
results strongly supports the hypothesis that solid 4He is indeed a super- could easily exchange their positions with neighbouring atoms through
solid, but the interpretation of the data is probably not as simple as was quantum tunnelling. Anyone who has tried to solve a 16-tile magic-
proposed 40 years ago. square puzzle knows that exchange is much easier in a lattice if there are
There is some consensus among theorists that supersolidity does empty sites. If the atoms are bosons, mobile vacancies should also obey
not exist in perfect crystals16,17 and that defects such as dislocations in Bose–Einstein statistics and could therefore undergo Bose–Einstein
single crystals, grain boundaries in polycrystals or glassy regions are condensation, thus giving rise to superfluid-like behaviour below a cer-
necessary. However, this consensus is not universal, as Anderson18–20 tain temperature (Box 1). Crucially, a flow of vacancies being an inverse
believes that supersolidity can be an intrinsic property of ideal crystals flow of mass, it should be possible to observe the coexistence of solid
that is only enhanced by disorder. The goal of this Review is to sum- properties (an elastic response to shear stress) and superfluid proper-
marize the present status of this intriguing field, to offer some possible ties (a fraction of the mass flowing without friction through the lattice).
interpretations of the observed phenomena and to raise some issues This was the original proposal for the emergence of a new ‘superstate of
whose investigation could help solve the enigma of supersolidity. Three matter’ — a supersolid. Shortly after this suggestion, Leggett explained
extensive review articles have already been published16,21,22, but this sub- that the rotation of this solid should be anomalous, like that of a super-
ject evolves quickly. fluid liquid25. The problem with this explanation is that it relies on the
assumption that there exist vacancies in the zero-temperature limit, and
From superfluids to supersolids that is far from obvious.
A classical liquid is made of atoms (or molecules) that move in a random
manner such as to make the liquid viscous (Box 1). In a superfluid, First sighting of supersolidity
however, atoms move coherently because together they form a macro- The suggestion of a new state of matter was attractive enough that many
scopic wave of matter. An important consequence of this coherence is research groups started looking for it26, but the first indication of its
that superfluids do not rotate like classical liquids: in a slowly rotating existence was found in only 2004, by Kim and Chan2,3. They used a well-
bucket, a superfluid stays at rest; it is not set in rotation by the moving documented method for the detection of anomalous rotation, which had
walls. If the rotation speed is increased beyond a critical value, then been used with superfluids. The experimental set-up consists of a tor-
some rotation appears inside the superfluid in the form of quantized sional oscillator containing a cylindrical cell with an annular space filled
vortices. The same is true for superconductors subjected to magnetic with a sample material, such as liquid helium, suspended from a torsion
fields: a small field is totally excluded from the superconductor but rod (Fig. 2a). At resonance, the period, τ, of the oscillator is related to
1
Laboratoire de Physique Statistique, Ecole Normale Supérieure and CNRS, 24 rue Lhomond, 75231 Paris, France.

176
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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

Box 1 | Superstates of matter

Superfluidity, superconductivity and now was explained in 1951by Penrose55.
supersolidity — the list of superstates of There is a close analogy with electrical
matter is growing. These quantum states conductors. In an ordinary metal, electrical
of matter differ significantly from classical resistance is a consequence of the incoherent
ones. In a classical fluid, particles (atoms or motion of electrons. However, Kamerlingh
molecules) move in a random manner and Onnes56 discovered in 1911 (that is, more
are distinguishable from each other. Owing than two decades before the discovery
to interparticle interactions, a classical fluid of superfluidity) that at a sufficiently
has a non-zero viscosity. However, in 1937 low temperature, some metals enter a
in Cambridge, UK, Allen and Misener49 ‘superconducting’ state in which the electrical
discovered that below a temperature of 2K, resistance vanishes. In 1957, Bardeen, Cooper
liquid 4He enters a new state, which moves and Schrieffer explained this phenomenon
without friction — a state named ‘superfluid’ as being due to the formation of a BEC of
by Kapitza50, who made similar observations electron pairs57,58 (the so-called BCS theory
at the same time in Moscow51. A few months of superconductivity) — single electrons
later, Allen and Jones52 made spectacular are fermions obeying Fermi statistics but
images of fountains of superfluid 4He. The become bosons when paired up. Similarly,
image (courtesy of J. F. Allen, University of St the superfluidity of liquid 3He was discovered
Andrews) shows the famous ‘fountain effect’ near 2mK by Osheroff, Gully, Richardson and
(photographed in 1972). The inviscid flow of Lee59,60 in 1972, and it was shown that because
superfluid 4He is produced by a small heater 3
He atoms are fermions, they also need to form
below the neck of the glass bottle. This motion pairs to achieve a superfluid state. During the
could not be explained using the classical past two decades, it has also been shown that
laws of thermodynamics and hydrodynamics, a similar BEC/BCS type of superfluidity can
and triggered the proposal by London53 and be realized in bosonic61,62 and fermionic atom
Tisza54 that superfluid 4He had to be a quantum gases. In fermionic atom gases, it has also been
fluid in which atoms are indistinguishable shown how the superfluid transition can be
and accumulate in the same state in such a tuned continuously from BCS pairing to a BEC
way that they all move together in a coherent of molecules as a function of the intensity of the
manner — a phenomenon called Bose–Einstein interactions between the atoms.
condensation (BEC) that pertains to bosons. As discussed in this Review, the great
Helium-4 atoms are bosons, and it is now surprise today has been to find that a quantum
generally accepted that superfluidity is solid may also be superfluid — a new state of
associated with a generalization of BEC that matter called a ‘supersolid’.

the rotational inertia, I, through the relationship τ = 2π(I/K)1/2, where Κ and Lifshitz24, or a phenomenon solely dependent on the presence of
is an elastic constant determined mainly by the rigidity of the rod. The defects in the studied samples. The crucial point thus became determin-
tell-tale sign of the superfluid transition is a shift in the resonance period ing whether there were vacancies in the ground state of a perfect 4He
of the oscillator because the inertia decreases when the material becomes crystal, that is, a crystal in equilibrium near T = 0 K, and whether the
superfluid — the superfluid liquid stays at rest while the walls of the box presence of such vacancies could explain the observed anomalies.
oscillate. The amplitude of the period shift thus varies with temperature, Several theorists looked at the energy cost and gain associated with
T, as the fraction of the liquid that is superfluid increases from zero to creating vacancies in a crystal. On the one hand, removing one atom
one as T decreases from the superfluid transition temperature to zero. from a crystal lattice costs potential energy because bonds between
Furthermore, this period shift is observed only for oscillation velocities atoms must be broken. On the other hand, if the vacancies are delocal-
smaller than a certain critical velocity (typically of the order of 1 cm s−1), ized, they are shared by all atoms instead of being confined inside a ‘cage’
beyond which dissipation starts and superfluidity is destroyed. made by nearest neighbours (Fig. 1). Heisenberg’s uncertainty principle
The remarkable discovery by Kim and Chan in 2004 was that they stipulates that a confinement in position space results in a large uncer-
observed a similar shift in the oscillator period in solid, not liquid, 4He. tainty in momentum space and, consequently, in a large kinetic energy.
At a temperature of the order of 100 mK, the oscillator period decreased Thus, a delocalization of the vacancies would entail fewer fluctuations
(Fig. 2b), a behaviour that was absent in control experiments with either of their velocity, meaning less kinetic energy; in short, the vacancies
an empty cell or a cell filled with 3He (recalling that 3He atoms are fermi- would gain energy by being delocalized. If the energy gain from the
ons, unlike 4He atoms, which are bosons (Box 1)). This was interpreted delocalization is larger than the potential energy cost of removing one
as a strong indication of supersolidity. The main difference from super- atom from the crystal lattice, the balance is negative and the ground
fluid liquid 4He was that, in Kim and Chan’s sample, the magnitude of state must contain vacancies. In addition, if vacancies repel each other,
the shift indicated a superfluid fraction of the order of 1% of the total they form a dilute gas, which can be superfluid. In such circumstances,
4
He mass, but it seemed that at a sufficiently low temperature, and in the crystal would still be periodic in position space, but the number of
agreement with Leggett’s prediction, solid 4He did not rotate like a clas- atoms would be less than the number of lattice sites; the crystal would
sical solid. be ‘incommensurate’, and some of its mass, in the form of atoms jump-
ing between the lattice sites owing to the presence of vacancies, could
The nature of supersolidity flow without friction. Below some superfluid transition temperature, it
Kim and Chan’s discovery2,3 triggered a period of intense activity, with would thus become supersolid.
several experimental groups trying to reproduce and better understand For a long time, the simple vacancy-based model of supersolidity
the data and many theorists trying to explain the observed phenomenon looked possible. However, post-2004 Monte Carlo simulations showed
(see, for example, refs 16, 21 and 22). One of the critical issues early on that the above energy balance is positive and large16,17,27; it was found
was the question of whether supersolidity could be an intrinsic property that the creation of vacancies costs more than 10 K (at the atomic level,
of 4He crystals, as originally considered by Thouless23 and by Andreev energies are often measured in kelvin, a temperature unit that translates
177
© 2010 Macmillan Publishers Limited. All rights reserved
INSIGHT REVIEW NATURE|Vol 464|11 March 2010

into energy according to E = kBT, where kB is the Boltzmann constant). In any event, it became clear that the magnitude of the supersolid frac-
Because 10 K is much larger than 0.1 K, the typical temperature at which tion strongly depends on the amount of disorder.
supersolidity was observed, the probability of vacancies existing under The other type of experiment was pursued by Sasaki et al.31,32, who,
experimental conditions should be negligible, and they could not be instead of investigating a.c. mass flow in torsional oscillator experiments,
responsible for the observed phenomena. decided to look for d.c. mass flow through a static, solid sample. To this
end, they built a two-part sample cell containing various types of 4He
Importance of disorder crystals in equilibrium with liquid 4He. Using an optical cryostat, they
Despite some disagreement18–20,22 about the validity of the Monte Carlo monitored the respective levels of the liquid–solid interface in the two
simulations mentioned above, it was then proposed that supersolidity parts of the cell and measured their relaxation to the same height in the
might instead be caused by the presence of defects in the solid samples cell. As any level change would necessitate mass flow through the solid
under study. Two possible types of defect were suggested, dislocation because of the different densities of liquid and solid 4He, such an equilibra-
cores and grain boundaries (Fig. 3). One reason for this conjecture was tion would signal supersolid behaviour. The experiments indeed provided
that these are regions of the crystal where local stresses naturally give evidence for superfluid d.c. mass flow, but only in polycrystals containing
rise to the formation of vacancies, thus allowing the exchange of atoms grain boundaries. No such flow was observed in single crystals. This then
and mass flow. Indeed, theoretical studies predicted28,29 that part of the seemed to imply that grain boundaries are necessary for supersolidity.
mass inside defects could, under the right circumstances, become super- However, as indicated below, the lowest temperature that the experi-
fluid and propagate along dislocations and grain boundaries connected ments by Sasaki et al.31,32 reached, 50 mK, today seems not to have been
in a three-dimensional network, thereby giving rise to the observed low enough to rule out the possibility of mass flow in single crystals.
supersolid behaviour at the macroscopic level. Following the experiments by Sasaki et al., more evidence was found
At this stage, two types of experiment seemed to confirm that disorder that the few atomic layers forming grain boundaries might be super-
does have a crucial role in the supersolid-like behaviour. One line of solid33; however, evidence was also found that some mass could flow
experiments continued the exploration of alternating (a.c.) mass flow along liquid channels forming at the interface between grain boundaries
using torsional oscillators. Although Rittner and Reppy4,5 reproduced and the cell walls or at points where grain boundaries intersect with each
Kim and Chan’s findings2,3, they also found that in annealed samples other34,35. In my opinion, the existence of d.c. mass flow inside single
(heat treatment typically reducing the defect density in crystals) the rota- crystals of 4He is not yet firmly established.
tion anomaly called ‘non-classical rotational inertia’ (NCRI) decreased As the growth of solid samples at constant volume can lead to the
to below the noise level in the measurements. By contrast, when per- formation of polycrystals with a high density of grain boundaries, in light
forming the same type of measurement on quench-frozen samples, that of the above discussion the question became that of whether the observed
is, in the presence of large disorder, the NCRI was found to be as large as supersolid behaviour in torsional oscillator experiments could also be
20% of the total helium inertia. Subsequent experiments by the group of attributed to mass flow along grain boundaries. To answer this question,
Chan30 later confirmed that annealing reduces the NCRI. However, this Chan’s group made new measurements30 using torsional oscillators in
group found that even in their best samples, a very small fraction of the which the cell was filled with single crystals of 4He. They found that the
mass, of the order of a few parts in 104, still decoupled from the oscillat- period shift is smaller than in the corresponding experiments on poly-
ing walls. Annealing did not completely suppress the rotation anomaly. crystals but is non-zero, indicating the presence of a supersolid fraction
of 0.04–0.4%. It is therefore possible that although grain boundaries could
be responsible for the supersolidity observed in polycrystals, supersolid-
ity could also be exhibited by single crystals, in which the origin of the
vacancy-induced mass flow more probably could be due to dislocations.
However, the interpretation of the data was to receive yet another twist.

Pinning at 3He impurities

The first experiments by Kim and Chan2 had already shown that the
phenomenon of supersolidity was very sensitive to the presence of tiny
amounts of 3He impurities. Helium-4 is extracted from oil wells where
it accumulates owing to the radioactive decay of uranium in the Earth.
In natural 4He, there are about 0.3 parts per million (3 × 10−7) of the
light isotope 3He. This may not seem much, but at low temperatures
the quantum statistical nature of particles (4He atoms being bosons
and 3He atoms being fermions) becomes important. This fundamental
difference in quantum behaviour makes 3He behave like a significant
impurity in a 4He system, even at such low concentrations.
The concentration of 3He in 4He can be controlled. In fact, it is possible
to prepare ultrapure 4He gas in which the concentration of 3He is only
one part per billion (10−9). Experiments have found that the supersolid
transition temperature depends on the presence of 3He, even at such
extremely low concentrations10,11,36. Because previous experiments had
shown that 3He impurities adsorb on dislocations at low temperatures37,38,
it was then suggested that the whole phenomenon could be attributed to
Figure 1 | Quantum tunnelling of vacancies. In the original model of how the properties of defects such as grain boundaries and dislocations,
supersolidity, the number of atoms is less than the number of lattice sites. discussed above, are modified by the presence of 3He atoms.
The lattice thus contains vacancies (red circles), which are able to exchange Unexpectedly, measurements in 2007 by Day and Beamish10 and Day,
their positions with neighbouring atoms (blue dots) so easily that they Syshchenko and Beamish11 found that the shear modulus, μ, increased
are delocalized throughout the whole system. These vacancies are bosons,
at the onset of supersolidity (Fig. 4) — when the mass started to flow,
which at low temperatures may form a Bose–Einstein condensate, that is,
a macroscopic wave that extends throughout the system. As a result, the they found that the solid 4He became stiffer. This was surprising, as the
atoms themselves are not localized at particular sites as in a classical lattice. naive expectation is that if part of the mass of a crystal starts flowing, the
Some of the mass can flow without friction through the rest, which remains quantum solid should somehow more closely resemble a liquid, such
a rigid solid. that its shear modulus should decrease, contrary to what was observed.
178
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

a Figure 2 | First experimental evidence of

Base plate: thermal contact
to the refrigerator supersolidity in 4He. a, Schematic view of the
Magnesium barrier experimental set-up in a typical torsional oscillator
(control experiment)
experiment by Kim and Chan2,3. The oscillating cell
Torsion rod Filling line contains helium in an annular channel where the
(and filling line)
circulation can be blocked by inserting a vertical
magnesium barrier. The helium is introduced in the
Magnesium cell through a filling line that runs vertically inside
disk the torsion rod. Electrodes are used to drive and
Oscillating cell
Aluminium
detect the oscillation of the cell, and consequently to
shell measure its resonant period. This period is expected
Electrodes to decrease when solid helium becomes supersolid
because a fraction of the helium mass stops moving
with the oscillating walls of the box. This fraction
Solid helium is found to be 0.01–20%, depending on sample
Detection
in annular channel quality. When the magnesium barrier is inserted,
Drive the period change disappears. b, Left: one of the first
measurements by Kim and Chan2, showing that the
b period, τ, of their torsional oscillator decreases at
Empty-cell background temperatures, T, below about 0.1 K when filled with
920 Solid helium (31 Nm s–1) solid 4He, relative to when the empty cell is used.
0.015
4 Nm s–1 The reference period, τ*, is chosen as 971,000 ns
6 Nm s–1 for convenience. The maximum velocity of the
910 0.012 14 Nm s–1 cell rim in the experiment with 4He is as indicated.
33 Nm s–1
t – t* (ns)

Right: measurements, made by the same authors3,

0.009 117 Nm s–1 where the period change is converted into the non-
NCRIF

900 420 Nm s–1 classical rotational inertia fraction (NCRIF) of the

4
0.006 He sample. At low temperatures, the NCRIF is of
the order of 1%. Its value depends on the maximum
890 velocity of the cell rim (as indicated) when this
0.003
velocity is greater than 6 μm s−1. (Panel courtesy
of M. H. W. Chan, Pennsylvania State University,
880 0 University Park, Philadelphia.)
0.02 0.04 0.1 0.2 0.4 1 2 0.02 0.04 0.1 0.2 0.4
T (K) T (K)

These experiments further found that μ increased at temperatures below Beamish collaborated to check this possibility41. They built a very strong
the same critical temperature at which the rotational inertia began to cell in which the helium contribution to K was negligible. They found
decrease in Kim and Chan’s2,3 experiments. In fact, the shape of the tem- a definite period shift that had to be the consequence of a decrease
perature variation of μ looks exactly the same as that of the NCRI, and in the rotational inertia41. Furthermore, they proved that the rotation
even the dependence of the transition temperature on 3He impurity anomaly is indeed associated with the quantum statistics of the 4He;
content was found to be the same (Fig. 4). they found a similar elastic anomaly in solid 3He, a fermionic system,
Thus, these experiments10,11 showed that the anomalies observed in but did not observe a rotation anomaly there (see ref. 41 for details on
both the rotational and elastic properties of solid 4He have a common the importance of the crystal structure). Supersolidity thus seemed to
origin, which prompted Day and Beamish10 to interpret the stiffen- have been confirmed.
ing as follows. In a quantum crystal, dislocations can be highly mobile The existence of supersolidity had in fact already been strongly
because they may have mobile kinks (Fig. 5). Kinks are regions where supported by two other types of measurement. One type was the
the dislocation lines shift from one atomic row to the next. Quantum so-called blocked annulus experiment. Kim and Chan3 and later Rit-
fluctuations may allow kinks to tunnel from one lattice site to the next, tner and Reppy42, with more accuracy, compared a torsional oscillator
and so induce motion of the whole line. Consequently, in measure- with a free annular space containing solid helium with a similar one in
ments of the stiffness the response of the system to an applied stress is which the annulus was blocked along a diameter so as to interrupt the
the sum of the usual lattice deformation plus a contribution due to the macroscopic circulation of mass (Fig. 2). In the experiments with a free
displacement of the dislocations. However, the mobility of dislocations annulus, a rotation anomaly signalling loss of inertia was found. In the
should depend on temperature and on the 3He concentration because blocked case, however, such an anomaly was not observed. Even though
3
He impurities should bind to dislocations and pin them locally below a Reppy recently presented some puzzling results from measurements in
certain temperature. Below this temperature, which could be of the order a more complicated cell geometry43, I believe that the rotation anomaly
of 0.1 K, the crystal should therefore be more rigid. This interpretation observed in torsional oscillator experiments in general is due to a mac-
looked likely to be correct, but it left open an important question, that roscopic superflow. The other supporting experiments were done by
of the mechanism by which supersolidity is associated to the pinning Lin and colleagues14,15. They found a peak in the specific heat of solid
of dislocations. helium at the temperature at which the rotation anomaly occurs. This
is a strong indication that the phenomenon is a true phase transition,
Torsional oscillator experiments revisited despite there being discussions about the magnitude of the peak relative
In view of the above developments, the very existence of supersolidity to that of the inertia change44.
was once again questioned. One proposal39,40 suggested that the anoma- At this point, I would like to mention a few interesting results related
lies seen in torsional oscillator experiments could in fact be an arte- to the response time and frequency dependence of the torsional oscil-
fact — it was suggested that the period shift perhaps was not due to a lator experiments. It has been found that as the frequency of the
reduction of the inertia, I, in the relationship τ = 2π(I/K)1/2, but to an oscillations is increased, the supersolid transition temperature increases
increase in the elastic constant, K. Naturally, the contribution to K of as well7. It also seems that the rotation velocity has a role similar to the
the helium stiffness would have been small in most experiments, but thermal agitation9. Furthermore, Aoki et al.7 and Penzev et al.8 have
the period shift itself was small. The respective groups of Chan and shown that their oscillators are hysteretic at low temperatures: the
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

a period decreases when cooling below 50 mK, but a subsequent warm-

ing does not bring the period to its first measured values. This obser-
vation leads to many questions, especially about the true value of the
critical velocity measured by various groups (which ranges from a few
micrometres per second to at least 100 times more). Eventually, Hunt
et al.9 showed that their oscillator has a response time that diverges
near the transition temperature in a way that, as far as I know, has not
been understood either.
It is not known what mechanisms are behind this set of observations.
It could be the network of dislocations (or grain boundaries), which
relaxes with a T-dependent time, or it could be the diffusion of 3He
impurities, which becomes slow at low T. A totally different picture was
proposed by Anderson18–20. If mass really flows it may form vortices,
Edge dislocation and Anderson invoked a ‘vortex liquid’, that is, a tangle of interacting
b vortices. Because such vortices should interact with the crystal lattice,
their proliferation near the supersolid transition temperature might lead
to an increase in the rigidity. Furthermore, the properties of a vortex
tangle should depend on frequency and temperature, but all these issues
are open problems and are difficult to solve, especially if the sample
disorder is unknown.

Towards a solution
Given the observations that I briefly describe above, I believe that even if
supersolidity remains mysterious, it has really been discovered. It appears
to be strongly dependent on disorder, and it seems that the supersolid
transition is accompanied by a surprising stiffening of the samples.
c To provide a better understanding of this set of observations, some
groups have tried to maximize the NCRI by preparing highly disordered
samples that in effect might be glassy. In my group, we have chosen an
opposite approach that seems promising. Having optical control of the
crystal growth34,45, we know how to prepare polycrystals, disordered
single crystals or very high-quality single crystals. The high-quality sin-
gle crystals have been shown to be free of screw dislocations if grown at
20 mK (ref. 46). Day and Beamish’s observations10,11 of stiffening were
done on polycrystals but not interpreted in terms of grain boundaries;
to clarify this, we have made new measurements of the elasticity of 4He,
this time in single crystals with variable concentration of 3He impuri-
d
ties13. Our most interesting result is on ultrapure single crystals (we have
discovered a simple method to eliminate all impurities47), in which we
also observed a stiffening13, this time below 40 mK. This temperature
is lower than has been observed in any other experiment. It is also the
temperature below which the search for d.c. mass flow through solid
4
He should be repeated. In view of these observations, the pinning of
dislocations by 3He impurities cannot be the only mechanism respon-
sible for the stiffening.
Interestingly, a new possible explanation for the observed stiffening in
the absence of 3He impurities was proposed by Kuklov and colleagues48
in 2009. They theoretically predicted that dislocations could change state
as a function of temperature. They calculated that at T = 0 K, a disloca-
tion is a straight line that is anchored by the lattice and does not fluctuate.
However, at a certain finite temperature, a transition occurs to a rough
Figure 3 | Various defects in solids. a, In a single crystal, an edge dislocation is
fluctuating state in which the dislocation line becomes mobile because
the end line of an interrupted lattice plane. b, Owing to their mutual attraction,
dislocation lines usually organize themselves as three-dimensional networks. it is invaded by kinks (Fig. 5). This ‘roughening transition’ could exist
For a detailed description, see the ParaDiS project (http://paradis.stanford. in the absence of 3He and thus provide an explanation for the change in
edu). (Image courtesy of the ParaDiS project code team: A. Arsenlis, V. V. stiffness observed in experiments. The sensitivity to the presence of 3He
Bulatov, W. Cai, R. D. Cook, M. Hiritani, M. Rhee and M. Tang, Lawrence impurities is a consequence of the fact that kinks become dressed with
3
Livermore National Laboratory, Livermore, California.) c, In polycrystals, the He atoms that attach to them (Fig. 5), such that a higher temperature is
boundaries (green) between crystal grains of different orientations are regions needed for their creation.
of atomic thickness where atoms are not ordered, owing to the competing To verify these new ideas experimentally, it is necessary to see whether
influences of the adjacent lattices. d, Photograph of a vertical grain boundary an ideal crystal free of impurities and dislocations shows stiffening at low
between two helium crystals in equilibrium with liquid helium above. The cell temperatures. This experiment is in progress in our group, but it is not
height and width are 11 mm. It is enclosed by two glass windows. Two crosses
are carved on the bottom right of the front window to help focus the camera.
easy to observe a single dislocation. In addition, these are very delicate
The photograph shows that a grain boundary is not a liquid region32,34. Indeed, experiments because helium crystals are extremely fragile, probably
as explained by Sasaki et al.32,34, the groove it makes where it meets the liquid owing to their quantum nature. If we succeed in showing that perfect,
phase has a non-zero opening angle (of about 30°). As a consequence, grain ultrapure crystals show no stiffening, the theory of Kuklov and col-
boundaries may be supersolid (that is, solid and partially superfluid) but not leagues48 will be strongly supported. If the crystals do show stiffening,
entirely superfluid. (Image reproduced, with permission, from ref. 32.) it will be necessary to search for another origin of the stiffening, which
180
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

a Figure 4 | Stiffness measurements and the role of 3He

1 p.p.b. 3He
1 85 p.p.b. 3He impurities. a, The original measurements by Day and Beamish10
0.3 p.p.m. 3He of the variation of the stiffness of 4He crystals as a function of 3He
0.8 impurities. For comparison, measurements by Kim and Chan3 of
0.3 p.p.m. 3He
% m/% m(18 mK)

(NCRI) the NCRI are shown on the same graph (see key). Both the stiffness
0.6 1 p.p.b. 3He and the NCRI are expressed as variation in shear modulus, μ, at
(NCRI)
0.4 temperature T relative to the variation at 18 mK. p.p.b., parts per 109;

p.p.m., parts per million. (Panel reproduced, with permission, from

0.2 ref. 10.) b, Further measurements have shown an even more striking
similarity between the temperature dependences of the shear
0 modulus and the NCRI: after adjustment of the vertical scales, the
0.02 0.03 0.04 0.06 0.1 0.2 0.3 0.4 two sets of data shown coincide (μo is the low-temperature value of
T (K) the stiffness). This is very strong evidence that the rotation anomaly
and the elastic anomaly have a common origin. (Panel courtesy of
b J. D. Beamish, University of Alberta, Edmonton, Canada.)
1.00 Shear modulus at 200 Hz 1.5
NCRI at 910 Hz

0.98
1.0

NCRI (%)
m/mo

0.96

0.5

0.94

0
0.92

0 0.1 0.2 0.3

T (K)

could perhaps be explained by Anderson’s vortex liquid picture18–20. function. When dislocations start fluctuating, phase fluctuations could
Naturally, we will also have to see whether the same ideal crystals show destroy the supersolidity. Conversely, if the dislocation is supersolid,
a rotation anomaly. For this, we need a transparent torsional oscillator the phase coherence should force the dislocation to be a smooth, non-
to be able to monitor the growth and measure the crystal orientation, fluctuating, straight line. If the rotation anomaly disappears in perfect
which is an important parameter that should be explored. This experi- crystals, this theory will receive strong support. Otherwise, the favoured
ment is also in progress in our group, now in collaboration with J. West theory will be Anderson’s, where supersolidity is an intrinsic crystal
and M. H. W. Chan. If the rotation anomaly disappears in ideal crystals, property that is only enhanced by disorder, and the elastic anomaly is
then the dislocation network theory will be strongly supported. due to the proliferation of vortices close to the supersolid transition.
We also have a suggestion for the connection between rigidity and In conclusion, I believe that supersolidity is a real phenomenon but
supersolidity: transverse fluctuations of dislocation lines should induce that it is not yet understood. The true origin of mass flow inside 4He
mass currents, which should manifest as phase fluctuations because, crystals is unclear, as are the origin of their stiffening at low temperatures
in a superfluid, a mass current is the gradient of the phase of the wave and the connection between the mechanical response of these quantum

Crystal lattice
Crysta

3He impurity

Kinks

Dislocation lines

Figure 5 | Dislocation lines. At the atomic scale and at a sufficiently high dislocation line is straight and immobile, such that the crystal rigidity is
temperature, dislocation lines show kinks where they shift from one atomic greater than it is above the roughening temperature. This roughening process
row to the next. In a quantum crystal such as 4He, these kinks may move may be at the origin of the stiffness anomaly of 4He crystals. The roughening
very easily by quantum tunnelling from one site to the neighbouring one. In temperature should depend on the presence of 3He impurities, which are
this case, the dislocation line itself is a mobile line whose presence reduces known to bind to the dislocation lines, where they probably dress kinks.
the crystal rigidity. However, Aleinikava et al.48 have proposed that below Even if this set of hypotheses is correct, the coupling between stiffness and
a certain roughening transition temperature, kinks disappear and the supersolidity remains to be clarified.

181
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

crystals and the onset of supersolidity. However, I expect experimental 29. Pollet, L. et al. Superfluidity of grain boundaries in solid 4He. Phys. Rev. Lett. 98, 135301
(2007).
results in the coming year that should help to discriminate between the 30. Clark, A. C., West, J. T. & Chan, M. H. W. Nonclassical rotational inertia in helium crystals.
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25. Leggett, A. J. Can a solid be superfluid? Phys. Rev. Lett. 25, 1543–1546 (1970).
26. Meisel, F. Supersolid 4He: an overview of past searches and future possibilities. Physica B Acknowledgements I acknowledge support from the Agence Nationale de la
178, 121–128 (1992). Recherche (grant BLAN07-1-215296).
27. Boninsegni, M. et al. Fate of vacancy-induced supersolidity in 4He. Phys. Rev. Lett. 97,
080401 (2006). Author Information Reprints and permissions information is available at www.
28. Boninsegni, M. et al. Luttinger liquid in the core of screw dislocation in helium-4.Phys. Rev. nature.com/reprints. The author declares no competing financial interests.
Lett. 99, 035301 (2007). Correspondence should be addressed to the author (balibar@lps.ens.fr).

182
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NATURE|Vol 464|11 March 2010|doi:10.1038/nature08914 PERSPECTIVE INSIGHT

Superconductivity gets an iron boost

Igor I. Mazin1

Superconductivity, the resistance-free flow of electrical charges, is one of the most exotic phenomena in
solid-state physics. Even though it was discovered almost a century ago, many questions remain unanswered,
in particular those concerning the physics of high-temperature superconductivity. The recent discovery of
iron-based superconductors was arguably the most important breakthrough in this field for more than two
decades and may provide new avenues for understanding this high-temperature phenomenon. Here I present
my view of the recent developments in this field that have led to the current understanding of this important
new class of superconductor.

A once popular joke asked how a physicist would interpret experimental conviction (a notable exception being Vitaly Ginzburg2), that a quantitative
data on odd numbers. As the first experiments reveal that 1, 3, 5 and theory of superconductivity ultimately would provide proof that Tc has a
7 are all prime numbers, the physicist becomes convinced that all odd fundamental limit of about 25–30 K. It is ironic that in the chemical cabi-
numbers are prime and that a correct theory of ‘primeness’ should be nets of many of these distinguished scientists was magnesium diboride
able to explain this experimental fact. Further studies, however, show (MgB2), which, as has been known since 2001, has a Tc of 40 K.
that 9 is not a prime number. The community initially disregards this as This conviction was radically challenged when copper-oxide-based
an experimental error; however, after more experiments, the research- superconductors with a high Tc were discovered in 1986. The rapid climb
ers are forced to admit that 9 is indeed not prime, making it a unique of the maximum known Tc up to 140 K created the first paradigm shift;
case. This view is reinforced when further experiments show that the it became clear that high-Tc superconductivity (with critical tempera-
next odd numbers in the series, 11 and 13, are both prime. Only after tures larger than the anticipated 25–30 K) was possible, even though the
it is found that, in violation of ‘conventional wisdom’, 15 is not a prime underlying mechanism remained unknown. The following two decades,
number, does the idea that there are infinitely many odd numbers, but however, brought little progress towards a further increase of Tc. No
not prime numbers, take root in researchers’ minds. new high-Tc materials were found, although some superconductors that
This joke can be seen as an allegory of the modern history of broke the old record of 23.2 K (but not by much) were discovered, for
superconductivity. During the 1960s and 1970s, the recorded transi- example (Ba,K)BiO3, doped fullerenes and MgB2. Inevitably, a grow-
tion temperature, Tc, for superconductors very slowly inched its way ing number of physicists started to look for the unique combination of
up, culminating in the 1976 finding of Tc = 23.2 K in Nb3Ge, largely physical properties that makes copper oxides such a spectacular excep-
thanks to the efforts of Berndt Matthias. At about that time, according tion in the materials universe.
to physicists’ folklore, Matthias formulated his famous six rules for a The new, iron-based, superconductors (initially called iron-pnictide
successful search for new superconductors1. One, a high symmetry is superconductors, before another, chalcogen-based subfamily was found)
good; cubic symmetry is the best. Two, a high density of electronic states were discovered in 2008 (ref. 3). Their epistemological value is that they
is good. Three, stay away from oxygen. Four, stay away from magnet- did not fit the same mould as copper oxides. Their discovery demonstrated
ism. Five, stay away from insulators. Six, stay away from theorists. All of that unconventional (that is, not phonon-mediated) high-Tc superconduc-
these rules, with the possible exception of rule six (I’ll leave that to the tivity, such as that found in copper oxides, is not unique and is probably
judgement of my experimental colleagues), not only have been proved just as ubiquitous as the conventional, ‘low-Tc’, kind, if researchers would
incorrect but also their exact opposite seems to be true. look in the right places. What is special about iron-based superconduc-
Following this stagnation in the search for superconductors with a tors is that the more they are studied, the less they look like copper oxides,
higher Tc than 23–24 K, between 1976 and 1986 an increasingly large suggesting that high-Tc superconductivity is not limited to copper oxides
number of physicists became convinced, and openly sounded their and might not even be limited to any particular class of materials. (This

Table 1 | Properties of different classes of superconductor

Property Conventional superconductors Copper oxides MgB2 Iron-based superconductors
Tc (maximum) <30K 134K 39K 56K
Correlation effects None (nearly-free electrons) Strong local electronic interaction None (nearly-free electrons) Long-range (non-local)
magnetic correlations
Relationship to magnetism No magnetism Parent compounds are magnetic No magnetism Parent compounds are
insulators magnetic metals
Order parameter One band, same-sign s wave One band, sign-changing d wave Two band, same-sign s wave Two band, presumably sign-
changing s wave
Pairing interaction Electron–phonon Probably magnetic (no consensus) Electron–phonon Presumably magnetic
Dimensionality Three dimensional Two dimensional Three dimensional Variable

1
Naval Research Laboratory, code 6390, 4555 Overlook Avenue Southwest, Washington DC 20375, USA.

183
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INSIGHT PERSPECTIVE NATURE|Vol 464|11 March 2010

may not be entirely true; as discussed below, there might be some common orbit each other with a particular angular momentum, avoiding close
conditions for high-Tc superconductivity, such as proximity to magnetism. contact with each other, and thereby reducing the effect of their mutual
But if these conditions exist, they are of a general character.) Coulomb repulsion. Many think that such ‘Coulomb avoidance’ is an
In this Perspective article, I discuss the commonalities and differences important component of high-Tc superconductivity8.
between the two previously known classes of high-Tc superconductors, In contrast to copper oxides, MgB2 has no trace of magnetism, has
copper oxides and MgB2, and what can be learned from this comparison. delocalized electrons (which are not subject to strong on-site correla-
I then take a look at how far the understanding of iron-based supercon- tions), and has a complex electronic structure, featuring two distinc-
ductors has advanced and what the remaining challenges and prospects tively different groups of electrons. These two groups form two kinds
are in this field. of electronic band (hence the term two-band superconductors), which
give rise to two separate sets of Fermi surfaces4.
True siblings or lookalikes Iron-based superconductors, on the one hand, resemble copper oxides
Eighteen months after the discovery of superconductivity in copper in that they are strong magnets in parts of their phase diagram (Fig. 1c)
oxides, there was almost no understanding of the underlying physics. and in that superconductivity develops when magnetism is destroyed
Eighteen months after superconductivity was found in MgB2, there was by doping. On the other hand, unlike copper oxides, the parent (mag-
almost a complete theory for it4. Some twenty months into the ‘iron netic) iron-based compounds are metallic, so the increase in the charge
age’, and the physics community’s understanding lies between these carrier concentration as a result of doping is not a decisive factor in the
limits: much is known, and there is a plausible theoretical hypothesis development of superconductivity. Indeed, magnetism can be suppressed
that the majority of researchers in the field seem to agree on (curiously, and superconductivity induced in these materials without charge dop-
proposed within days of the original discovery5,6). However, there is not ing. This can be achieved by applying pressure, by partially substituting
a complete theory, and there are several facts that are difficult to explain arsenic with phosphorus, or by simply diluting the iron layer with a non-
and to reconcile with one another7. magnetic species, such as ruthenium. With few exceptions, one of the
In many respects, iron-based superconductors sit between copper main characteristic features of iron-based superconductors is that when
oxides and MgB2 (Table 1). Undoped (‘parent’) copper oxides are strong magnetism is destroyed (by any means), superconductivity emerges9.
magnets and insulators. This is because two electrons located on the Another property that sets iron-based superconductors apart from cop-
same copper ion are subject to strong Coulomb repulsion, leading to per oxides is the strength of the Coulomb correlations. The latest calcula-
strong correlations and hence electron localization8. The undoped tions, as well as estimates from spectroscopic experiments, seem to be in
compounds have exactly one valence electron for each copper atom, agreement that the correlation strength is weak to moderate10,11, although
which makes them strong magnets — each electron has a spin of ½ and not as weak as in MgB2, in which Coulomb correlations are almost absent.
therefore a magnetic moment. When extra charge carriers are intro- Another important property, which iron-based superconductors have in
duced by way of doping, these carriers are free to move about and screen common with MgB2, is that electrons form a multi-sheet Fermi surface
the Coulomb repulsion. Given enough doping, the static magnetism that can be separated into two distinct sets of surfaces (Fig. 3). But, as dis-
disappears, and the electron states form a single band, as is the case in cussed below, there seem to be significant differences between these two
simple metals. Crucially, there is a critical range of doping in which a classes of materials regarding the nature of the superconducting states.
superconducting phase appears within a characteristic ‘superconduct-
ing dome’ (Fig. 1b). Many, but not all, researchers think that the ‘glue’ Ironing out the last wrinkles
that binds electrons into Cooper pairs, which give rise to superconduc- So what is known about these materials, and how has this information
tivity, is provided by the exchange of magnetic fluctuations. Another been uncovered? It is known that iron-based superconductors
important characteristic of copper oxides is that the wavefunction of the comprise a broad variety of materials9 and that they all have similar
Cooper pairs — also known as the superconducting order parameter phase diagrams (Fig. 1c) and presumably have the same mechanism
— has d-wave symmetry (Fig. 2b). This means that the paired electrons of superconductivity. All iron-based superconductors have the same

Temperature Temperature
a b c
Paramagnetic
Paramagnetic

Phase separation
Super-
Su Coexistence
conducting
cond
Super-
Antiferro- conducting Antiferro-
Pseudo-
magnetic magnetic
gap

Hole Doping Electron Hole Doping Electron

fraction fraction
Figure 1 | Iron-based superconductors and copper-oxide-based induce superconductivity under the characteristic superconducting dome.
superconductors. a, Generic crystal structure of iron-based The green lines between the paramagnetic and pseuodogap phases (green
superconductors. Fe atoms are shown in red, and pnictogens (As or P) or shading) represent crossover transitions, black lines between paramagnetic
chalcogens (Se or Te) are indicated in blue. The filler layer, shown without and antiferromagnetic phases are well-defined transitions. c, Schematic
atomic detail, can contain any of the following constituents (for each two phase diagram for the 122 family of iron-based superconductors. An
iron atoms): one alkaline earth atom; two alkali atoms; two rare-earth example is BaFe2As2, in which Ba atoms can be substituted with K atoms,
atoms and two oxygen atoms; or more complex layers with various atoms. thereby doping the parent compound with holes; Fe atoms can be substituted
It is also possible for there to be no filler layer. b, Schematic phase diagram with Co atoms, doping the parent compound with electrons; or Fe atoms
of copper-oxide-based superconductors. Note how both electron doping can be substituted with Ru atoms (or As atoms by P atoms), to suppress
and hole doping suppress the magnetism of the parent compounds and magnetism without changing the carrier concentration.

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NATURE|Vol 464|11 March 2010 PERSPECTIVE INSIGHT

a b It is tempting to assume (and in fact almost the entire community has

succumbed to this temptation) that the features that such disparate iron-
based superconductors have in common reflect a common origin for the
observed superconductivity. Adopting this path, it can be concluded that
proximity to a magnetic quantum critical point (as is seen in the phase
diagram) signals that magnetic (spin) fluctuations play an important
role. The fact that neutron-scattering measurements always uncover
magnetic excitations with a particular wavevector, Qm, also suggests
that these excitations are instrumental for mediating the pairing of elec-
trons. Note that the two electrons in a singlet Cooper pair have the same
c d charge but opposite spins. A corollary of this is that magnetic excitations
lead to pairing only if the corresponding wavevector spans parts of the
Fermi surface with order parameters (that is, the pair wavefunction) of
opposite sign (see ref. 14 for further explanation). Now, noting that there
are two sets of Fermi surfaces that are roughly separated by the same
wavevector Qm (Fig. 3), the so-called s± superconductivity is derived, in
which the sign of the order parameter is switched between the two sets
Figure 2 | Superconducting order parameter. A schematic representation
of Fermi surfaces (Fig. 2).
of the superconducting order parameter in different cases: a conventional,
uniform, s wave, such as in an ‘old-fashioned’ superconductor (for example In the previous paragraph, I describe how the s± superconductivity
aluminium) (a); a d wave, as is the case in copper oxides (b); a two-band model could have been arrived at, by using data from ARPES and neutron-
s wave with the same sign, as in MgB2 (c); an s± wave, as is thought to be the scattering experiments that became available roughly one year after the
case in iron-based superconductors (d). In a and b, the two-dimensional initial discovery. It is gratifying that theorists were able to come up with
Fermi surface is approximated by one circle. In c and d, the Fermi surface this model within a few weeks of the initial discovery, solely on the basis
is approximated by a small circle in the centre (the first band) surrounded of electronic structure calculations and theoretical models5,6.
by four larger circles (to comply with the tetragonal symmetry; the second
band). In all cases, the height of the ‘rubber sheet’ is proportional to the Farther down the road
magnitude of the order parameter (including its sign).
It is still not clear beyond a reasonable doubt that the superconducting
symmetry realized in iron-based superconductors is s± symmetry and
that pairing is due to spin fluctuations. The jury is still out. This is
crystallographic motif (Fig. 1a), with the main component being a a jury that is deeply convinced by the prosecution but is reluctant to
square lattice of iron atoms sandwiched between two square lattices base its verdict solely on circumstantial evidence. However, circum-
of pnictogen (arsenic or phosphorus) — hence the initial name — or stantial evidence is plenty in this case, and physicists might be en route
chalcogen (selenium or tellurium) atoms. Between these crucial trilay- to uncovering a direct proof.
ers, various ‘filler slabs’ can be placed (although this is not essential): for It is known (from nuclear-magnetic-resonance spectroscopy data)
example, a single crystallographic layer of sodium, barium, strontium that the Cooper pairs in iron-based superconductors are spin singlets
or calcium; a trilayer consisting of a layer of oxygen between layers of (formed by electrons with antiparallel spins). In this class, three possible
a rare-earth element (the highest Tc so far, 56 K, has been observed in symmetries of the order parameter are compatible with the tetragonal
this family); or an even more complex filler slab.
Superconductivity can be induced in all materials by chemical doping
or pressure, or a combination of both, as long as this results in the sup-
pression of magnetism. In contrast to copper oxides, which have very
low electrical conductivity along the direction perpendicular to the
copper-oxide layers, none of these systems is truly two dimensional.
This is beneficial in terms of practical applications, because in polycrys-
talline two-dimensional materials superconductivity can be destroyed
Electron momentum (ky)

by a relatively small current.

What can be inferred from the large number of iron-based
superconductors and the known properties of these materials? So far,
no simple correlation has been noticed between anisotropy, the distance
between planes in the crystal lattice, the temperature at which antifer-
romagnetic ordering occurs in the parent phase (or even the pattern of
the magnetic order of this phase) and the superconducting Tc. Initially, it
seemed as though Tc is optimized when the four anions around an iron ion
form an ideal tetrahedron12. However, after more iron-based supercon-
ductors had been uncovered, this finding seemed not to be universal13.
By contrast, phase diagrams of essentially all iron-based
superconductors have both superconducting phases and a strongly anti-
ferromagnetic phase (see Fig. 1, which is a schematic view of the generic Electron momentum (kx)
features of the phase diagram). All iron-based superconductors contain
iron in a valence state that is close or equal to Fe2+. All materials that have
been studied so far show a peak in inelastic neutron-scattering spectra Figure 3 | A typical calculated Fermi surface of an iron-pnictide
superconductor. The Fermi surface (projected onto the kx–ky plane, where
that corresponds to magnetic excitations at a particular wavevector, Qm
k is electron momentum) shown is calculated for 10% electron-doped
(even though for at least one family, FeTexSe1−x, the static magnetic order LaFeAsO. Experimentally observed Fermi surfaces show similar geometries.
occurs at a different wavevector). In cases in which the Fermi surface has The momentum connecting the two sets of Fermi surfaces, Qm, is shown by
been mapped by angle-resolved photoemission spectroscopy (ARPES), the arrow. Spin fluctuations with this moment were predicted theoretically
two sets of Fermi surfaces, roughly separated by the same wavevector, and found experimentally, and they are now thought to be instrumental for
Qm, have been revealed (Fig. 3). creating high-Tc superconductivity in iron-based superconductors.

185
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INSIGHT PERSPECTIVE NATURE|Vol 464|11 March 2010

symmetry of iron-based superconductor crystals: a d-wave symmetry; quantum critical point ensures the abundance of spin fluctuations that
an s-wave symmetry without sign change; or the s±-symmetry model, may provide the necessary glue. It also seems important to have a Fermi
which combines s-wave symmetry with a sign change of the order surface topology that matches the structure of magnetic excitations.
parameter. All of these models are illustrated in Fig. 2. A layered crystal structure may be another not-so-accidental com-
As can be seen in Fig. 2, an s-wave order parameter (of either flavour) ponent. One great advantage of a quasi-two-dimensional system is that
and a d-wave order parameter differ by their rotational symmetry: the the density of electronic states that are available for superconductiv-
s wave does not change under a 90° rotation, whereas the d wave changes ity depends very weakly on the carrier concentration. Although it is
sign. This difference allows the design of ‘phase-sensitive’ experiments15 unclear why a low carrier concentration is beneficial, the possibility
that can distinguish between the two. The preliminary indications are of changing this parameter provides additional flexibility that is not
strongly against iron-based superconductors having d-wave symmetry. present in three-dimensional systems. In addition, low-dimensional
However, arguably the most convincing evidence against the d-wave systems generally fluctuate more, which may also be helpful for super-
model (or any finite angular momentum model) is that such a state, conducting pairing.
according to its symmetry, has nodes in the order parameter, which The following is my attempt to concoct a set of rules that could replace
implies that there is a zero minimal excitation gap in the superconduct- Matthias’s rules. One, layered structures are good. Two, the carrier den-
ing state. In other words, excitations with arbitrarily small energy exist. sity should not be too high (compared with, say, conventional metals).
Because this is mandated by the symmetry, such zero minimal excitation Three, transition metals of the fourth period (vanadium, chromium,
gaps should be present in all iron-based superconductors, but numerous manganese, iron, cobalt, nickel and copper) are good. Four, magnetism
experiments have established that some iron-pnictide superconductors is essential. Five, proper Fermi surface geometry is essential (it must
have a finite minimal gap. By contrast, an s state (whether s or s±) may match the structure of the spin excitations). Six, enlist theorists, at least
or may not have nodes (depending on the particular combination of to compute the Fermi surfaces (I hope that theorists are more useful
material-dependent parameters). Indeed, some of iron-pnictide super- than this but do not dare insist). Note that there is one corollary to these
conductors (KFe2As2, LaFePO and BaFe2As2−xPx) show clear indications rules. Materials of interest are likely to be complex chemical compounds
of nodes, whereas others are fully gapped. — work closely with solid-state chemists. ■
Taken together, the above considerations lead to the idea that the
1. Pickett, W. E. The other high-temperature superconductors. Physica B 296, 112–119 (2001).
overall symmetry of the superconducting state in iron-based super- 2. Mazin, I. I. Vitaly Ginzburg and high temperature superconductivity: personal
conductors is s-wave symmetry. It still remains to be experimentally reminiscences. Physica C 468, 105–110 (2008).
established whether this is a conventional s-wave state (such as is in 3. Kamihara, Y., Watanabe, T., Hirano, M. & Hosono, H. Iron-based layered superconductor
La[O1−xFx]FeAs (x=0.05−0.12) with Tc=26K. J. Am. Chem. Soc. 130, 3296–3297 (2008).
MgB2), which in some cases becomes disrupted (by magnetic or Cou- 4. Mazin, I. I. & Antropov, V. P. Electronic structure, electron–phonon coupling, and multiband
lomb interactions) and develops nodes, or an s± state, which also may effects in MgB2. Physica C 385, 49–65 (2003).
exist with or without nodes, depending on the material. At present, the 5. Mazin, I. I., Singh, D. J., Johannes, M. D. & Du, M. H. Unconventional sign-reversing
superconductivity in LaFeAsO1−xFx. Phys. Rev. Lett. 101, 057003 (2008).
preponderance of experimental evidence7,9 favours an s± state, which is 6. Kuroki, K. et al. Unconventional superconductivity originating from disconnected Fermi
also favoured theoretically7. surfaces in LaO1−xFxFeAs. Phys. Rev. Lett. 101, 087004 (2008).
Reproducibly confirming the proposed s± symmetry remains the main 7. Mazin, I. I. & Schmalian, J. Pairing symmetry and pairing state in ferropnictides: theoretical
experimental challenge. Phase-sensitive experiments15 have provided overview. Physica C 469, 614–627 (2009).
8. Lee, P. A., Nagaosa, N. & Wen, X. G. Doping a Mott insulator: physics of high-temperature
such confirmation for the superconductivity in copper oxides, which superconductivity. Rev. Mod. Phys. 78, 17–85 (2006).
has a d-wave symmetry. Given that the s and the s± state belong to the 9. Chu, P. C. W. et al. (eds) Superconductivity in iron-pnictides. Physica C 469 (special issue),
same rotational symmetry group, it is far more challenging to devise 313–674 (2009).
10. Anisimov, V. I., Kurmaev, E. Z., Moewes, A. & Izyumov, I. A. Strength of correlations in
a phase-sensitive experiment to distinguish between them. But, hav- pnictides and its assessment by theoretical calculations and spectroscopy experiments.
ing excluded d-wave symmetry, researchers need to prove only that the Physica C 469, 442–447 (2009).
order parameter in iron-based superconductors changes sign (while not 11. Yang, W. L. et al. Evidence for weak electronic correlations in iron pnictides. Phys. Rev. B 80,
014508 (2009).
becoming zero anywhere on the Fermi surface). Several experimental 12. Lee, C.-H. et al. Effect of structural parameters on superconductivity in fluorine-free
designs along these lines have been proposed, and several groups are LnFeAsO1−y (Ln=La, Nd). J. Phys. Soc. Jpn 77, 083704 (2008).
working to implement these. Within a year or two, the final experimen- 13. Hosono, H., Matsuishi, S., Nomura, T. & Hiramatsu, H. Iron-based superconducting
materials. Bull. Phys. Soc. Jpn 64, 807 (2009).
tal answer is likely to have been revealed. 14. Scalapino, D. J. Superconductivity and spin fluctuations. J. Low Temp. Phys. 117, 179–188
So what comes next? Can we invent a new set of rules, Matthias’s rules (1999).
for the twenty-first century? It might be preposterous to think about a 15. Van Harlingen, D. J. Phase-sensitive tests of the symmetry of the pairing state in the high-
temperature superconductors — evidence for dx2−y2 symmetry. Rev. Mod. Phys. 67, 515–535
general strategy for finding new high-Tc superconductors, given that (1995).
so far only two such families have been found. But this might be the
time to start thinking about it. There are good reasons to believe that Acknowledgements I dedicate this article to the memory of Vitaly Ginzburg, a
proximity to a magnetic transition is essential: magnetism is basically relentless enthusiast of high-temperature superconductivity and my former teacher,
the only quantum coherence phenomenon that commonly occurs at who passed away while this article was being written.
room temperature. The magnetic exchange interaction is basically the Author Information Reprints and permissions information is available at www.
only interaction that is strong (on the order of the Fermi energy) and nature.com/reprints. The author declares no competing financial interests.
yet does not conflict with electronic itineracy. Proximity to a magnetic Correspondence should be addressed to the author (mazin@dave.nrl.navy.mil).

186
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NATURE|Vol 464|11 March 2010|doi:10.1038/nature08915 REVIEW INSIGHT

Non-Abelian states of matter

Ady Stern1

Quantum mechanics classifies all elementary particles as either fermions or bosons, and this classification
is crucial to the understanding of a variety of physical systems, such as lasers, metals and superconductors.
In certain two-dimensional systems, interactions between electrons or atoms lead to the formation of
quasiparticles that break the fermion–boson dichotomy. A particularly interesting alternative is offered by
‘non-Abelian’ states of matter, in which the presence of quasiparticles makes the ground state degenerate,
and interchanges of identical quasiparticles shift the system between different ground states. Present
experimental studies attempt to identify non-Abelian states in systems that manifest the fractional quantum
Hall effect. If such states can be identified, they may become useful for quantum computation.

Electrons are fermions; photons are bosons. Although seemingly a to noise and decoherence, and this requirement is amply satisfied by the
mundane statement about the symmetries of a quantum mechani- insensitivity of the effect of interchange of non-Abelian quasiparticles to
cal wavefunction when two identical particles are interchanged (for noise and perturbations. The topological aspects of these interchanges
instance with respect to degrees of freedom such as position or momen- motivate the name ‘topological quantum computation’.
tum), this statement is in fact a pillar of the understanding of nature, There are real-life settings in which existing theory predicts
and a basis of the understanding of the periodic table and the existence non-Abelian states of matter to exist, but they are not numerous2. Sys-
of metals, to list just two examples. Such quantum statistical consid- tems that manifest the fractional quantum Hall effect are believed to
erations also affect the behaviour of composites of quantum particles: have a series of states that are non-Abelian9–27. Closely related states may
helium-4 atoms are bosons and form superfluids at low temperatures; be realized in cold atoms28,29, superconductors of p-wave pairing symme-
and pairs of electrons are effectively bosons, which is how Cooper pairs try2 and hybrid systems of superconductors with so-called topological
of electrons make the phenomenon of superconductivity possible. insulators30–32 (see page 194) and/or semiconductors33. Non-Abelian
Non-Abelian systems1,2 contain composite particles that are neither lattice spin models have been proposed19 but are far from experimental
fermions nor bosons and have a quantum statistics that is far richer than realization. Among all of these, the most prominently studied candidate
that offered by the fermion–boson dichotomy. The presence of such for an experimentally accessible non-Abelian state is the ν = ⁄ quantum
quasiparticles manifests itself in two remarkable ways. First, it leads to Hall state9–27.
a degeneracy of the ground state that is not based on simple symmetry In this Review, I discuss the properties of non-Abelian states, the systems
considerations and is robust against perturbations and interactions with in which they are expected to emerge, the possible ways of identifying them
the environment. Second, an interchange of two quasiparticles does not and the status of the experimental attempts to find them. I focus mainly
merely multiply the wavefunction by a sign, as is the case for fermi- on the ν = ⁄ quantum Hall state but also touch on hybrid systems that
ons and bosons. Rather, it takes the system from one ground state to combine the effects of spin–orbit interaction with superconductivity.
another. If a series of interchanges is made, the final state of the system
will depend on the order in which these interchanges are being carried Non-Abelian quasiparticles
out, in sharp contrast to what happens when similar operations are per- Non-Abelian quasiparticles appeared on the quantum mechanical stage
formed on identical fermions or bosons. It is this ‘ordering’ dependence after several decades of engagement with the quantum mechanics of
that justifies the name ‘non-Abelian’ (‘non-commutative’ in mathemati- identical particles. The originators of quantum mechanics distinguished
cal terms). Just as the minus sign that accompanies the interchange of fermions from bosons through the symmetry restrictions imposed
two fermions is independent of details such as their interaction or envi- on their many-body wavefunctions. A wavefunction, Ψ(r1, …, rN), of
ronment, the effect of the interchange of two non-Abelian quasiparticles N identical spin-polarized particles whose coordinates are r1, …, rN must
is insensitive to noise from the environment around them. be odd under the interchange of the positions of two of the particles if
Non-Abelian states have generated great interest recently1–5, for three they are fermions, and even if they are bosons.
main reasons. The first is the theory behind them. Understanding of In the past three decades, it has been realized that in two spatial dimen-
their origin and properties is in its infancy. The second is the challenge to sions the statistics of quasiparticles formed as composites of the elemen-
observe them in an experiment. There are some systems in which strong tary particles of a system is not limited to the fermion–boson dichotomy.
theoretical arguments suggest the existence of non-Abelian quasiparti- As a first break from that dichotomy, the interchange of two quasiparticles
cles, and that motivates a search for their experimental discovery. Third, if may multiply the wavefunction by a phase. That phase may take any value
non-Abelian states were shown to exist in realizable systems, they would and, as a consequence, the quasiparticles are known as ‘anyons’.
be ideal candidates for constructing topological quantum computers6–8. The second break from the fermion–boson dichotomy, embodied
A quantum computer needs a set of quantum states that is well separated by so-called non-Abelian quasiparticles, is much more surprising: an
from the rest of the world. The degenerate ground states of a non-Abelian interchange of two quasiparticles does not merely multiply the ground-
system, separated by an energy gap from the rest of the spectrum, deliver state wavefunction by a phase factor; rather, it shifts the system to a
that. Furthermore, a quantum computer needs a minimum sensitivity different ground state.
1
Department of Condensed Matter Physics, Weizmann Institute of Science, Rehovot 76100, Israel.

187
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

Defining characteristics of non-Abelian states quasiparticles’ trajectory, that is, on their windings around one another and
To introduce the four defining characteristics of non-Abelian states of their mutual interchanges of position. It does not depend on the detailed
matter, it is useful to think of a two-dimensional (2D) fluid in which geometry of the trajectory. The transformation that corresponds to a set of
several vortices are localized at large distances from one another. Four consecutive braidings is a product of such unitary matrices. Matrix product
conditions need to be satisfied for these vortices to become non-Abelian is non-commutative, hence the term ‘non-Abelian’ and the naming of the
quasiparticles. First, there must be an energy gap separating the fluid’s quasiparticles as non-Abelian anyons. The degeneracy of the ground state
ground state from its excitations (Fig. 1a). Second, the ground state of and the non-Abelian statistics of the quasiparticles therefore go together.
the system must be degenerate, with the degeneracy being exponentially Degeneracies in quantum spectra usually originate from a symmetry
large in the number of vortices. Third, the degeneracy should be robust, of the underlying Hamiltonian, and are removed when the symmetry is
and insensitive to weak noise and perturbations. Fourth, when vortices violated, for instance by the application of perturbations such as impuri-
are made to ‘braid’, that is, to encircle one another or interchange their ties or applied electric or magnetic fields. The degeneracy of the ground
positions (Fig. 1b), the system should transform from one ground state state in a non-Abelian state is different. It does not originate from a simple
to another, with the transformation being determined only by the topol- symmetry of the system, and as such it is robust. Small changes in the
ogy of the quasiparticles’ trajectory: two braiding trajectories that may details of the system — varied interactions or varied disorder — do not
be deformed into one another without the quasiparticles ever getting lift the degeneracy. The degeneracy is only removed if a change to the
close should apply the same transformation to the system, up to a phase system is large enough to close the energy gap between the ground state
factor. It is the combination of an energy gap, ground-state degeneracy, and the excitations (Fig. 1a), which would take the system to another
robustness and topology — all of which are elaborated on below — that state of matter, or if the quasiparticles are brought close to one another. In
defines non-Abelian states of matter. the second case, the degeneracy is lifted by terms that are exponentially
Although energy gaps are common in many-body systems, the small in the ratio of the spatial separation between the quasiparticles and
combination of an energy gap with the three other characteristics is unique a microscopic length, the coherence length of the underlying fluid.
to non-Abelian states. To elaborate on these characteristics, it is necessary
to be slightly more formal. The fluid in the picture above is made up of a The inner makings of a non-Abelian state
large number of microscopically identical particles (usually electrons, but The question of what it is in the microscopic interactions between the
they could also be atoms, for instance) whose set of degrees of freedom is constituents of a system that gives rise to a non-Abelian state of matter
{ri}. The vortices in turn are collective degrees of freedom, created by the is a question of great interest and scarce understanding. Consider, for
collective behaviour of the microscopic particles. Their number is much example, the fractional quantum Hall state (Box 1), which so far is the
smaller (Fig. 1b) than the total number of particles forming the back- main arena for the study of non-Abelian states. A partially filled Landau
ground fluid. Assume that they are localized at the coordinates {Rj}. The level is characterized by its ‘filling fraction’, ν, and by the type of interac-
wavefunctions of the set of degenerate ground states, |Ψα(Rj)〉, enumerated tion between its electrons. For a wide range of interaction parameters,
by the index α, are thus functions of the particles’ coordinates, {ri}, and the degeneracy of the ground state is insensitive to the details of the
depend on the vortices’ coordinates, {Rj}, as parameters. interaction but is exponentially sensitive to ν. For example, the ground-
A braiding — a quasiparticle interchange operation — is carried out state degeneracy is exponential in (small) |ε| for ν = ⁄ + ε, thus making
when the vortices slowly move along a trajectory, {Rj(t)}, that starts and the ν = ⁄ case non-Abelian (at least for a certain range of interactions),
ends in the same configuration, {Rj} (Fig. 1b). If there is just a single, non- whereas no such degeneracy appears for ν = ⁄ + ε.
degenerate, ground state, the slowness (‘adiabaticity’) of the motion and A hint to a possible origin of non-Abelian systems may lie in the
the existence of an energy gap guarantee that the final state of the system observation that most theoretical constructs of these systems are fluids
is identical to the initial state, up to a possible phase factor. A series of in which microscopic particles of arbitrary quantum statistics group to
such braidings results in a product of phase factors. Such a product is form clusters whose statistics is effectively bosonic1,18. These clusters
commutative and, hence, the quasiparticles are Abelian anyons. then form a Bose–Einstein condensate. Generally, breaking of a clus-
By contrast, if the ground state is degenerate, a braiding may transform ter in that condensate costs energy, at least as much as the energy gap.
one ground state, |Ψα(Rj)〉, into a different one, |Ψβ(Rj)〉. The transforma- Crudely speaking, vortices formed in these Bose–Einstein condensates
tion, Uαβ, which is a unitary matrix, depends only on the topology of the become non-Abelian quasiparticles when their presence allows clusters
to be broken at no energy cost, leading to ground-state degeneracy.
In the simplest example, the cluster is a Cooper pair of fermions and the
a condensate is a Bardeen–Cooper–Schrieffer superconductor (see page 176
for a discussion on the connections between superfluidity, superconduc-
tivity and Bose–Einstein condensation in systems of quantum particles).
Generally, the spectrum of a superconductor has excited states in which
Energy

some Cooper pairs are broken, and these states are separated by an energy
gap from the ground state. In the language of ‘second quantization’, these
|Ya(Rj)° excitations are created and annihilated by a set of creation and annihila-
tion operators. For some superconductors, in which the two fermions that
constitute the Cooper pair have a relative angular momentum equal to an
b R1 odd multiple of " (Planck’s constant divided by 2π), the presence of vortices
makes the energy cost for some of the excitations vanish, thus leading to
a degeneracy of the ground state. The simplest of these superconductors,
Fluid
which has an angular momentum of " per pair, is known as a p-wave super-
conductor. The operators that create and annihilate the cost-free excitations
R2 in this type of superconductor are known as Majorana operators. They are
Hermitian operators formed by equal-weight superposition of fermionic
creation and annihilation operators, and as such are electrically neutral
Figure 1 | Characteristics of non-Abelian systems. In the presence of (see Box 2 and a recent review of Majorana fermions34).
vortices — non-Abelian quasiparticles — the spectrum includes a set
of degenerate ground states, |Ψα(Rj)〉, separated by an energy gap from a
continuous spectrum of excitations. a, Spectrum. b, Vortices and possible Candidate systems
braidings of their positions. The two winding trajectories on the right are Given the well-developed understanding of Cooper pairs in supercon-
topologically equivalent. ductors, it is not surprising that the best-understood candidate systems
188
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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

Box 1 | Integer and fractional quantum Hall systems

Ohmic
contacts
0.50 ½(h/e2)

0.45

Rxy (h/e2)
2D electron fluid Vy (h/e2)
0.40

Ix
0.35
⅓(h/e2)

Edge
states 4.5 5.0 5.5
Magnetic field Magnetic field (T)
out of page

Two-dimensional electronic systems subject to a perpendicular magnetic Furthermore, these states are characterized by an energy gap in the bulk
field show the Hall effect, in which a current flow, Ix, is accompanied by a and a chiral flow of the current along the edges.
voltage, Vy, perpendicular both to the current and to the magnetic field (see The integer quantum Hall states, for which ν is an integer, may be
figure, left, which shows a schematic of an experimental quantum Hall understood from a simple picture of non-interacting electrons under the
system). The ratio of this voltage to the current, the Hall resistance, Rxy, may effect of a magnetic field and a disordered potential. For clean systems,
be written as h/e2ν, where h is Planck’s constant, e is the electron charge ν is roughly the number of filled Landau levels, that is, the number of highly
and ν is a dimensionless number. Classical considerations predict that degenerate energy states of non-interacting electrons in a magnetic field.
ν = nΦ0/B, where n is the electron density, Φ0 = hc/e is the flux quantum, c is The interaction between electrons is essential to the understanding of the
the speed of light and B is the magnetic field. At low enough temperature fractional quantum Hall states, for which ν is a fraction (see figure, right).
and for clean enough samples, quantum Hall states are formed. These A useful tool for analysing how this interaction leads to the formation of
states are characterized by quantized ν values, leading to plateaux in fractional quantum Hall states is composite fermion theory, as discussed
the Hall resistance as a function of magnetic field (see figure, right). in the main text. (Right panel reproduced, with permission, from ref. 11.)

of non-Abelian states are based on the model of Majorana operators in spin-polarized composite fermions, pairing is possible only if the relative
a superconductor. Realizations of this model have been proposed in angular momentum per pair is odd, with the simplest example being
fractional quantum Hall systems12,17, in p-wave superconductors and p-wave pairing. Vortices may be induced into the composite fermion
in hybrid systems of superconductors with materials in which spin– superconductor by variation of the magnetic field B such that the filling
orbit coupling has a dominant role30–33. Non-Abelian states that are fraction is shifted slightly away from ν = ⁄. These vortices carry Majo-
not described by this model are believed to exist in certain fractional rana operators and are thus non-Abelian quasiparticles.
quantum Hall states18,28, where they are based on clusters larger than Numerical works20–27 support these theories in several ways, of which
two fermions, and in certain spin models19. I describe two. First, the ground state has been calculated numerically
for a small number of particles, and the Pfaffian wavefunction has been
Fractional quantum Hall systems found to approximate it remarkably well for a wide range of microscopic
The first system in which non-Abelian quasiparticles were predicted to interactions. Second, the spectrum has been obtained for a series of
exist12, the quantum Hall system (Box 1), is still the richest arena for the Hamiltonians that slightly differ from one another in the microscopic
search for non-Abelian states of matter. The fractional quantum Hall interactions between the electrons. At one end of this series, the Ham-
effect occurs in a 2D electronic system subject to a strong magnetic field, iltonian was artificially constructed such that the interaction between
and is a consequence of the combination of the magnetic field and the its electrons makes the Pfaffian wavefunction its exact ground state. At
interaction between the electrons. It is characterized by an energy gap in the other end, the Hamiltonian had the realistic Coulomb interaction
the bulk of the system, a gapless mode at its edge, a dissipationless flow of between the electrons built in. The intermediate Hamiltonians adiabati-
current along the edges and a quantization of the Hall resistivity. cally connect between these two extremes. The energy gap was found
There are good reasons to believe that the ν = ⁄ fractional quantum not to vanish in all these cases, at least for the small systems that are
Hall state is non-Abelian9–27 and that the non-Abelian quasiparticles computationally tractable. Because topological properties are robust
carry one-quarter of an electron charge. Originally, deep considerations as long as the energy gap does not close, this conclusion, if correct also
of conformal field theories led Moore and Read12 to propose a trial wave- for an infinite system, implies that the exact ground state has the same
function (known as the ‘Pfaffian’ wavefunction) for the ν = ⁄ state and topological properties as the Pfaffian state.
to conclude that the state may be non-Abelian. If the ν = ⁄ is indeed a non-Abelian state, the first topological qubit
A more intuitive picture that leads to the same understanding is based may be realized8, by means of an electronic analogue to the Fabry–Pérot
on so-called composite fermion theory17,35,36. In this theoretical descrip- interferometer, as described below. It should be noted, however, that
tion, the problem of interacting electrons in a magnetic field B is mapped a Majorana-based qubit has a limited potential (it is ‘non-universal’),
onto a problem of composite fermions in a magnetic field Bʹ = B − 2Φ0n, as the unitary transformations that can be applied to this type of qubit
where Φ0 = hc/e is the flux quantum and n is the electron density. A com- through braiding of quasiparticles do not allow the system to reach
posite fermion is an electron to which two quanta of magnetic flux are to all possible ground states7. It may be possible to realize a universal
attached. This mapping modifies the interaction between the particles: topological qubit using other non-Abelian quantum Hall states, in
composite fermions interact both through their charges and through which the clusters consist of more than two electrons.
the magnetic flux they carry.
The ν = ⁄ state is composed of two filled and inert Landau levels p-wave superconductors
and a half-filled level. Composite fermion theory maps the half-filled If a p-wave superconductor of composite fermions such as the ν = ⁄
Landau level (ν = ½) of interacting electrons onto spin-polarized com- state is a good candidate for a non-Abelian state of matter, a p-wave
posite fermions at zero magnetic field (Bʹ = 0). These composite fer- superconductor of less exotic particles, such as electrons or atoms, might
mions may then form a gapped state by pairing into Cooper pairs. For also be expected to be one. Although relatively rare, superconductors of
189
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

Box 2 | Majorana operators Detecting non-Abelian states

Superconductivity results from the pairing of fermions into Cooper pairs. The difficulty of demonstrating that a many-body state is non-Abelian
When described in the formalism of second quantization, a fermion at a is almost inherent in its nature. In experiments, the system under study
point r in space is created by a creation operator ψ̂+(r) and is annihilated is usually perturbed, and then how it responds to perturbation is exam-
by an annihilation operator ψ̂(r). The spectrum of a superconductor has ined. However, the local perturbations used in experiments, such as
excitations in which some Cooper pairs are broken. These excitations, electric fields, do not couple different ground states of a non-Abelian
which are separated by an energy gap from the ground state, are created system, and therefore do not allow the multitude of ground states to be
by a set of creation operators of the form Γ+ = ∫[g1(r)ψ̂(r) + g2(r)ψ̂+(r)] dr, probed. Nevertheless, several experiments have been proposed as ways
where g1(r) and g2(r) are functions determined by the details of the to probe non-Abelian states. Of these, I focus on those probing the ν = ⁄
system. The excitations are annihilated by a corresponding set of fractional quantum Hall state.
operators, Γ, that are the Hermitian conjugates of Γ +. These excitations
are unique to superconductors: to create an excitation one needs a Predicted signatures of the ν = 5⁄2 quantum Hall state
superposition of operators creating a fermion and annihilating one. Most of the experimental effort directed at studying non-Abelian states
For some superconductors, the presence of vortices allows Cooper has so far been focused on the ν = ⁄ state. The motivation behind this
pairs to be broken at no energy cost. When that happens, the operators effort is twofold. The first is the interest in the nature of the ν = ⁄ state;
that create and annihilate these zero-energy excitations have some the non-Abelian Moore–Read phase is but one of several candidate
unique properties. First, they are Majorana operators, meaning that
descriptions for this state, with Abelian and non-Abelian contenders37.
Γ + = Γ or, equivalently, g1(r) = g2*(r). Creating such an excitation is the
The second is the desire to observe non-Abelian quasiparticles and see
same as annihilating it. Consequently, the zero-energy excitations
that are described by these operators cannot be counted in the way
topological qubits realized.
particles are. Second, the functions g1(r) and g2(r) are localized within The first of the four defining properties of a non-Abelian state, an
the cores of the vortices. Third, the phases of the functions g1(r) and g2(r) energy gap between the ground state and the excitations, characterizes all
localized within the core of one vortex depend on the positions of all quantum Hall states, including that with ν = ⁄. The second characteristic
other distant vortices in a multiply valued way. As a consequence, when to probe is the degeneracy of the ground state38,39. Being a property of
one vortex encircles another, the Majorana operators associated with the spectrum, it may in principle be observed in thermodynamic meas-
both are modified. These zero-energy excitations are the reason for the urements, such as that of the heat capacity. In practice, the electronic
degeneracy of the ground state. Their dependence on the position of the contribution to the heat capacity is overwhelmed by non-electronic,
vortices is what makes them sensitive to braiding. low-energy degrees of freedom, such as phonons, nuclear spins and
so on. The electronic entropy, which is related to the degeneracy of the
ground state, would be better accessed through its effect on the tempera-
this symmetry are formed in helium-3 under certain conditions, and ture dependence of the electronic magnetization and chemical potential,
possibly also in electronic materials, such as SrRuO4. They have also as dictated by Maxwell relations. Measurements of these two thermo-
been conjectured to form from certain cold fermionic atoms29. Vortices dynamic quantities in 2D electronic systems have already been carried
in these superconductors may then be good candidates in which to out40,41, albeit not for the ν = ⁄ state.
observe non-Abelian statistics.
Unfortunately, none of these superconductors has so far been realized Braiding and interferometry
in two dimensions. The vortices that carry Majorana modes in these The most intriguing — and challenging — characteristic to probe is
superconductors are generally not the lowest-energy vortices. Finally, the unitary transformation applied by the braiding of quasiparticles.
the energy difference between the zero-energy Majorana states and other Quasiparticles winding around one another may be observed most
excitations within the cores of the vortices is likely to be very small, being directly through interferometry. The ‘holy grail’ of this line of study
of the order of Δ2/EF, where Δ is the superconductor’s energy gap and is the realization of a qubit8, but an essential step along the way is a
EF is its Fermi energy. definitive experimental demonstration of the quasiparticles being non-
Abelian. Generally, an interferometer interferes pairs of wave trajecto-
Hybrid systems ries that enclose an interference loop. When the interfering particles are
Another set of candidate systems for which Majorana modes are non-interacting electrons in a strong magnetic field, the relative phase
proposed as a means of observing non-Abelian statistics is based on a between the paths is 2π times the number of flux quanta enclosed by the
2D conductor in which spin–orbit coupling — a term in the Hamiltonian interference loop, as dictated by the Aharonov–Bohm effect.
that couples the orbital and spin degrees of freedom of electrons moving In interferometers in the fractional quantum Hall regime (Fig. 2), the
in the crystal field potential of a solid — has a crucial role30–33. This 2D relative phase between the trajectories is affected also by the presence
conductor is to be positioned in proximity to a three-dimensional (3D) of localized quasiparticles trapped within the loop42,43. In particular, for
superconductor in such a way that the latter induces superconductivity the ν = ⁄ Moore–Read state44–47, no interference takes place when a non-
in the former through the so-called proximity effect. The p-wave Cooper Abelian quarter-charged quasiparticle interferes around an interference
pairing originates in this case from the spin–orbit coupling in the 2D loop that encloses an odd number of similar quasiparticles. Interfer-
conductor. Conveniently, the 3D superconductor may be of the very ence does take place when that number is even, in which case one of
common s-wave or d-wave pairing, rather than the rare p-wave type. two interference patterns will be seen, mutually shifted by a phase of π.
Perhaps the most intriguing example for this idea is one in which The choice between these two patterns is determined by the particular
the normal conductor is the 2D metal formed on the surface of a 3D ground state that the localized quasiparticles are in.
topological insulator (see page 194 for a discussion of these newly dis- These general statements become concrete when two types of
covered materials). In this metal, spin–orbit coupling makes the elec- interferometer are considered. A Fabry–Pérot interferometer42–47 is
tron spin lie in the plane of the metal and fixes its angle relative to the shown schematically in Fig. 2a. In the limit where the two constrictions
electron’s linear momentum. Because Cooper pairs in a superconduc- introduce only a small amplitude for tunnelling between the edges, the
tor always comprise pairs of electrons with opposite linear momenta, a probability of the incoming current being backscattered involves an
superconductor formed at the surface of a topological insulator would interference of two trajectories only, one corresponding to each constric-
have Cooper pairs with electrons having a relative angular momentum tion. Ideally, the number of localized quasiparticles in the interference
of ". The superconductor would therefore be of p-wave symmetry and loop is controlled by the magnetic field, and the interference pattern is
vortices formed therein would carry Majorana operators, as discussed probed as a function of the loop’s area, which is controlled by the appli-
above. A similar situation should occur in semiconductors with strong cation of voltage to a side gate. Therefore, if the ν = ⁄ state is indeed the
enough spin–orbit coupling27. non-Abelian Moore–Read state, interference should be turned on and
190
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

off periodically as the magnetic field is varied and the number of trapped a
quasiparticles alternates between even and odd. For the even case, the D2
phase of the interference pattern may assume one of two possible values,
depending on the ground state occupied by the trapped quasiparticles.
When the inter-edge coupling is made stronger, the interferometer
becomes a quantum dot and the sinusoidal interference pattern becomes S D1
a series of so-called Coulomb blockade peaks. These are resonances in
the transmission of the current through the dot that take place when
the dot can accommodate another electron at no electrostatic energy b
cost. The signature of the Moore–Read state is then in two patterns of
peaks: equally spaced peaks when an odd number of quasiparticles is
trapped and peaks that are bunched into pairs when an even number S D2 D1
of quasiparticles is trapped. Again, for the even case there are two pos-
sible patterns of peaks, depending on the ground state of the trapped
quasiparticles44,47.
A Fabry–Pérot interferometer enclosing two quasiparticles is the Figure 2 | Interferometric measurement setups in quantum Hall
proposed ν = ⁄ qubit8 and a measurement of its interference pattern or systems. a, In the context of the quantum Hall effect, the Fabry–Pérot
the positions of its Coulomb blockade peaks as a function of its area serves interferometer is a Hall bar perturbed by two constrictions (‘quantum point
as a read-out of its state. The strength of this qubit is in its insensitivity to contacts’, each represented by a pair of triangles). Current (black) flows
noise, such as that from phonons or photons. As long as the noise is too chirally along the edges. The edge along which it moves to the right is put at
weak to expel one of the localized quasiparticles from the interferometer, a higher chemical potential than the edge along which it moves to the left.
it will have no effect on the ground state occupied by these quasiparticles. The point contacts introduce amplitudes t1 and t2 for inter-edge tunnelling.
The presence of two point contacts makes the probability of backscattering
To change this state, a winding is needed, this time a winding of an edge the quantum Hall analogue of the probability of reaching the interference
quasiparticle around an odd number of the localized ones. screen in a double-slit interference experiment. The backscattered current
A Mach–Zehnder interferometer48,49 is shown schematically in is, to lowest order, proportional to |t1 + t2|2. The relative phase between the
Fig. 2b. In contrast to the Fabry–Pérot interferometer, its interference two amplitudes may be varied by varying the magnetic field and/or the area
loop encloses one of the edges that separate the ν = ⁄ state from the of the interference loop. The latter is implemented using a side gate that, to
vacuum. The current enters the interferometer from the source located the crudest approximation, ‘moves’ the walls of the cell without introducing
at the exterior edge, and ends up in one of the two drains. One drain is quasiparticles into the bulk. By contrast, the variation of the magnetic
located at the interior edge and the other at the exterior edge. Whenever field leads to the introduction of localized quasiholes or quasiparticles.
a quasiparticle tunnels from the exterior edge to the interior edge, it As explained in the text, if the ν = ⁄ state is non-Abelian, the interference
will be turned off when the number of quasiparticles localized in the bulk
changes the parity of the number of quasiparticles within the interfer-
is odd, and turned on when it is even. S, source; D1, D2, drains. b, The
ence loop, and affects the tunnelling probability for the quasiparticles Mach–Zehnder interferometer is another device that allows interference of
that follow. The measured current is then a proper average over tunnel- trajectories of particles that are backscattered through two point contacts.
ling rates that are history dependent. As a striking consequence, in the In the Fabry–Pérot interferometer, the interference loop encloses only the
limit of weak tunnelling through the constrictions the current flows in cell between the two point contacts, whereas here the entire internal edge is
batches of quasiparticles. The average charge in each batch oscillates part of the interference loop. The image shows the two trajectories leading
with the magnetic flux through the interferometer, and varies between from the source to one of the drains.
one-quarter and about three electron charges. This average, which may
be observed in noise measurements, is limited to be less than or equal constraints on the sample quality and temperature needed to observe these
to one electron charge for single-edge quantum Hall states, such as states are less stringent than those needed for the ν = ⁄ state, primarily as a
the ν = 1 and ν = ⅓ states. Because the number of quasiparticles in the result of their larger energy gaps. These studies showed that the magnetic
loop inherently varies in time, its thermal fluctuations do not affect the field and the voltage on the side gate affect the interference in a way that is
noise. Experiments carried out using a Mach–Zehnder interferom- more complicated than that predicted by the idealized model described
eter in the regime of the integer quantum Hall effect conform closely above (Fig. 3a). For most of the interferometers studied, the magnetic
to the ideal model, at least in the limit of small currents through the field and the gate voltage affect both the area of the interference loop and
interferometer (the ‘linear response limit’)50,51. Technically, however, the number of localized quasiparticles62. Although these difficulties have
the Mach–Zehnder interferometer is complicated to realize, and has to be understood in detail, they do not obscure the distinction, described
so far been used only for integer filling fractions, which are easier to above, between the types of interference corresponding to even and odd
study (see Fig. 3). numbers of localized quasiparticles.
The non-Abelian nature of the ν = ⁄ state may also be probed by a A Fabry–Pérot interferometer has also been realized for the ν = ⁄ state.
direct measurement of the edge, independent of the bulk and the local- Recent studies yield encouraging, yet not definitive, results63,64 (Fig. 3). The
ized quasiparticles that it contains. Possible probes include tunnelling of backscattering probability of the current, reflected in the interferometer’s
electrons into the edge from the outside, tunnelling between two edges resistance, was observed to oscillate as a function of the voltage on a side
and thermal transport17,37,52–55. The current–voltage characteristics of gate, and the periodicity of the oscillations was observed to switch between
such transport, the associated current noise and the thermal Hall con- two values. The oscillations were attributed to the effect of the side gate
ductance all carry signatures of the nature of the ν = ⁄ state. on the interferometer’s area, and the switching between periodicities was
attributed to its effect on the number of localized quasiparticles. In this
Present experimental status of the ν = 5⁄2 quantum Hall state interpretation, one periodicity corresponds to the interference of non-
A realization of the various experimental tests described here is being Abelian quarter-charged edge quasiparticles, and dominates when the
vigorously attempted at present by many experimenters. Preliminary number of localized bulk quasiparticles is even. The other periodicity cor-
results support the identification of the ν = ⁄ quasiparticles as carriers responds to the interference of Abelian half-charged edge quasiparticles,
of one-quarter of an electron charge, a necessary condition for non- and dominates when the number of localized bulk quasiparticles is odd
Abelian phenomena to be observed56,57. These results are, however, and quarter-charged quasiparticles do not interfere65.
non-definitive, and are not fully understood. Although the observed switching of periodicities is suggestive, it is not
A Fabry–Pérot interferometer has already been thoroughly studied for yet unambiguous. To make a conclusive identification, a two-pronged
quantum Hall states with integer ν values, ν = ⅓ and ν = ⁄ (refs 58–61). The effort is likely to be required. On the experimental side, a mapping of
191
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

the backscattered current in the two-parameter plane of gate voltage and Outlook
magnetic field is probably the best way to establish the interpretation of the The experiments reviewed in the previous sections, those proposed and
oscillations. On the theoretical side, a connection should be made between those attempted, are aimed at the observation of a non-Abelian state of
the ideal picture and the real world. In the ideal picture, quasiparticles are matter. While this observation is awaited, researchers in the field actively
kept very far away from one another, their number is controlled and their study its theory.
positions may be braided at will. In the real world none of these fully holds. In the context of known candidates for non-Abelian states of matter,
Moreover, energy splittings that are exponentially small cannot always much is still to be learned about probing states whose main defining prop-
be neglected, temperature may not be assumed to be zero and localized erty is their insensitivity to perturbations. The list of possible definitive
bulk quasiparticles may have tunnel couplings to the edge. Some of these tests for the identification of non-Abelian states is likely to be extended by
issues have already been addressed in theoretical works66–71, and others future proposals, and the subtleties associated with interpreting experi-
still need to be understood. mental results will be better understood.
An example of the latter is the issue of spin polarization (refs 22, 72, 73 In the general context of non-Abelian states of matter, the theoretical
and T. D. Rhone and A. Pinczuk, personal communication). The original challenge is wide open — it is the search for physical systems that may
Moore–Read state is fully spin polarized, and numerical works support form non-Abelian states. Among the plethora of interacting systems that
the assumption of full polarization of the ground state. Full polarization is, condensed-matter physics makes it possible to realize, it is desirable to
however, not a condition for non-Abelian quasiparticles to exist. Experi- identify those in which topology dominates the physics. It is to be hoped
ments indicate that in real samples spin polarization may not be complete, that, armed with such an understanding, the fine control that experimen-
either as a property of the ground state or as a finite-temperature conse- talists have over the synthesis of new materials, the fabrication of elec-
quence of low-energy spin-reversed excitations. The effect of incomplete tronic systems and the interaction between cold atoms in condensates
spin polarization on the attempts to study non-Abelian statistics in the can be used to design non-Abelian states of matter.
ν = ⁄ state and to use it for topological quantum computation is not fully In the field of topological quantum computation, the potential of the
understood yet. special properties of non-Abelian states to help quantum computers over-

a 2.0-Nm2 device b d
1.0
6 0.8
5
4 0.7
0.8
0
2 0.6

%B (G)
0.6 –5 0 0.5
V (mV)

–2 0.4
%R (10–3h/e2)

0.4 0.3
–4
0.2
–6
0.2 0.1
6 8 10 12 14 16 18
V (mV)
0.0
1.763 1.764 1.765 1.766 c
B (T)
Near filling fraction 5/2
e/2 e/2
0.01
18-Nm2 device
4

2 0.00
2
%R t200 (8)

0
0 –2 –0.01
0.3
V (mV)

–2 e/4 e/4 e/4

Amplitude

0.2
–0.02
%R (10–3h/e2)

–4
0.1 Period, 15.5 mV
e/2
–6 0
–0.03 0 0.1 0.2
Frequency (mV–1)
–8
0.544 0.545 0.546 0 40 80 120 160 200 240
B (T) Change in side-gate voltage V (mV)

Figure 3 | Experimental results from interferometers. a, Fabry–Pérot interferometer. The change in the resistance is shown as a function of
interferometers were realized first in the integer59,61 and Abelian fractional61 the change in side-gate voltage. The switching of periodicities may be an
quantum Hall states. The plots show the backscattered current (expressed observation of effects associated with non-Abelian statistics. Inset, Fourier
in terms of the change in the sample’s resistance, ΔR) as a function of transform of the data in the main panel. (Panel reproduced from ref. 63.)
magnetic field, B, and voltage on a side gate, V, for small and large areas d, Mach–Zehnder interferometers have already been realized, but only for
of interference loops, for an integer quantum Hall state. The different states of the integer quantum Hall effect. The plot shows the probability
directions of the constant phase lines are due to the variation of the of the current reaching from the source to the drain at the exterior as a
interferometer area with magnetic field, which is crucial for small devices function both of the variation of magnetic field, ΔB, around a value of 9 T,
and negligible for large ones. (Image reproduced, with permission, from and of voltage applied to a side gate. (Data are unpublished data associated
ref. 59.) b, Image of a ν = ⁄ Fabry–Pérot interferometer. Scale, 1 μm. with ref. 50; panel courtesy of N. Ofek and M. Heiblum, Weizmann Institute
(Image reproduced from ref. 63.) c, Data from the ν = ⁄ Fabry–Pérot of Science, Rehovot, Israel.)
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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

come decoherence is all but exhausted, even theoretically. 35. Jain, J. Composite Fermions (Cambridge Univ. Press, 2007).
36. Heinonen, O. (ed.) Composite Fermions (World Scientific, 1998).
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(2000). quantum Hall interferometer. Phys. Rev. Lett. 100, 226803 (2008).
25. Tőke, C., Regnault, N. & Jain, J. K. Nature of excitations of the 5⁄2 fractional quantum Hall 67. Overbosch, B. J. & Wen, X. G. Dynamical and scaling properties of ν=5⁄2 interferometer.
effect. Phys. Rev. Lett. 98, 036806 (2007). Preprint at 〈http://arxiv.org/abs/0706.4339〉 (2007).
26. Wójs, W. & Quinn, J. J. Landau level mixing in the ν=5⁄2 fractional quantum Hall state. Phys. 68. Rosenow, B., Halperin, B., Simon, S. & Stern, A. Exact solution for bulk-edge coupling in the
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27. Feiguin, A. E., Rezayi, E., Nayak, C. & Das Sarma, S. Density matrix renormalization group 69. Bishara, W. & Nayak, C. Odd–even crossover in a non-Abelian ν=5⁄2 interferometer. Phys.
study of incompressible fractional quantum Hall states. Phys. Rev. Lett. 100, 166803 (2008). Rev. B 80, 155304 (2009).
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Einstein condensates. Phys. Rev. Lett. 87, 120405 (2001). quantum Hall droplets. Phys. Rev. B 78, 195321 (2008).
29. Gurarie, V. & Radzihovsky, L. Resonantly paired fermionic superfluids. Ann. Phys. 322, 2–119 71. Overbosch, B. J. & Bais, F. A. Inequivalent classes of interference experiments with non-
(2007). Abelian anyons. Phys. Rev. A 64, 062107 (2001).
30. Fu, L. & Kane, C. L. Superconducting proximity effect and Majorana fermions at the surface 72. Feiguin, A. E., Rezayi, E., Yang, K., Nayak, C. & Das Sarma, S. Spin polarization of the ν=5⁄2
of a topological insulator. Phys. Rev. Lett. 100, 096407 (2008). quantum Hall state. Phys. Rev. B 79, 115322 (2009).
This paper proposes a setting in which Majorana fermions are created in a hybrid system 73. Dean, C. R. et al. Contrasting behavior of the ν=5⁄2 and 7⁄3 fractional quantum Hall effect in a
of a topological insulator and a superconductor. tilted field. Phys. Rev. Lett. 101, 186806 (2008).
31. Fu, L. & Kane, C. L. Probing neutral Majorana fermion edge modes with charge transport.
Phys. Rev. Lett. 102, 216403 (2009).
32. Nilsson, J., Akhmerov, A. R. & Beenakker, C. W. Splitting of a Cooper pair by a pair of Acknowledgements This work was supported by the US–Israel Binational Science
Majorana bound states. Phys. Rev. Lett. 101, 120403 (2008). Foundation, the Minerva foundation and Microsoft’s Station Q.
33. Sau, J. D., Lutchyn, R. M., Tewari, S. & Das Sarma, S. A generic new platform for topological
quantum computation using semiconductor heterostructures. Preprint at 〈http://arxiv.org/ Author Information Reprints and permissions information is available at www.nature.
abs/0907.2239〉 (2009). com/reprints. The author declares no competing financial interests. Correspondence
34. Wilczek, F. Majorana returns. Nature Phys. 5, 614–618 (2009). should be addressed to the author (adiel.stern@weizmann.ac.il).

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INSIGHT PERSPECTIVE NATURE|Vol 464|11 March 2010|doi:10.1038/nature08916

The birth of topological insulators

Joel E. Moore1,2

Certain insulators have exotic metallic states on their surfaces. These states are formed by topological
effects that also render the electrons travelling on such surfaces insensitive to scattering by impurities. Such
topological insulators may provide new routes to generating novel phases and particles, possibly finding uses
in technological applications in spintronics and quantum computing.

Many aspects of condensed-matter physics are concerned with twisted, without being cut. Nevertheless, these invariants do change
understanding how order emerges when a very large number of simple in crossing the interface between topological and ordinary insulators,
constituents, such as ions, magnetic moments or electrons, interact with so by contradiction the surface cannot remain insulating, which is
each other. In ordered phases such as crystals and magnets, the order is analogous to cutting the knot.
described through symmetry breaking: in a crystal, ions are arranged In a topological insulator, the component that is ‘knotted’ is the
periodically owing to their electrostatic interactions, thereby breaking electron’s wavefunction as it moves through momentum space. Associ-
the continuous symmetry of space under rotations and translations; ated with this knotting are topological invariants (usually expressed as
in typical magnets, some of the rotational symmetry of spin space is integrals involving the wavefunction) that cannot change as long as the
broken, together with the time-reversal symmetry. material remains insulating. Even when the boundary between ordi-
A major discovery in the 1980s was that electrons that are confined nary insulators and topological insulators is atomically sharp and the
to two dimensions and subject to a strong magnetic field show a com- continuous description using topology might not seem to be applica-
pletely different, topological, type of order, which underlies the quantum ble, there are nevertheless metallic delocalized wavefunctions at this
Hall effect. Consequences of this order include dissipationless transport boundary. In Fig. 1b, the simplest case of knotting in a 3D electronic
and emergent particles with fractional charge and statistics. One of the structure is illustrated: for one occupied electronic band and one empty
important discoveries of the past few years is that topological order also one, each point in 3D momentum space is associated with a unit vector
occurs in some three-dimensional (3D) materials; in these materials, the representing the occupied state, and the Hopf map shown in Fig. 1b is a
role of the magnetic field is assumed by the mechanism of spin–orbit topologically non-trivial example.
coupling, an intrinsic property of all solids. These materials have been
named topological insulators because they are insulators in the ‘bulk’ Lessons from the past
but have exotic metallic states present at their surfaces owing to the The appearance of the unusual metal when the topology changes at a
topological order. surface is the main experimental signature that an insulator is indeed
In this Perspective article, I provide an overview of the basic concepts topological. To explain the properties of the insulator, I will begin with
underlying topological insulators and recent studies of these remark- a short review of the historical developments that led to the theoretical
able new materials. After an explanation of what makes some insulators predictions that topological insulators exist. A simpler version of this
‘topological’ and a brief history of this rapidly developing field, I discuss metal occurs at the edge of a quantum Hall droplet, the first reported
recent advances made in experiments on topological insulators — for example of two-dimensional (2D) topological order. Quantum Hall
both bulk and nanostructured materials — and in the theoretical under- edges are perfect quantum wires wrapping around the insulating droplet
standing of these materials. I conclude by explaining why many research (Fig. 2a), and they result from the topological properties of the elec-
groups are seeking to use topological insulators to generate new particles tronic wavefunctions when the constituent electrons are confined to
and phases, which has possible applications to quantum computing. two dimensions and subject to a strong magnetic field perpendicular
to the plane to which the electrons are confined (see page 187 for more
A primer on topological insulators details on the quantum Hall effect).
The easiest way to describe a topological insulator is as an insulator Work on topological insulators grew out of the idea that the quantum
that always has a metallic boundary when placed next to a vacuum Hall effect that arises in such 2D systems in the presence of a magnetic
or an ‘ordinary’ insulator. These metallic boundaries originate from field could occur even for electrons moving on a lattice in the absence
topological invariants, which cannot change as long as a material of a macroscopic magnetic field1. Instead of being driven by such a mag-
remains insulating. An intuitive illustration showing why the metallic netic field, it was predicted in the late 1980s that electrons could, in
surfaces exist is presented in Fig. 1a: a trefoil knot is used to represent principle, form a quantum Hall state driven by forces that result from
a topological insulator and a closed loop to represent an ordinary one. their motion through the crystal lattice. Recent developments are based
Topology is a branch of mathematics that studies the properties of on spin–orbit coupling, a relativistic effect in which the spin and orbital
objects that are invariant under smooth deformations, a classic exam- angular momentum degrees of freedom of electrons are coupled; this
ple being a doughnut transforming into a coffee cup. In contrast to the coupling causes electrons that are moving through a crystal to feel a
doughnut/coffee cup pair, the trefoil knot and the closed loop have spin-dependent force, even in non-magnetic materials.
different topological invariants and thus neither can be deformed to Although spin–orbit coupling does not have the symmetry required
become the other, no matter how the string (or wire) is stretched or to induce the quantum Hall effect (that is, it does not break time-reversal

1
Department of Physics, 366 Le Conte #7300, University of California, Berkeley, California 94720, USA. 2Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
California 94720, USA.

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NATURE|Vol 464|11 March 2010 PERSPECTIVE INSIGHT

a b

Figure 1 | Metallic states are born when a surface unties ‘knotted’ electron defined. If the topological invariants are always defined for an insulator,
wavefunctions. a, An illustration of topological change and the resultant then the surface must be metallic. b, The simplest example of a knotted 3D
surface state. The trefoil knot (left) and the simple loop (right) represent electronic band structure (with two bands)35, known to mathematicians as
different insulating materials: the knot is a topological insulator, and the the Hopf map. The full topological structure would also have linked fibres
loop is an ordinary insulator. Because there is no continuous deformation on each ring, in addition to the linking of rings shown here. The knotting
by which one can be converted into the other, there must be a surface where in real topological insulators is more complex as these require a minimum
the string is cut, shown as a string with open ends (centre), to pass between of four electronic bands, but the surface structure that appears is relatively
the two knots; more formally, the topological invariants cannot remain simple (Fig. 3).

symmetry as an applied magnetic field would), in simplified models in the crystal) will always contain a quantum wire like that at the edge
introduced in around 2003 it can lead to a quantum spin Hall effect, of the quantum spin Hall effect (discussed earlier), which may allow 2D
in which electrons with opposite spin angular momentum (commonly topological insulator physics to be observed in a 3D material9.
called spin up and spin down) move in opposite directions around the There is also, however, a ‘strong’ topological insulator, which has a
edge of the droplet in the absence of an external magnetic field2 (Fig. 2b). more subtle relationship to the 2D case; the relationship is that in two
These simplified models were the first steps towards understanding dimensions it is possible to connect ordinary insulators and topologi-
topological insulators. But it was unclear how realistic the models were: cal insulators smoothly by breaking time-reversal symmetry7. Such a
in real materials, there is mixing of spin-up and spin-down electrons, continuous interpolation can be used to build a 3D band structure that
and there is no conserved spin current. It was also unclear whether the respects time-reversal symmetry, is not layered and is topologically non-
edge state of the droplet in Fig. 2b would survive the addition of even trivial. It is this strong topological insulator that has protected metallic
a few impurities. surfaces and has been the focus of experimental activity.
In 2005, a key theoretical advance was made by Kane and Mele3. Spin–orbit coupling is again required and must mix all components of
They used more realistic models, without a conserved spin current, the spin. In other words, there is no way to obtain the 3D strong topologi-
and showed how some of the physics of the quantum spin Hall effect cal insulator from separate spin-up and spin-down electrons, unlike in
can survive. They found a new type of topological invariant that could the 2D case. Although this makes it difficult to picture the bulk physics of
be computed for any 2D material and would allow the prediction of the 3D topological insulator (only the strong topological insulator will be
whether the material had a stable edge state. This allowed them to show discussed from this point), it is simple to picture its metallic surface6.
that, despite the edge not being stable in many previous models, there are The unusual planar metal that forms at the surface of topological
realistic 2D materials that would have a stable edge state in the absence of insulators ‘inherits’ topological properties from the bulk insulator.
a magnetic field; the resultant 2D state was the first topological insulator The simplest manifestation of this bulk–surface connection occurs at
to be understood. This non-magnetic insulator has edges that act like a smooth surface, where momentum along the surface remains well
perfectly conducting one-dimensional electronic wires at low tempera- defined: each momentum along the surface has only a single spin state
tures, similar to those in the quantum Hall effect. at the Fermi level, and the spin direction rotates as the momentum
Subsequently, Bernevig, Hughes and Zhang made a theoretical moves around the Fermi surface (Fig. 3). When disorder or impurities
prediction that a 2D topological insulator with quantized charge con- are added at the surface, there will be scattering between these surface
ductance along the edges would be realized in (Hg,Cd)Te quantum states but, crucially, the topological properties of the bulk insulator do
wells4. The quantized charge conductance was indeed observed in this not allow the metallic surface state to vanish — it cannot become local-
system, as a quantum-Hall-like plateau in zero magnetic field, in 2007 ized or gapped. These two theoretical predictions, about the electronic
(ref. 5). These experiments are similar to those on the quantum Hall structure of the surface state and the robustness to disorder of its metallic
effect in that they require, at least so far, low temperature and artificial behaviour, have led to a flood of experimental work on 3D topological
2D materials (quantum wells), but they differ in that no magnetic field insulators in the past two years.
is needed.
Experimental realizations
Going 3D The first topological insulator to be discovered was the alloy BixSb1−x,
The next important theoretical development, in 2006, was the the unusual surface bands of which were mapped in an angle-resolved
realization6–8 that even though the quantum Hall effect does not general- photoemission spectroscopy (ARPES) experiment10,11. In ARPES exper-
ize to a genuinely 3D state, the topological insulator does, in a subtle way. iments, a high-energy photon is used to eject an electron from a crystal,
Although a 3D ‘weak’ topological insulator can be formed by layering and then the surface or bulk electronic structure is determined from an
2D versions, similar to layered quantum Hall states, the resultant state analysis of the momentum of the emitted electron. Although the surface
is not stable to disorder, and its physics is generally similar to that of the structure of this alloy was found to be complex, this work launched a
2D state. In weak topological insulators, a dislocation (a line-like defect search for other topological insulators.
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INSIGHT PERSPECTIVE NATURE|Vol 464|11 March 2010

A graphene lookalike
a Figure 3 shows the measured surface state of Bi2Se3 and a theoretical
idealization of its state, including the electron spin. The surface state of
e– the next-generation topological insulators is closely related to the Dirac
electronic structure of graphene, which has a linear energy–momentum
relationship like that of a relativistic particle (and is known as a Dirac
cone). Graphene, which consists of a single layer of carbon atoms, has
Integer quantum Hall state been an extremely active subject of research in recent years15: it is inter-
(n = 1) esting both structurally, because it is the most 2D material possible, and
electronically, because of its linear energy–momentum relationship. The
main difference between the surface of a topological insulator and that
of graphene is that the topological insulator has only one Dirac point (or
valley) and no spin degeneracy, whereas graphene has two Dirac points
and is spin degenerate. This difference has far-reaching consequences,
Ordinary insulator including the possibility of generating new particles that have applica-
tions in quantum computing.
Another remarkable consequence of the absence of the extra
b degeneracies was detected by scanning tunnelling microscopy experi-
ments16–18: the interference patterns near defects or steps on the surface
show that electrons are never completely reflected when scattered. Even
if the disorder becomes much stronger and a description in terms of
well-separated scattering events is invalid, the surface remains metallic19.
This protection of the surface metal from Anderson localization (that
is, formation of an insulating state as a result of strong disorder20) is one
Idealized quantum spin Hall state of the key differences between the surface of the topological insulator
and the ‘accidental’ surface states present in other materials, such as the
noble metals. Beyond just being stable to disorder, it is now understood
that the topological insulator, at least in some 2D models, forms as a
result of disorder21,22. In graphene, there is an approximate version of
this protection if the scattering has a very smooth potential, but real
graphene is likely to become localized with strong disorder.
Ordinary insulator There was, however, initially a disadvantage in using a topological
insulator for some purposes as opposed to graphene. In graphene, the
chemistry of the carbon atoms naturally locates the Fermi level at the
Figure 2 | Topological order in two dimensions. a, Edge of an integer
quantum Hall state. The electrons (e–) are confined to a 2D insulating
Dirac point (that is, the point at which the two cones intersect), where
droplet with a metallic edge. Along the edge, electrons propagate only in the density of states vanishes. This means that the density of carriers in
one direction, which is determined by the sign of the applied magnetic graphene is highly tunable using an applied electrical field and allows
field perpendicular to the droplet. One integer, n, the topological invariant, applications of graphene in both basic science and microelectronics. The
determines the Hall conductance and the number of propagating edge surface Fermi level of a topological insulator does not have any particu-
modes. b, Edge of an idealized quantum spin Hall state (that is, a 2D lar reason to sit at the Dirac point; however, through a combination of
topological insulator). Along the edge, spin-up electrons move clockwise, surface and bulk chemical modification, tuning to the Dirac point in
whereas spin-down electrons move anticlockwise. Spin-up and spin-down Bi2Se3 was recently demonstrated23. This control of chemical potential is
electrons are independent and are in oppositely directed quantum Hall important for applications, as well as for a proposal to create a topologi-
states. An applied electrical field generates a spin current but no charge
cal exciton condensate by biasing a thin film of topological insulator24.
current. Each droplet is surrounded by an ordinary insulator.
This condensate is a superfluid state of electron–hole pairs that would
have a bound electronic state around superfluid vortices.
For a topological insulator to form, spin–orbit coupling must be strong
enough to modify the electronic structure significantly, which suggests Materials challenges
that heavy-element, small-bandgap semiconductors are the most promis- Two recent advances in the nanostructuring of topological insulators
ing candidates. This suggestion stems from two points. First, spin–orbit also deserve a mention. Nanoribbons of Bi2Se3 have been synthesized,
coupling is a relativistic effect and is only strong for heavy elements. Sec- allowing observation of the Aharonov–Bohm effect in the metallic
ond, if the bandgap is much larger than the energy scale of spin–orbit surface state when a magnetic field is applied along the long direction
coupling, then spin–orbit coupling will not be able to change the phase. of the nanoribbon25, and molecular beam epitaxy has been used to
The search for topological insulators culminated in the recent discovery grow thin films of Bi2Se3 with controlled thickness down to a single
of topological insulator behaviour in Bi2Se3 and Bi2Te3 (refs 12–14). These unit cell26. Such nanostructures are essential for many of the proposed
‘next-generation’ materials both show topological insulator behaviour up applications of topological insulators to spintronics and other fields.
to higher temperatures than does the original material (BixSb1−x), with For example, the spin structure in Fig. 3 means that a charge current
bulk bandgaps of more than 0.1 eV, and have the simplest surface state along the surface of the topological insulator automatically yields a
that is allowed. Beyond providing further confirmation of the theory non-zero spin density; a heterostructure that combines a topologi-
of topological insulators, the simplicity of the surface state in these cal insulator with a ferromagnet could allow the ferromagnet to be
materials opens up the possibility of many experiments, some of which switched by passing a current through the topological insulator’s sur-
are described below. Furthermore, the large bandgap means that these face, and this would be a new type of spin torque device for magnetic
experiments do not need to be carried out at extremely low temperatures. memory applications27. The next important steps are to carry out
The main remaining complication about these materials, especially when direct transport and optical experiments on the metallic surface state
using experimental techniques that do not distinguish directly between to measure its conductivity and spin properties, and for these experi-
bulk states and surface states (unlike ARPES), is that in the bulk state they ments improved materials with reduced residual bulk conductivity
have residual conductivity arising from impurities. might be required.
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NATURE|Vol 464|11 March 2010 PERSPECTIVE INSIGHT

A nascent field should be possible to observe the effect in topological insulators.

Several experiments that are under way are based on a different picture Another recent theoretical advance was understanding how phases
of the essential properties of a topological insulator, a picture that is con- similar to the 3D topological insulator might appear in systems with
nected to particle physics research from the 1980s. It has been known different symmetries than those of the non-magnetic solids discussed
for many years that the magnetic field and electric field are coupled in above33–35. Finding experimental examples of these other types of topo-
the interior of some insulators. Topological insulators contain a special logical insulator and superconductor would be a major accomplish-
type of such coupling that is quantized and was originally discussed by ment. Work in this area also led to a reconsideration of the properties of
particle physicists who were analysing the coupling of an axion particle superfluid 3He, in which neutral fermionic atoms form Cooper pairs: the
to ordinary electric and magnetic fields. quasiparticles in this superfluid have some remarkable properties that
are closely connected to those of electrons in the topological insulator,
Particle physics and superfluidity and finding direct experimental signatures of these quasiparticles is an
Condensed-matter physicists often seek to define a phase of matter important challenge.
in terms of its response to some influence. For example, a solid is a
material with a non-zero stiffness in response to applied shear force, Emergent particles and quantum computing
and a superconductor is defined in terms of the Meissner effect (that The interface between a topological insulator and a superconductor
is, by its expulsion of applied magnetic fields). Such definitions are may allow the creation of an ‘emergent’ particle that neither material
connected directly to experimentation and are independent of details supports by itself. Like any other metal, the metallic surface of a topo-
on the microscopic level. It turns out that the defining response of a logical insulator becomes superconducting, by way of the proximity
topological insulator was almost worked out in the 1980s in an effort to effect, when placed next to an ordinary superconductor. However, the
understand the properties of axion electrodynamics, which differs from superconducting surface of a topological insulator is novel: if a vortex
ordinary electrodynamics by the addition to the Lagrangian of a term line runs from the superconductor into the topological insulator, then
that is proportional to the scalar product of the electric field and the a zero-energy Majorana fermion is trapped in the vicinity of the vor-
magnetic field, E•B (ref. 28). There are only two values of the coefficient tex core36,37. Unlike vortex core states in ordinary superconductors, the
of this term that are compatible with time-reversal symmetry, and these Majorana fermion has quantum numbers that differ from those of an
correspond to ordinary insulators and topological insulators. Hence, ordinary electron: it is its own antiparticle, is electrically neutral and
topological insulators are materials whose internal structure generates is, in most respects, ‘half ’ of an ordinary electron38. There are several
the non-zero value of the axion-like coupling, in the same way that reasons for the excitement about this proposal and others of a simi-
insulators modify the dielectric constant, which is the coefficient of E2 lar spirit39. First, these proposals may allow a Majorana fermion to be
in the Lagrangian29. observed directly, a long-sought goal in particle physics and condensed-
There are many predicted consequences of this term30,31, including matter physics. Second, Majorana fermions are one step towards a topo-
monopole-like behaviour and surface states with continuously vari- logical quantum computer, which would be exceptionally well protected
able Hall conductivity. The most important for applications may be the from errors40 because the quasiparticles obey a special kind of quantum
simple magnetoelectric effect in which an applied electrical field gen- statistics that is non-Abelian (for a more in-depth discussion of non-
erates a magnetic dipole and vice versa. The potential advantage of the Abelian states of matter, see page 187). More complex materials might
magnetoelectric effect in topological insulators over that in multiferroic even allow Majorana fermions as point excitations in the bulk of the
materials32 is an increase not in strength but instead in speed and repro- 3D material41.
ducibility without fatigue, because this effect results purely from orbital More generally, there are several aspects of the topological order in
motion of the electrons. Whether a given material is a topological insu- quantum Hall states that have not yet been realized in topological insu-
lator can be theoretically defined simply by asking how much its bulk lators, and the most important is the emergence of quasiparticles with
polarization changes in an applied magnetic field, and measurements modified charge and statistics. The traditional wellspring of topological
of this magnetoelectric polarizability in other materials suggest that it order, the 2D electron gas in a strong magnetic field, has not dried up;

a b E
ky
0.1 High
M ( M

0.0 SS kx
EF

–0.1 ky
EB (eV)

Low
–0.2 kx
Surface Bulk
band bandgap
–0.3
Dirac
Bulk point
–0.4 bands
–0.15 0 0.15
ky (Å–1)

Figure 3 | Signatures of the exotic metallic surface states in topological is indicated by white arrows. The direction of electron spin is indicated by
insulators. a, The electronic structure of Bi2Se3, as measured by blue arrows. (Panel modified, with permission, from ref. 12; data taken
ARPES. Measured energy electron energy, EB, is plotted against electron from refs 12 and 23.) b, Theoretical idealization of the electronic structure
momentum, ky. High intensity (red and yellow areas) indicates a non-zero of Bi2Se3, showing the rotation of the spin degree of freedom (red arrows)
electronic density of states. The surface bands crossing the bulk bandgap as an electron (with energy E) moves around the Fermi surface (with
–
enclose a single Dirac point at the Brillouin-zone centre (Γ), which is the Fermi energy EF). Scattering of the surface electrons by non-magnetic
signature that this material is a topological insulator. —
M indicates the disorder will modify the details of the electronic wavefunctions but will not
centre of an edge of the Brillouin zone, and the path in the Brillouin zone eliminate the metallic surface.
197
© 2010 Macmillan Publishers Limited. All rights reserved
INSIGHT PERSPECTIVE NATURE|Vol 464|11 March 2010

recent results from studies of this gas include the first observation of 14. Chen, Y. L. et al. Experimental realization of a three-dimensional topological insulator,
Bi2Te3. Science 325, 178–181 (2009).
the fractional quantum Hall effect in graphene and suggest the possible 15. Castro Neto, A. H., Guinea, F., Peres, N. M., Novoselov, K. S. & Geim, A. K. The electronic
observation of non-Abelian statistics in interferometry experiments. properties of graphene. Rev. Mod. Phys. 81, 109–163 (2009).
Topological states have also been proposed to occur in frustrated spin 16. Roushan, P. et al. Topological surface states protected from backscattering by chiral spin
systems (such as those discussed on page 201). The variety of topological texture. Nature 460, 1106–1109 (2009).
17. Alpichshev, Z. et al. STM imaging of electronic waves on the surface of Bi2Te3:
phenomena observed in semiconductor heterostructures over the past topologically protected surface states and hexagonal warping effects. Preprint at 〈http://
three decades suggests that the topological insulator materials that have arxiv.org/abs/0908.0371〉 (2009).
been uncovered so far are just the first examples and that new types of 18. Zhang, T. et al. Experimental demonstration of the topological surface states protected by
the time-reversal symmetry. Preprint at 〈http://arxiv.org/abs/0908.4136〉 (2009).
topological order still await discovery. 19. Nomura, K., Koshino, M. & Ryu, S. Topological delocalization of two-dimensional
massless Dirac fermions. Phys. Rev. Lett. 99, 146806 (2007).
Looking forward 20. Anderson, P. W. Absence of diffusion in certain random lattices. Phys. Rev. 109, 1492–1505
(1958).
The recent discovery of topological insulators, like other advances in 21. Li, J. et al. Topological Anderson insulator. Phys. Rev. Lett. 102, 136806 (2009).
basic condensed-matter physics, allows new applications that build on 22. Groth, C. W. et al. Theory of the topological Anderson insulator. Phys. Rev. Lett. 103,
our new understanding. The unusual metallic surfaces of these insula- 196805 (2009).
23. Hsieh, D. et al. A tunable topological insulator in the spin helical Dirac transport regime.
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more, in combination with superconductors, topological insulators 24. Seradjeh, B., Moore, J. E. & Franz, M. Exciton condensation and charge fractionalization in
could lead to a new architecture for topological quantum bits. These a topological insulator film. Phys. Rev. Lett. 103, 066402 (2009).
25. Peng, H. et al. Aharonov–Bohm interference in topological insulator nanoribbons. Preprint
insulators have already had a considerable impact on ‘pure’ condensed- at 〈http://arxiv.org/abs/0908.3314〉 (2009).
matter physics, making it clear that topological effects — which were 26. Zhang, Y. et al. Crossover of three-dimensional topological insulator of Bi2Se3 to the two-
long thought to be restricted to low temperatures, reduced dimensional- dimensional limit. Preprint at 〈http://arxiv.org/abs/0911.3706〉 (2009).
ity and high magnetic field — can determine the physics of seemingly 27. Garate, I. & Franz, M. Inverse spin-galvanic effect in a topological-insulator/ferromagnet
interface. Preprint at 〈http://arxiv.org/abs/0911.0106〉 (2009).
ordinary bulk materials under ordinary conditions. ■ 28. Wilczek, F. Two applications of axion electrodynamics. Phys. Rev. Lett. 58, 1799–1802
(1987).
1. Haldane, F. D. M. Model for a quantum Hall effect without Landau levels: condensed- 29. Qi, X.-L., Hughes, T. L. & Zhang, S.-C. Topological field theory of time-reversal invariant
matter realization of the ‘parity anomaly’. Phys. Rev. Lett. 61, 2015–2018 (1988). insulators. Phys. Rev. B 78, 195424 (2008).
2. Murakami, S., Nagaosa, N. & Zhang, S.-C. Spin-Hall insulator. Phys. Rev. Lett. 93, 156804 30. Qi, X.-L. et al. Inducing a magnetic monopole with topological surface states. Science 323,
(2004). 1184–1187 (2009).
3. Kane, C. L. & Mele, E. J. Z2 topological order and the quantum spin Hall effect. Phys. Rev. 31. Essin, A. M., Moore, J. E. & Vanderbilt, D. Magnetoelectric polarizability and axion
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This paper explains the theoretical requirements for a non-magnetic material to be a 32. Ramesh, R. & Spaldin, N. A. Multiferroics: progress and prospects. Nature Mater. 6, 21–27
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has a quantum spin Hall effect. magnetic insulators. Phys. Rev. Lett. 101, 186805 (2008).
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106803 (2007). surface of a topological insulator. Phys. Rev. Lett. 100, 096407 (2008).
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Phys. Rev. B 75, 121306(R) (2007). (1980).
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topological insulators with lattice dislocations. Nature Phys. 5, 298–303 (2009). 40. Collins, G. P. Computing with quantum knots. Sci. Am. 294, 57–63 (2006).
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970–974 (2008). Phys. Rev. Lett. 104, 046401 (2009).
By using ARPES experiments, this study observed a 3D topological insulator, the
theoretical predictions for which were made in refs 6–8. Acknowledgements I have benefited from conversations about topological
11. Hsieh, D. et al. Observation of unconventional quantum spin textures in topological insulators with L. Balents, B. A. Bernevig, A. Essin, M. Franz, D. Haldane, Z. Hasan,
insulators. Science 323, 919–922 (2009).
C. Kane, D.-H. Lee, A. Ludwig, L. Molenkamp, S. Ryu, D. Vanderbilt, A. Vishwanath,
12. Xia, Y. et al. Observation of a large-gap topological-insulator class with a single Dirac cone
X.-G. Wen, C. Xu and S.-C. Zhang. My work on topological insulators is supported
on the surface. Nature Phys. 5, 398–402 (2009).
13. Zhang, H. et al. Topological insulators in Bi2Se3, Bi2Te3 and Sb2Te3 with a single Dirac cone by the US National Science Foundation.
on the surface. Nature Phys. 5, 438–442 (2009).
References 12 and 13 report experiments and theory on next-generation topological Author Information Reprints and permissions information is available at www.
insulator materials, which have a large bandgap and a single surface Dirac cone; these are nature.com/reprints. The author declares no competing financial interests.
the most promising materials for future experiments. Correspondence should be addressed to the author (jemoore@berkeley.edu).

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NATURE|Vol 464|11 March 2010|doi:10.1038/nature08917 REVIEW INSIGHT

Spin liquids in frustrated magnets

Leon Balents1

Frustrated magnets are materials in which localized magnetic moments, or spins, interact through competing
exchange interactions that cannot be simultaneously satisfied, giving rise to a large degeneracy of the
system ground state. Under certain conditions, this can lead to the formation of fluid-like states of matter,
so-called spin liquids, in which the constituent spins are highly correlated but still fluctuate strongly down to
a temperature of absolute zero. The fluctuations of the spins in a spin liquid can be classical or quantum and
show remarkable collective phenomena such as emergent gauge fields and fractional particle excitations. This
exotic behaviour is now being uncovered in the laboratory, providing insight into the properties of spin liquids
and challenges to the theoretical description of these materials.

Sometimes, a little frustration can make life interesting. This is true in the result is a quantum spin liquid (QSL), which is a superposition state
physics, where frustration often leads to exotic properties of materi- in which the spins simultaneously point in many different directions. In
als. In physics, frustration refers to the presence of competing forces a QSL, the spins are highly entangled with one another in a subtle way
that cannot be simultaneously satisfied. The concept has been applied that involves entanglement between widely separated spins. QSLs are
broadly, from magnetism, which I discuss here, to negative thermal more elusive than their classical counterparts but offer a much greater
expansion of solids1 and to soft materials2. Studies of frustration began conceptual pay-off. From theory, it is predicted that they should show
with antiferromagnets, in which frustration usually has a simple geo- extremely exotic phenomena such as hosting exotic excitations with
metric origin. Such geometric frustration occurs in systems of spins fractional quantum numbers and artificial gauge fields. They may also
on lattices that involve triangular motifs (Fig. 1), in which the nearest- be associated with exotic forms of superconductivity4. Researchers are
neighbour interactions favour anti-aligned spins (Box 1). On a triangle, seeking QSL states in solid-state materials known as Mott insulators (in
the three spins cannot all be antiparallel. Instead, depending on the which electrons are localized to individual atomic or molecular orbit-
circumstances, the spins fluctuate, or order, in a less obvious manner. als but maintain their spin degree of freedom) and recently in artificial
An antiferromagnet consisting of a two-dimensional (2D) triangular frustrated lattices that have been created optically for ultracold atoms.
lattice of Ising spins (which point either upward or downward along In this Review, I survey how the discovery of new materials and
a single axis) provided one of the prototypes of frustration. Wannier improved experimental probes, together with complementary advances
showed in 1950 that this model has a very large degeneracy of ground in theory, have reinvigorated the study of spin liquids and frustrated mag-
states3. Now, such degeneracy is considered to be a key, or even defining, netism in general. I begin with the best-understood example of spin ice,
characteristic of frustration. In the triangular Ising antiferromagnet, the a classical spin liquid, whose properties are far better understood after
ground-state entropy is extensive and equals 0.323kBN, where kB is the progress made in the past year. I then turn to quantum frustrated systems.
Boltzmann constant and N is the number of spins. At low temperatures, After describing their basic physics, I discuss the experimental situation
the spins continue to fluctuate thermally, although in a correlated man- and its challenges. I uncover some unexpected physics in ‘failures’ to find
ner because they are restricted to the ground states of the Ising anti- QSLs. Finally, I discuss promising directions for QSL research.
ferromagnet. By analogy to an ordinary liquid, in which the molecules
form a dense, highly correlated state that has no static order, the spins in Spin ice
the triangular Ising antiferromagnet form a ‘spin liquid’, or cooperative To begin the survey of these developments, I start with a simple exam-
paramagnet. The ‘frustration’ (or ‘fluctuation’) parameter, f (Box 1), pro- ple, in which the spins can be regarded as classical: spin-ice materials5,6,
vides a quantitative measure of the depth of the spin-liquid regime; f = ∞ which were first studied by Harris and colleagues in 1997 (ref. 7). In spin
if the spins remain liquid down to a temperature, T, of absolute zero. ices — for example Dy2Ti2O7, Ho2Ti2O7 and Ho2Sn2O7 — only the ions
Fluctuations of the spins in a spin liquid can be classical or quantum. of the rare-earth atoms (Dy and Ho) are magnetic, and these reside on
In quantum mechanics, the magnitude of a spin is quantized in half- a pyrochlore lattice, which is a network of corner-sharing tetrahedra
integer units of " (where " denotes Planck’s constant divided by 2π), (Fig. 1c). Their f-electron spins are large and classical, and they behave
the quantum of angular momentum. Classical fluctuations dominate as Ising doublets aligned with the local 〈111〉 axis, which connects the
for large spins (those with a size, S, much larger than the minimum centres of the two tetrahedra shared by that spin. Their interactions are
size of ½) and are driven by thermal energy. Spins can be thought of as predominantly long range and dipolar, but much of their physics can be
reorienting randomly with time, cycling through different microstates. understood solely from an effective nearest-neighbour exchange energy,
When the energy kBT becomes too small, classical fluctuations cease and Jeff, which is ferromagnetic8,9 (Box 2). This ferromagnetic interaction
the spins either freeze or order. For small spins, with S comparable to is frustrated, owing to the different Ising axes of the spins. Indeed,
½, the quantum mechanical uncertainty principle produces zero-point the states that minimize the energy of a single tetrahedron are highly
motions comparable to the size of the spin itself, which persist down to degenerate, consisting of all six configurations in which two spins point
T = 0 K. Although they are similar to thermal fluctuations in some ways, inward and two spins point outward from the centre of the tetrahedron
quantum fluctuations can be phase coherent. If they are strong enough, (Fig. 2a). The name spin ice originates from a direct analogy between
1
Kavli Institute for Theoretical Physics, University of California, Santa Barbara, Santa Barbara, California 93106, USA.

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these configurations and the positions of protons in the tetrahedrally because of the two-in, two-out rule is divergence free. In this sense, the
coordinated O2− framework of water ice10. vectors define an ‘artificial’ magnetic field, b, on the lattice (the field can
When kBT << Jeff, the system fluctuates almost entirely within the be taken to have unit magnitude on each link, with the sign determined
two-in and two-out manifold of states. It turns out that the number of by the arrows). Because the spins are not ordered but fluctuating, the
such states is exponentially large, so a low-temperature entropy remains magnetic field also fluctuates. However, because the magnetic field lines
even within this limit. This entropy, first estimated by Pauling in 1935 do not start or end, these fluctuations include long loops of flux (Fig. 2b)
(ref. 11), has been measured in spin ice10. Although the spins remain that communicate spin correlations over long distances.
paramagnetic in this regime, the ‘ice rules’ imply strong correlations: The nature of the long-distance spin correlations was derived by Young-
for instance, if it is known that two spins on a tetrahedron are pointing blood and colleagues in a mathematically analogous model of a fluctuating
out, then the other two spins must point in. The correlated paramagnet ferroelectric, in which the electric polarization is similarly divergence-
is a simple example of a classical spin liquid. A key question is whether less12; this was subsequently rederived for spin ice13. The result is that the
the local constraints have long-range consequences: is the spin liquid artificial magnetic field, at long wavelengths, fluctuates in equilibrium just
qualitatively distinguishable from an ordinary paramagnet? Interest- as a real magnetic field would in a vacuum, albeit with an effective mag-
ingly, the answer for spin ice is ‘yes’. netic permeability. For the spins in spin ice, this implies power-law ‘dipo-
lar’ correlations that are anisotropic in spin space and decay as a power
Analogies to electromagnetism law (~1/r3, where r is the distance between the spins) in real space. It is
To understand how long-range effects arise from a local constraint, remarkable to have power-law correlations without any broken symmetry
it is helpful to use an analogy to electromagnetism. Each spin can be and away from a critical point. After Fourier transformation of these cor-
thought of as an arrow pointing between the centres of two tetrahedra relations on the lattice, a static spin structure factor with ‘pinch points’ at
(Fig. 2b). This defines a vector field of flux lines on the lattice, which reciprocal lattice vectors in momentum space is obtained12–14.
Such dipolar correlations have recently been observed in high
resolution neutron-scattering experiments by Fennell and colleagues15.
a At the pinch points, if the ice rules are obeyed perfectly, a sharp singular-
J
ity is expected, as well as a precise vanishing of the scattering intensity
J’ J’ along lines passing through the reciprocal lattice vectors. The rounding
of this singularity gives a measure of the ‘spin-ice correlation length’,
which is estimated to grow to 2–300 Å (a large number) at a temperature
of 1.3 K. In the future, it may be interesting to see how this structure
changes at even lower temperatures, at which spin ices are known to
freeze and fall out of equilibrium. Although the argument of Young-
blood and colleagues12 and the model outlined above rely on equilib-
rium, arguments by Henley suggest that the pinch points could persist
even in a randomly frozen glassy state14.
b
Magnetic monopoles
Interestingly, the magnetostatic analogy goes beyond the equilibrium
spin correlations. One of the most exciting recent developments in this
area has been the discovery of magnetic monopoles in spin ice16. These
J
arise for simple microscopic reasons. Even when kBT << Jeff, violations of
the two-in, two-out rule occur, although they are costly in energy and,
J’ J’
hence, rare. The simplest such defect consists of a single tetrahedron
with three spins pointing in and one pointing out, or vice versa (Fig. 2c).
This requires an energy of 2Jeff relative to the ground states. From a
magnetic viewpoint, the centre of this tetrahedron becomes a source or
sink for flux, that is, a magnetic monopole. A monopole is a somewhat
non-local object: to create a monopole, a semi-infinite ‘string’ of spins
c must be flipped, starting from the tetrahedron in question (Fig. 2c).
Nevertheless, when it has been created, the monopole can move by
single spin flips without energy cost, at least when only the dominant
nearest-neighbour exchange, Jeff, is considered.
Remarkably, the name monopole is physically apt: this defect carries
a real ‘magnetic charge’16. This is readily seen because the physical mag-
netic moment of the rare-earth atom is proportional to the pseudo-
magnetic field, M = gμBb, where g is the Landé g factor and μB is the Bohr
magneton. Thus, a monopole with the non-zero divergence =•b also
has a non-zero =•M. The actual magnetic charge (which measures the
strength of the Coulomb interaction between two monopoles) is, how-
ever, small: at the same distance, the magnetostatic force between two
monopoles is approximately 14,000 times weaker than the electrostatic
force between two electrons. Nevertheless, at low temperatures, this is
Figure 1 | Frustrated magnetism on 2D and 3D lattices. Two types of 2D still a measurable effect.
lattice are depicted: a triangular lattice (a) and a kagomé lattice (b). The 3D
A flood of recent papers have identified clear signatures of magnetic
lattice depicted is a pyrochlore lattice (c). In experimental materials, the
three-fold rotational symmetry of the triangular and kagomé lattices may monopoles in new experiments and in previously published data. Jaubert
not be perfect, allowing different exchange interactions, J and Jʹ, on the and Holdsworth showed that the energy of a monopole can be extracted
horizontal and diagonal bonds, as shown. Blue circles denote magnetic ions, from the Arrhenius behaviour of the magnetic relaxation rate17. They
arrows indicate the direction of spin and black lines indicate the shape of the found that in Dy2Ti2O7 the energy of a monopole is half that of a single
lattice. In b, ions and spins are depicted on only part of the illustrated lattice. spin flip, reflecting the fractional character of the magnetic monopoles.
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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

They also identified deviations from the Arrhenius form at lower tem- Box 1 | Elements of frustration
peratures as a result of Coulomb interactions between monopoles. In
an intriguing paper, Bramwell and colleagues applied an old theory put
forward by Onsager18 for the electric-field dependence of thermal charge
dissociation in electrolytes — the Wien effect — to the magnetic analogue
in spin ice, that is, to the disassociation of monopole–antimonopole pairs
with a magnetic field19. The theory allows an extraction of the absolute
value of the magnetic charge of a monopole from the dependence of the
magnetic relaxation rate on the magnetic field. The authors measured
muon spin relaxation in Dy2Ti2O7 to obtain the rate, and from this they
extracted a magnetic charge in near perfect agreement with theoretical
expectations. In addition to these quantitative measures of the energy
and charge of a monopole, two recent papers presented neutron-scat-
tering measurements in magnetic fields, interpreting them as evidence
of monopoles and the ‘strings’ emanating from monopoles20,21. Many
more experiments in which the monopoles in spin ice are studied and
manipulated are likely to emerge soon.

Quantum spin liquids

A triangle of antiferromagnetically interacting Ising spins, which must
In spin ice, as the temperature is lowered, the spins themselves fluctuate
point upward or downward, is the simplest example of frustration. All
ever more slowly, eventually falling out of equilibrium and freezing
three spins cannot be antiparallel. As a result, instead of the two ground
below about 0.5 K (from theory, it is predicted that, in equilibrium, states mandated by the Ising symmetry (up and down), there are six
the spins should order at T = 0.1−0.2 K (ref. 6)). This is a consequence ground states (see figure; blue circles denote magnetic ions, arrows
of the large energy barriers between different ice-rule configurations, indicate the direction of spin, and black and red lines indicate the shape
which require the flipping of at least six spins, and the weak quantum of the triangular lattice, with red lines denoting the axis on which the
amplitude for such large spins to cooperatively tunnel through these spins are parallel). On 2D and 3D lattices, such degeneracies can persist.
barriers. By contrast, for materials with spins of S = ½ and approximate When they do, fluctuations are enhanced and ordering is suppressed.
Heisenberg symmetry, quantum effects are strong, and there are no On the basis of this fact, Ramirez introduced a simple empirical measure
obvious energy barriers. I now turn to such materials in the search for of frustration that has become widely used50. At high temperatures, the
a QSL ground state, in which spins continue to fluctuate and evade spin (or magnetic) susceptibility of a local-moment magnet generally
order even at T = 0 K. Such a QSL is a strange beast: it has a non-mag- has a Curie–Weiss form, χ≈ C/(T − ΘCW), where T is temperature and
netic ground state that is built from well-formed local moments. The C is the Curie constant. This allows extraction of the Curie–Weiss
theoretical possibility of QSLs has been hotly debated since Anderson temperature, ΘCW, from a plot of 1/χ versus T. |ΘCW| provides a natural
proposed them in 1973 (ref. 22). estimate for the strength of magnetic interactions (ΘCW<0 for an
antiferromagnet) and sets the scale for magnetic ordering in an
Wavefunctions and exotic excitations unfrustrated material. By comparing the Curie–Weiss temperature with
A natural building block for non-magnetic states is the valence bond, the temperature at which order freezes, Tc, the frustration parameter, f, is
a pair of spins that, owing to an antiferromagnetic interaction, forms a obtained: f = |ΘCW|/Tc. Typically, f>5–10 indicates a strong suppression
spin-0 singlet state (Fig. 3a). A valence bond is a highly quantum object, of ordering, as a result of frustration. For such values of f, the temperature
range Tc<T<|ΘCW| defines the spin-liquid regime.
the two spins being maximally entangled and non-classical. If all of the
spins in a system are part of valence bonds, then the full ground state has
spin 0 and is non-magnetic. One way in which this can occur is by the
partitioning of all of the spins into specific valence bonds, which are static spins33. If a valence bond is formed from spins that are far apart, then
and localized. Mathematically, such a ground state is well approximated as those spins are only weakly bound into a singlet and the valence bond can
a product of the valence bonds, so that each spin is highly entangled with be ‘broken’ to form free spins with relatively little energy. So states that
only one other, its valence-bond partner. This is known as a valence-bond have a significant weight from long-range valence bonds have more low-
solid (VBS) state (Fig. 3a) and occurs in several materials23–25. VBS states energy spin excitations than states in which the valence bonds are mainly
are interesting because, for instance, they provide an experimental way short range (see, for example, ref. 34). There are also other excitations that
of studying Bose–Einstein condensation of magnons (which are triplet do not break the valence bonds but simply rearrange them. Such excita-
excitations of the singlet valence bonds) in the solid state26. tions can have low energy even in short-range RVB states.
A VBS state is not, however, a true QSL, because it typically breaks Given the possibility of different QSL states, it is interesting to attempt
lattice symmetries (because the arrangement of valence bonds is not to classify these states. This problem is only partly solved, but it is clear
unique) and lacks long-range entanglement. To build a QSL, the valence that the number of possible states is huge, if not infinite. For instance,
bonds must be allowed to undergo quantum mechanical fluctuations. Wen has classified hundreds of different ‘symmetrical spin liquids’
The ground state is then a superposition of different partitionings of (QSLs with the full space-group symmetry) for S = ½ antiferromagnets
spins into valence bonds (Fig. 3b, c). If the distribution of these partition- on the square lattice35. Finding the correct QSL ground state among all
ings is broad, then there is no preference for any specific valence bond of the many possible RVB phases, many of which have similar energies,
and the state can be regarded as a valence-bond ‘liquid’ rather than a is a challenge to theory, reminiscent of the landscape problem in string
solid. This type of wavefunction is generally called a resonating valence- theory, in high-energy physics.
bond (RVB) state22. RVB states became subjects of intense theoretical Even with this diversity of possible states, one feature that theorists
interest when, in 1987, Anderson proposed that they might underlie the expect QSLs to have in common is that they support exotic excitations.
physics of high-temperature superconductivity4. Only relatively recently, What is meant by exotic? In most phases of matter, all of the excitations
however, have RVB wavefunctions been shown to be ground states of can be constructed from elementary excitations that are either electron-
many specific model Hamiltonians27–32. like (spin S = ½ and charge ±e) or magnon-like (spin S = 1 and charge
It is now understood that not all QSLs are alike. Generally, different neutral). Only in rare examples, such as the fractional quantum Hall
states have different weights of each valence-bond partition in their states, do the elementary excitations differ from these, in this case by
wavefunctions. The valence bonds need not be formed only from nearby carrying fractional quantum numbers. The magnetic monopoles in spin
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

Box 2 | Theoretical frameworks is in equilibrium, corrections to this form would be expected to lead to
Theoretical models of magnets with localized electrons typically start monopole confinement at low temperatures.
with the Heisenberg Hamiltonian, for example: In a true 2D or 3D QSL, the string associated with a spinon remains
robustly tensionless even at T = 0 K, owing to strong quantum fluctua-
tions (Fig. 4c). This can be understood from the quantum superposition
1
H=− —
2
∑J S •S
ij
ij i j (1) principle: rearranging the spins along the string simply reshuffles the
various spin or valence-bond configurations that are already super-
posed in the ground state. Detailed studies of QSL states have shown
where Jij are exchange constants, and Si and Sj are quantum spin-S that higher-dimensional spinons can have varied character. They may
operators. They obey Si2 = S(S + 1) and canonical commutation relations obey Fermi–Dirac36, Bose–Einstein37 or even anyonic statistics (see page
[Siμ, Sjν] = iєμνλSiλδij, where єμνλ is the Levi-Civita symbol. In spin ice, owing 187). They may be gapped (that is, require a non-zero energy to excite)
to the large S (S=15/2 for Dy and 8 for Ho), the spins can be regarded or gapless, or they may even be so strongly interacting that there are no
simply as classical vectors (conventionally they are taken to be of unit sharp excitations of any kind38.
length, with factors of S absorbed into the exchange constants). For
spin ice, equation (1) above mimics the short-range effect of dipolar Experimental search for QSLs
exchange, by taking ferromagnetic Jij = Jeff > 0 on nearest neighbours, In contrast to the apparent ubiquity and variety of QSLs in theory, the
as is discussed in the main text. In addition, in spin ice, strong Ising
experimental search for QSLs has proved challenging. Most Mott insu-
anisotropy must be included by way of the crystal field term
lators — materials with localized spins — are ordered magnetically or
freeze into spin-glass states. A much smaller set of Mott insulators form
Hcf = −D ∑ (S • n̂ )
i
i i
2
VBS states23–25. A clear identification of RVB-like QSL states has proved
more elusive.
where n̂i is a unit vector along the Ising 〈111〉 axis of the ith spin and D is How is a QSL identified in experiments? One good indication is a
the Ising anisotropy. The positive D >> J requires the spins to point along large frustration parameter, f > 100–1,000 (f may be limited by material
this axis. In low-spin systems of transition metal spins, the exchange is complications, such as defects or weak symmetry-breaking interac-
antiferromagnetic, Jij < 0, crystal field effects are much less significant tions, or the ability to cool). A more stringent test is the absence of static
and S is much smaller. For the QSL candidates discussed in the main moments, even disordered ones, at low temperatures, a feature that can
text, Stakes its minimum value,½. Thus, when the assumption of be probed by nuclear magnetic resonance (NMR) and muon spin reso-
localized electrons is valid, equation (1) itself is a good starting point. If nance experiments. Specific-heat measurements give information on the
this assumption is not valid, a better description, which includes charge low-energy density of states of a putative QSL, which can be compared
fluctuations, is the Hubbard model: with those of theoretical models. Thermal transport can determine
whether these excitations are localized or itinerant. Elastic and inelastic
HHubbard = − ∑t c c
ij,α
†
ij iα jα ∑
+ U ni(ni − 1)
i
neutron-scattering measurements, especially on single crystals, provide
crucial information on the nature of correlations and excitations, and
these could perhaps uncover spinons. All told, this is a powerful arsenal
where c†iα and ciα are creation and annihilation operators, respectively, of experimental tools, but the task is extremely challenging. At the heart
for electrons with spin α = ±½ on site i, and ni = ∑α c†iαciα is the number of the problem is that there is no single experimental feature that identi-
of electrons on the site. The electron operators obey canonical anti-
fies a spin-liquid state. As long as a spin liquid is characterized by what it
commutation relations {c†iα, cjβ} = δαβδij. The coefficient tij gives the
is not — a symmetry-broken state with conventional order — it will be
quantum amplitude for an electron to hop from site j to site i, and U is
the Coulomb energy cost for two electrons to occupy the same site.
much more difficult to identify conclusively in experiments.
When U>> t, and there is on average one electron per site, charge Nevertheless, in recent years, increased activity in the field of highly
fluctuations become negligible. Then the Hubbard model reduces to the frustrated magnetism has led to a marked increase in the number of
Heisenberg one, with Jij≈ −4tij2/U. However, when U/t is not very large, candidate QSL materials, giving reason for optimism39,40. A partial list
the two models are inequivalent. For a small enough U/t, the Hubbard of the materials that have been studied is presented in Table 1.
model usually describes a metal, in which electrons are completely
delocalized. As U/t is increased from zero, there is a quantum phase QSL materials
transition from a metal to an insulator; this is known as the Mott The last two entries in Table 1 (Cs2CuCl4 and FeSc2S4) have already
transition. For U/t larger than this critical value, the system is known as been ruled out as true QSLs, and I return to these later (in the section
a Mott insulator. ‘Weak’ Mott insulators are materials with U/t close to ‘Unexpected findings’). All of the remaining materials are promising
the Mott transition. candidates, with S = ½ quantum spins on frustrated lattices. They show
persistent spin dynamics down to the lowest measured temperatures,
with every indication that the dynamics persist down to T = 0 K. With
ice are examples of this: the elementary magnetic dipole ‘splits’ into a the exception of Cu3V2O7(OH)2•2H2O, no signs of phase transitions
monopole–antimonopole pair. However, these are classical excitations are seen on cooling from the high-temperature paramagnet to a low
and not true coherent quasiparticles. In QSLs, the most prominent exotic temperature. The characteristics of these materials are now beginning
excitations are in the form of spinons, which are neutral and carry spin to be understood.
S = ½ (Fig. 4). Spinons are well established in one-dimensional (1D) sys- Surveying them, it is clear that there is a variety of structures and
tems, in which they occur as domain walls (Fig. 4a). A spinon can thus properties. There are organic compounds — κ-(BEDT-TTF)2Cu2(CN)3
be created similarly to a monopole in spin ice, by flipping a semi-infinite and EtMe3Sb[Pd(dmit)2]2 — and a variety of inorganic compounds. The
string of spins. A key difference, however, is that in one dimension the spins form several different structures: 2D triangular lattices; 2D kagomé
only boundary of such a string is its end point, so the string is guar- lattices; and a hyperkagomé lattice, which is 3D. In some cases, these
anteed to cost only a finite energy from this boundary. By contrast, in are ideal, isotropic forms. In other cases, there are asymmetries and,
two or three dimensions, the boundary of a string extends along its full consequently, spatial anisotropy. Existing samples are compromised
length. A string would naturally be expected to have a tension (that by varying degrees of disorder, from as much as 5–10% free defect
is, there is an energy cost proportional to its length). String tension spins and a similar concentration of spin vacancies in ZnCu3(OH)6Cl2
represents confinement of the exotic particle, as occurs for quarks in (ref. 41) to as little as 0.07% impurity in recently improved samples of
quantum chromodynamics. This is avoided in spin ice by the special Cu3V2O7(OH)2•2H2O (ref. 42). In several materials, the nature and
form of the nearest-neighbour Hamiltonian. However, when spin ice degree of disorder have not been well characterized.
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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

A key physical distinction among the candidates is their degree of a

charge localization — this is important for understanding the mech-
anism of the spin-liquid behaviour. The kagomé materials can be
regarded as strong Mott insulators, in which the electrons constituting
the spins are approximately localized to a single atomic orbital. This can
be seen from the small exchange couplings (proportional to the Curie–
Weiss temperature, ΘCW) relative to the Mott charge gap, which is of the
order of electronvolts (eV) in such copper oxides. For these materials, a
microscopic description purely in terms of Heisenberg spins is justified.
By contrast, for the organic compounds and Na4Ir3O8, the exchange
energies are larger and the charge gaps are smaller. These materials are
therefore weak Mott insulators, in which the electronic spins are much b
less well localized, so charge fluctuations may have an important role.
In the organic compounds, the Hubbard repulsion, U (Box 2), which
sets the Mott gap, is much smaller than in inorganic compounds, owing
to the extended nature of the molecular orbital constituting each spin.
In Na4Ir3O8, U is likewise reduced because 5d orbitals are significantly
larger than 3d ones. The fact that metal–insulator transitions can be
induced by physical pressure or chemical pressure in all three cases
(for the two organic compounds and Na4Ir3O8)43,44, and the fact that
in κ-(BEDT-TTF)2Cu2(CN)3 no clear Mott gap is seen spectroscopi-
cally45, is direct experimental evidence that these materials are weak
Mott insulators. The applicability of a Heisenberg spin description to
these materials is thus open to question.
Thermodynamic studies of these materials are also important because
c
they reveal the spectrum of low-energy states, an abundance of which
is expected in frustrated systems. At very low temperatures, the func-
tional form of the specific heat and sometimes the spin (or magnetic)
susceptibility can distinguish between QSLs. The general thermody-
namic features of the materials are remarkably similar. In all cases in
which it has been measured, the magnetic specific heat shows a peak
well below the Curie–Weiss temperature46–49, indicating that significant
spin entropy is maintained above this peak temperature. This specific
heat then gradually decreases on further lowering of the temperature, in
a manner that can roughly be fitted to a quadratic behaviour, Cv ≈ AT2,
where Cv is molar specific heat at constant volume and A is a constant.
This non-exponential form clearly indicates the absence of an energy
gap. However, some caution is in order before interpreting this as char-
acteristic of QSLs: such approximately quadratic behaviour of Cv is com-
mon in frustrated magnets, even ones that are classical and for which
Figure 2 | Spins, artificial magnetic fields and monopoles in spin
QSL physics is clearly irrelevant50. At very low temperatures, the can- ice. a, A ground-state configuration of spins is shown in a pyrochlore
didate QSL materials generally show a linear behaviour, Cv ≈ γT, where lattice. The spins obey the constraint of the ice rules that mandates two
γ is the Sommerfeld coefficient (refs 46–49, 51), which varies widely inward-pointing spins and two outward-pointing ones on each tetrahedron.
(between 1 and 250 mJ K−2 mol−1). This is evidence of a large density of b, For the same lattice type, some of the loops of ‘magnetic flux’ are shown
low-energy states. (red lines), as defined by the mapping of spins to an artificial magnetic field.
Ideally, the spin susceptibility, χ, is a useful measure of the available c, For the same lattice type, a monopole (green) and antimonopole (red)
low-energy spin excitations. All of the candidate QSL materials also show are shown. These are created by flipping the ‘string’ of spins connected by
a large Pauli-like paramagnetic susceptibility, χ0, in the zero-temperature the yellow line (compare with the spins in b). For simplicity, the particles
limit. This susceptibility is smallest for the organic compounds: for these, themselves are not depicted. Note that the spins on the tetrahedron
containing a monopole (antimonopole) orient three-in, one-out (three-out,
χ0 ≈ 3 × 10−4 e.m.u. mol−1 (refs 52, 53), whereas it is 3–10 times larger
one-in), violating the ice rules.
for the inorganic compounds41,42,46,47,49. Before these values of χ0 can be
interpreted as additional evidence for gapless excitations in QSLs, it is
important to consider the influence of spin–orbit coupling. Spin–orbit larger than 1, with the exception of ZnCu3(OH)6Cl2 (for which an
coupling generally leads to a non-zero χ0 value irrespective of the pres- estimate of the intrinsic γ value, uncontaminated by impurities, is
ence or absence of an energy gap. An empirical indicator of the impor- not available) and κ-(BEDT-TTF)2Cu2(CN)3. The value of the Wilson
tance of spin–orbit coupling is the Wilson ratio, which is defined as ratio for ZnCu3(OH)6Cl2 is probably suppressed by the high degree
of disorder. The extremely large value for Na4Ir3O8 (R = 70) indicates
4π2kB2χ0 very strong spin–orbit effects, which are not surprising given the large
R = _________ (1) atomic number of iridium.
3(gμB)2γ The most direct evidence for a lack of magnetic ordering comes from
local probe measurements, by using techniques such as NMR and muon
and should be, at most, of order 1 in the absence of strong ferromagnetic spin resonance, in which local fields resulting from static moments affect
tendencies or spin–orbit coupling. Because all of the candidate QSL the nuclear or muon spins and can be readily detected by their influence
materials are strongly antiferromagnetic, the ferromagnetic interpreta- on the muon spins. Such measurements have confirmed the absence of
tion is untenable. In fact, most (but not all) QSL theories, in the absence local static moments down to T = 32 mK in κ-(BEDT-TTF)2Cu2(CN)3
of spin–orbit coupling, predict that R << 1. By contrast, the measured (ref. 52), T = 1.37 K in EtMe3Sb[Pd(dmit)2]2 (ref. 54) and T = 50 mK in
Wilson ratios for all of the QSL candidates in Table 1 are significantly ZnCu3(OH)6Cl2 (ref. 55). However, in Cu3V2O7(OH)2•2H2O, magnetic
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

order and/or freezing is observed, by using NMR spectroscopy, at T < 1 K for these materials are much more limited. In all cases, the comparison
(ref. 56). Moreover, recent experiments show that this compound has a of theory with experiment has, so far, been indirect. I return to this
complex series of low-temperature phases in an applied magnetic field56. problem in the subsection ‘The smoking gun for QSLs’.
Given the exceptionally high purity of Cu3V2O7(OH)2•2H2O, an expla-
nation of its phase diagram should be a clear theoretical goal. Unexpected findings
In the course of a search as difficult as the one for QSLs, it is natural for
Theoretical interpretations there to be false starts. In several cases, researchers uncovered other
I now turn to the theoretical evidence for QSLs in these systems and interesting physical phenomena in quantum magnetism.
how the experiments can be reconciled with theory. Theorists have
attempted to construct microscopic models for these materials (Box 2) Dimensional reduction in Cs2CuCl4
and to determine whether they support QSL ground states. In the case Cs2CuCl4 is a spin-½ antiferromagnet on a moderately anisotropic
of the organic compounds, these are Hubbard models, which account triangular lattice69,70. It shows only intermediate frustration, with f ≈ 8,
for significant charge fluctuations. For the kagomé materials, a Heisen- ordering into a spiral Néel state at TN = 0.6 K. However, neutron-scat-
berg model description is probably appropriate. There is general theo- tering results for this compound reported by Coldea and colleagues
retical agreement that the Hubbard model for a triangular lattice has suggested that exotic physical phenomena were occurring69,70. These
a QSL ground state for intermediate-strength Hubbard repulsion near experiments measure the type of excitation that is created when a neu-
the Mott transition57–59. On the kagomé lattice, the Heisenberg model tron interacts with a solid and flips an electron spin. In normal mag-
is expected to have a non-magnetic ground state as a result of frus- nets, this creates a magnon and, correspondingly, a sharp resonance is
tration60. Recently, there has been growing theoretical support for the observed when the energy and momentum transfer of the neutron equal
conjecture that the ground state is, however, not a QSL but a VBS with that of the magnon. In Cs2CuCl4, this resonance is extremely small.
a large, 36-site, unit cell61,62. However, all approaches indicate that many Instead, a broad scattering feature is mostly observed. The interpreta-
competing states exist, and these states have extremely small energy dif- tion of this result is that the neutron’s spin flip creates a pair of spinons,
ferences from this VBS state. Thus, the ‘real’ ground state in the kagomé which divide the neutron’s energy and momentum between them. The
materials is probably strongly perturbed by spin–orbit coupling, dis- spinons were suggested to arise from an underlying 2D QSL state.
order, further-neighbour interactions and so on63. A similar situation A nagging doubt with respect to this picture was the striking similar-
applies to the hyperkagomé lattice of Na4Ir3O8 (ref. 64). ity between some of the spectra in the experiment and those of a 1D
These models are difficult to connect directly, and in detail, to spin chain, in which 1D spinons indeed exist71. In fact, in Cs2CuCl4 the
experiments, which mainly measure low-energy properties at low tem- exchange energy along one ‘chain’ direction is three times greater than
peratures. Instead, attempts to reconcile theory and experiment in detail along the diagonal bonds between chains (that is, Jʹ ≈ J/3 in Fig. 1a).
have relied on more phenomenological low-energy effective theories Experimentally, however, the presence of substantial transverse disper-
of QSLs. Such effective theories are similar in spirit to the Fermi liquid sion (that is, dependence of the neutron peak on momentum perpendic-
theory of interacting metals: they propose that the ground state has a ular to the chain axis in Cs2CuCl4), and the strong influence of interchain
certain structure and a set of elementary excitations that are consistent coupling on the magnetization curve, M(H), seemed to rule out a 1D
with this structure. In contrast to the Fermi liquid case, however, the origin, despite an early theoretical suggestion72.
elementary excitations consist of spinons and other exotic particles, In the past few years, it has become clear that discarding the idea of
which are coupled by gauge fields. A theory of this type — that is, pro- 1D physics was premature73,74. It turns out that although the interchain
posing a ‘spinon Fermi surface’ coupled to a U(1) gauge field — has coupling is substantial, and thus affects the M(H) curve significantly,
had some success in explaining data from experiments on κ-(BEDT- the frustration markedly reduces interchain correlations in the ground
TTF)2Cu2(CN)3 (refs 65, 66). Related theories have been proposed for state. As a result, the elementary excitations of the system are simi-
ZnCu3(OH)6Cl2 (ref. 67) and Na4Ir3O8 (ref. 68). However, comparisons lar to those of 1D chains, with one important exception. Because the

a
1 ( + )
–2

+ +…

+…

Figure 3 | Valence-bond states of frustrated antiferromagnets. In a VBS superposition of many different pairings of spins. The valence bonds may
state (a), a specific pattern of entangled pairs of spins — the valence bonds be short range (b) or long range (c). Spins in longer-range valence bonds
— is formed. Entangled pairs are indicated by ovals that cover two points (the longer, the lighter the colour) are less tightly bound and are therefore
on the triangular lattice. By contrast, in a RVB state, the wavefunction is a more easily excited into a state with non-zero spin.

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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

Figure 4 | Excitations of quantum antiferromagnets. a, In a quasi-1D number of spins, in this case all of the red spins and their counterparts on
system (such as the triangular lattice depicted), 1D spinons are formed as the next chain. b, A bound pair of 1D spinons forms a triplon. Because a
a domain wall between the two antiferromagnetic ground states. Creating finite number of spins are flipped between the two domain walls (shown
a spinon (yellow arrow) thus requires the flipping of a semi-infinite line in red), the triplon can coherently move between chains, by the flipping of
of spins along a chain, shown in red. The spinon cannot hop between spins along the green bonds. c, In a 2D QSL, a spinon is created simply as an
chains, because to do so would require the coherent flipping of an infinite unpaired spin, which can then move by locally adjusting the valence bonds.

spinons are inherently 1D, they are confined to the chains, and to take tration but to a competition between spin–orbit coupling and magnetic
advantage of the transverse exchange, they must be bound into S = 1 exchange84. Microscopic estimates of the spin–orbit interaction, λ,
‘triplon’ bound states (Fig. 3b). These triplons move readily between indeed show that its strength, λ/kB = 25–50 K, is comparable to the Curie–
chains and are responsible for the transverse dispersion observed in Weiss temperature, 45 K. As a result, the material is serendipitously close
the experiment. Thus, the observation of triplons provides a means to to a quantum critical point between a magnetically ordered state and
distinguish 1D spinons from their higher-dimensional counterparts. a ‘spin–orbital singlet’, induced by spin–orbit coupling84 (Fig. 5). This
A quantitative theory of this physics agrees well with the data, with no picture seems to explain data from a variety of experiments, including
adjustable parameters. It is therefore understood that Cs2CuCl4 is an NMR81, neutron-scattering82, spin susceptibility83 and specific-heat83
example of ‘dimensional reduction’ induced by frustration and quan- measurements. Most notably, the anomalously small excitation gap of
tum fluctuations. This phenomenon was unexpected and might have a 2 K that was measured in neutron-scattering82 and NMR81 experiments
role in other correlated materials. Perhaps it is related to the cascade of is understandable — this gap vanishes on approaching the quantum
phases that is observed in the isostructural material Cs2CuBr4 in applied critical point. If the theory is correct, FeSc2S4 can be viewed as a kind of
magnetic fields75. spin–orbital liquid with significant long-distance entanglement between
spins and orbitals. Because the material is not precisely at the quantum
Spin–orbital quantum criticality in FeSc2S4 critical point, however, there is a finite correlation length; therefore, this
Among the entries in Table 1, FeSc2S4 stands out as a material that has entanglement does not persist to arbitrarily long distances, as would be
not only spin degeneracy but also orbital degeneracy. This is common expected in a true RVB state.
in transition-metal-containing compounds76,77. It is possible to imagine
a quantum orbital liquid78–80, analogous to a QSL. Like the more familiar Future directions
(theoretically) QSL, the quantum orbital liquid is experimentally elu- I have only touched the surface of the deep well of phenomena to
sive. Nevertheless, experimentalists have observed that FeSc2S4, which be explored, experimentally and theoretically, in frustrated magnets
has a twofold orbital degeneracy, evades order down to T = 50 mK, and and spin liquids. In spin ice, there are subtle correlations, collective
on this basis it was characterized as a spin–orbital liquid81–83. excitations and emergent magnetic monopoles, all of which are highly
Recently, it was suggested that this liquid behaviour is due not to frus- amenable to laboratory studies. In several frustrated magnets with spin

Table 1 | Some experimental materials studied in the search for QSLs

Material Lattice S ΘCW (K) R* Status or explanation
κ-(BEDT-TTF)2Cu2(CN)3 Triangular† ½ −375‡ 1.8 Possible QSL
EtMe3Sb[Pd(dmit)2]2 Triangular† ½ −(375–325)‡ ? Possible QSL
Cu3V2O7(OH)2•2H2O (volborthite) Kagomé† ½ −115 6 Magnetic
ZnCu3(OH)6Cl2 (herbertsmithite) Kagomé ½ −241 ? Possible QSL
BaCu3V2O8(OH)2 (vesignieite) Kagomé† ½ −77 4 Possible QSL
Na4Ir3O8 Hyperkagomé ½ −650 70 Possible QSL
Cs2CuCl4 Triangular† ½ −4 0 Dimensional reduction
FeSc2S4 Diamond 2 −45 230 Quantum criticality
BEDT-TTF, bis(ethylenedithio)-tetrathiafulvalene; dmit, 1,3-dithiole-2-thione-4,5-ditholate; Et, ethyl; Me, methyl. *R is the Wilson ratio, which is defined in equation (1) in the main text. For EtMe3Sb[Pd(dmit)2]2
and ZnCu3(OH)6Cl2, experimental data for the intrinsic low-temperature specific heat are not available, hence R is not determined. †Some degree of spatial anisotropy is present, implying that Jʹ≠J in Fig. 1a. ‡A
theoretical Curie–Weiss temperature (ΘCW) calculated from the high-temperature expansion for an S=½ triangular lattice; ΘCW=3J/2kB, using the J fitted to experiment.

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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

FeSc2S4 The smoking gun for QSLs

The diversity of QSL candidates and the progress being made towards
their theoretical description are encouraging, but the research com-
munity is still in the unsatisfactory situation in which the evidence for
QSL states is circumstantial and heavily depends on theoretical inter-
Temperature

Quantum pretation. For spin ice, by contrast, there is increasingly direct experi-
critical mental evidence that magnetic monopoles exist. For QSLs, so far, such
region a ‘smoking gun’ is lacking. A conventional experimental technique that
could be a candidate for identifying QSLs is inelastic neutron scattering,
Spin–orbital
Antiferromagnet which the experiments on Cs2CuCl4 clearly show can reveal spinons
singlet
under appropriate conditions69. This is an important avenue for future
experiments, but it would be advantageous to design new experiments
Quantum critical point specifically to identify QSLs. Some proposals have been made. It has
Competition between magnetic exchange and spin–orbit interaction, J/l
been suggested that a particular class of QSL could display a vortex
memory effect, which would probe the existence of the ‘vison’ excitation
of such a spin liquid93, but it is extremely difficult to measure this effect
Figure 5 | Spin–orbital quantum criticality. Schematic phase diagram of
FeSc2S4, representing the competition between spin–orbit interaction, in most materials94. For QSLs with a spinon Fermi surface or Fermi
λ, and magnetic exchange energy, J. For large values of J/λ, the spins points, it might be possible to measure ‘2kF’ oscillations, which would
magnetically order into an antiferromagnetic state. For small J/λ, the be considerable if observed in an insulator. Such observations could be
spins and orbitals combine into a non-magnetic spin–orbital singlet made in neutron-scattering measurements, or they could be made in
state. The two ground states are separated by a quantum critical point, real space by way of analogues of Friedel oscillation measurements, by
which governs the quantum critical region (in the ‘fan’ above it) at T > 0. using local probes or composite structures95.
The dashed line indicates the value of J/λ for FeSc2S4, which has a large
correlation length and small gap as a consequence of its proximity to the Connections
quantum critical point.
The physics of frustrated magnetism and spin liquids has intriguing
connections to diverse exotic phenomena, many of which are discussed
S = ½, researchers seem finally to have uncovered QSL ground states, in other articles in this Insight. For example, the modern interest in spin
which have long eluded them, but the detailed structures of these liquids grew largely from Anderson’s proposed connection between
have not yet been elucidated. Explaining them will be a theoretical RVB states and high-temperature copper-oxide-based superconduc-
and experimental challenge. In addition, in the process of searching tivity4. The idea is that, in an RVB state, electrons bound into valence
for QSLs, examples of dimensional reduction and quantum criticality bonds are paired even though the state is non-superconducting. Thus,
were unexpectedly found. part of the physics of superconductivity has already been achieved in
this state, and if the material can be made conducting and phase coher-
Beyond spin ice ent, for example by doping with mobile charges, it will naturally become
The recent observation of magnetic monopoles, in agreement with superconducting. Despite the elegance of this suggestion and decades of
theory, suggests that there is a good understanding of the interactions theoretical work, RVB superconductivity remains unproven in experi-
and dynamics of the canonical spin-ice materials. The challenge is now ments, although the idea is still actively discussed96.
to improve this understanding by making a more detailed and accurate More generally, the problem of frustration and spin-liquid physics in
comparison of theory and experiment and perhaps by ‘engineering’ new systems with mobile charges is relatively unstudied. There are several
phenomena through the control of magnetic monopoles. For instance, it interesting materials in which this type of physics can be explored exper-
might be possible to make spectral measurements of magnetic monopo- imentally. I have discussed materials in which such physics might be
les by exciting them with neutrons. Some theoreticians have suggested involved, including the triangular-lattice organic compounds, Na4Ir3O8,
another consequence of emergent electromagnetism in spin ice. If the and Pr2Ir2O7 (a type of metallic spin ice). Another interesting material in
material can be driven through certain types of phase transition, then this context is 2H-AgNiO2, a semi-metallic material that shows charge
exotic critical behaviour in violation of the standard Landau–Ginzburg– ordering on a triangular lattice97. It has been suggested that a novel,
Wilson paradigm might be observed85–87. continuous Mott transition — rather than the usual discontinuous one
Spin ice certainly proves that there is more to be learned about clas- — might be observed in such a situation98,99.
sical spin liquids, and that these materials are much simpler to under- From the above discussion, it is clear that spin–orbit coupling seems
stand than their quantum counterparts. In fact, many other materials to have an important role in frustrated systems and QSLs. Even in the
have classical spin-liquid regimes, and perhaps the ideas that have organic compounds, in which spin–orbit effects are expected to be mini-
arisen from studies of spin ice will inspire an understanding of their mized by the extended molecular orbital states, spin–orbit effects are not
dynamics. Well-known classical spin liquids include gadolinium gal- obviously negligible. Experiments on κ-(BEDT-TTF)2Cu[N(CN)2]Cl,
lium garnet and SrCr9–xGa3+xO19, both of which have large, predomi- which is a magnetically ordered organic compound, demonstrated a
nantly classical spins with very large frustration parameters50. Another Dzyaloshinskii–Moriya (DM) coupling, D, of order D/kB ≈ 5 K (ref. 100).
set of examples is the spinel chromites, ACr2O4 (where A = Zn, Cd or If κ-(BEDT-TTF)2Cu2(CN)3 had a similar magnitude of DM coupling
Hg), which form S = 3⁄2 Heisenberg pyrochlore antiferromagnets and it would probably affect many of the low-temperature measurements,
show a spin-liquid regime with unusual ‘loop’ structures seen in dif- such as specific heat and thermal conductivity. In the inorganic materi-
fuse neutron scattering88. More recently, the A-site spinels MnSc2S4 als, DM interactions and other spin–orbit effects are clearly significant
(ref. 83) and CoAl2O4 (ref. 89) have been interpreted in terms of a and are even dominant in the case of Na4Ir3O8.
‘spiral spin liquid’90. For such materials with strong spin–orbit coupling, it will be interest-
Another way to move ‘beyond’ spin ice is to introduce quantum ing to explore the connection to topological insulators (see page 194),
mechanics. Quantum effects are seen in Tb2Ti2O7, which in a magnetic which are known to occur in situations with strong spin–orbit coupling
field shows many of the anisotropies that are characteristic of spin ice. It and weak correlation. How does the topological insulator evolve with
avoids freezing or ordering down to T = 50 mK and remains an intrigu- increasing electron localization by Coulomb interactions? Ultimately,
ing theoretical puzzle6,91. The material Pr2Ir2O7 combines features of spin with very large values of U, the strong Mott insulating limit is reached
ice with metallic conduction92 and has a zero-field anomalous Hall effect and the system is described by a Heisenberg model, as I have discussed
that is indicative of a ‘chiral spin liquid’ phase. here. It is also interesting to ask how far the physics of topological insula-
206
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NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

tors survives in this direction and whether anything qualitatively new 32. Levin, M. A. & Wen, X. G. String-net condensation: a physical mechanism for topological
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71. Lake, B., Tennant, D. A., Frost, C. D. & Nagler, S. E. Quantum criticality and universal scaling 89. Tristan, N. et al. Geometric frustration in the cubic spinels MAl2O4 (M=Co, Fe, and Mn).
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84. Chen, G., Balents, L. & Schnyder, A. P. A. Spin–orbital singlet and quantum critical point on Acknowledgements I am grateful to countless friends and collaborators, especially
the diamond lattice: FeSc2S4. Phys. Rev. Lett. 102, 096406 (2009). O. Starykh and M. Fisher for many stimulating interactions. I would also like to
85. Powell, S. & Chalker, J. T. Classical to quantum mappings for geometrically frustrated thank R. Coldea, H. Takagi, A. Loidl and P. Mendels for sharing their insights into
systems: spin-ice in a [100] field. Phys. Rev. B 78, 024422 (2008). experiments on frustrated magnets. My research is supported by the US National
86. Saunders, T. E. & Chalker, J. T. Structural phase transitions in geometrically frustrated Science Foundation, the US Department of Energy, the David and Lucile Packard
antiferromagnets. Phys. Rev. B 77, 214438 (2008). Foundation and the US Army Research Office.
87. Bergman, D. L., Fiete, G. A. & Balents, L. Ordering in a frustrated pyrochlore
antiferromagnet proximate to a spin liquid. Phys. Rev. B 73, 134402 (2006). Author Information Reprints and permissions information is available at www.
88. Lee, S. H. et al. Emergent excitations in a geometrically frustrated magnet. Nature 418, nature.com/reprints. The author declares no competing financial interests.
856–858 (2002). Correspondence should be addressed to the author (balents@kitp.ucsb.edu).

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NATURE|Vol 464|11 March 2010|doi:10.1038/nature08918 REVIEW INSIGHT

Electron liquids and solids in one dimension

Vikram V. Deshpande1, Marc Bockrath2, Leonid I. Glazman3 & Amir Yacoby4

Even though bulk metallic systems contain a very large number of strongly interacting electrons, their properties
are well described within Landau’s Fermi liquid theory of non-interacting quasiparticles. Although many higher-
dimensional systems can be successfully understood on the basis of such non-interacting theories, this is not
possible for one-dimensional systems. When confined to narrow channels, electron interaction gives rise to such
exotic phenomena as spin–charge separation and the emergence of correlated-electron insulators. Such strongly
correlated electronic behaviour has recently been seen in experiments on one-dimensional carbon nanotubes
and nanowires, and this behaviour challenges the theoretical description of such systems.

According to Landau’s Fermi liquid theory1, a bulk metallic system of phonons. Despite the strength of the Coulomb forces (the corresponding
interacting electrons has low-energy excitations called quasiparticles energy is in the range of 10 eV), the classification of the low-energy
that behave much like weakly interacting fermions. Therefore, many excitations, suggested by Landau’s Fermi liquid theory1, is remarkably
experiments on such systems can be understood on the basis of theories universal. In the language of this theory, the electric charge carriers in
of non-interacting quasiparticles. The theoretical picture is qualitatively a conventional metal can be viewed as almost-free quasiparticles with
different in materials in which the electrons are confined to one-dimen- Fermi statistics, the Coulomb interaction ultimately determining the
sional (1D) channels, or wires. In such wires, interactions cause electrons crystalline structure of a material and the specific form of the quasi-
to behave in a highly cooperative way, and the Fermi liquid theory no particles’ dispersion relationship (their energy–momentum relation-
longer applies. Progress in obtaining 1D materials realized as individual ship). The residual interaction between quasiparticles, stemming from
nanostructures has provided highly tunable systems that allow the study the Coulomb repulsion, leads to the quasiparticle states having a finite
of the effects of interactions in many-electron systems. Other 1D tunable energy uncertainty; the uncertainty for a given quasiparticle, however,
systems, such as those based on cold atoms2,3, are also being developed is small in comparison with its energy.
and keep producing interesting results (see, for example, refs 4 and 5). Box 1 | Luttinger liquid and bosonization in one dimension
Here, we discuss the many ways in which interacting electrons in one
dimension often produce surprising results that illuminate the role of the In our discussion about the inapplicability of the Fermi liquid theory to
describe 1D interacting electron systems, shifts of the electron fluid
dimension of an interacting many-body system. After discussing the basic
have a crucial role. The creation of a particle at some point, x, along the
manifestations of 1D cooperative electron behaviour in simple tunnelling
wire may be viewed as a result of a shift of all the fluid to the left, say,
experiments, we focus on a number of new developments in 1D physics of x by the interparticle distance, while the fluid to the right of x stays
both in theory and in experiments on nanowires and carbon nanotubes. put; the ‘bump’ of density created at point x in this way will contain just
We first discuss the 1D phenomenon of spin–charge separation and exper- one extra particle. The displacements of the fluid may be viewed as a
iments to observe it using the technique of momentum-conserved tun- superposition of harmonic waves. The quantized displacement waves
nelling between parallel nanowires6,7. The same kind of experiment raises are bosons that can be thought of as quanta of sound waves. At low
the question of how a charge with a definite momentum is accommodated energies, the interaction between bosons can be disregarded, which
in a correlated 1D system8; this question is discussed in a section devoted allows a low-energy excitation of a 1D fluid to be considered a collection
to the apparent charge ‘break-up’. The above physics can be understood of free bosons.
using a theory valid at sufficiently low energies; however, the question of In one dimension, it is the above-mentioned bosons, rather than the
how 1D systems behave at higher energies is interesting as well. We discuss Fermi quasiparticles of the Fermi liquid theory, that are the well-defined
the recently developed ‘nonlinear Luttinger liquid’ theory addressing this elementary excitations. This simple representation of the low-energy
question9–13 and possible experiments aimed at its verification. excitations of a 1D many-body quantum system is known as a Luttinger
The availability of clean, freely suspended carbon nanotubes has ena- liquid. The lower the energy scale, the better this representation is
bled experiments to be performed in the low-electron-density regime. supposed to work. It becomes exact for the so-called Tomonaga–
We discuss this limit, in which electrons organize themselves in a state of Luttinger model38,75,76, which is an idealized system of interacting
electron matter resembling a solid rather than a liquid14. Finally, we discuss fermions with a linear dispersion relation (this may be pictured as a
recent observations of electrons in carbon nanotubes showing behaviour model of electrons with some fixed Fermi velocity but with an infinitely
similar to that of a Mott insulator15, which is a special kind of insulating deep conduction band). The operator that creates an electron at point x
in a Luttinger liquid is, in the ‘bosonized’ representation, proportional to
state induced by electron–electron interactions.
an operator that causes the finite displacement of a huge portion (one-
half) of the liquid. Thus, in terms of the bosonic variables, the electron
From Fermi liquids to Luttinger liquids operator is a very non-local one. That inconvenience is a small price to
Coulomb forces acting between electrons and ions hold in place the pay to be able to recast a Hamiltonian of interacting electrons in the
atoms forming a metal and ultimately determine the low-energy form of non-interacting particles38,75,76 (bosons).
spectra of its elementary excitations, electrons in conduction bands and

1
Department of Physics, Columbia University, New York, New York 10027, USA. 2Department of Physics and Astronomy, University of California, Riverside, California 92521, USA. 3Departments
of Physics and Applied Physics, Yale University, New Haven, Connecticut 06520, USA. 4Department of Physics, Harvard University, Cambridge, Massachusetts 02138, USA.

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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

a b
10
10–6

Current (A)
10–8
5

10–10 35 K
55 K
1/T 1+α (a.u.)

70 K
10–12 95 K

α
0.01 0.1 1 105 K
Bias (V) 125 K
140 K
160 K
180 K 1
200 K
Fit

0.5
0.001 0.01 0.1 1 10 100 0 5 10 15 20
γeV/kBT Diameter (nm)

Figure 1 | Power-law behaviour of electron tunnelling with energy for between wire segments in series between the electrodes. a.u., arbitrary
molybdenum selenide wires of various diameters. a, Current–voltage data units; e, elementary charge; kB, Boltzmann constant. b, Power-law exponent,
at different temperatures, T, for a molybdenum selenide wire bundle, scaled α, as a function of wire diameter; 1/α scales as the square root of the number
to a universal curve. Inset, the same data, but unscaled29. The parameter γ of channels, in agreement with theory19. Error bars, diameter uncertainty.
is a constant that indicates how the voltage drop across the device is shared (Figure reproduced, with permission, from ref. 29.)

Although Landau’s Fermi liquid theory provides a satisfactory account tool that makes the theory remarkably simple (Box 1).
of bulk three-dimensional (3D) metals, the situation is qualitatively In experiments, the zero-bias anomaly can be observed in the
different in materials in which the electrons are confined to channels differential conductance, dI/dV, where I is the current and V the volt-
that are narrow relative to the electron wavelength and as a result effec- age, of a tunnel junction attached to a 1D wire; a signature of Luttinger
tively act as 1D wires for the electrons. Because the electrons are con- liquid behaviour would be a deep minimum at zero bias (V = 0). This
strained to move in only two directions, right or left along the wire, any discussion also applies to tunnelling into (or from) an end of a wire.
perturbations will affect a large number of electrons collectively. For The only difference is that now the electron liquid must shift away from
instance, a charged particle trying to tunnel into the wire may create a the end by the entire interparticle distance; tunnelling is therefore sup-
short-wavelength density perturbation, forcing the liquid of interacting pressed more strongly.
electrons to shift quickly away from the incoming particle. To accom- A specific signature of Luttinger liquid behaviour predicted by the-
modate the extra particle, however, ultimately all electrons to the left of it ory16–19 is that the zero-bias anomalies of dI/dV for tunnelling into a
must shift by one-half of the average interparticle distance along the wire point at an end or into a point within the ‘bulk’ of a wire take the form
— in analogy to displacing equidistant beads on a string to make room of a power law in V (ref. 16): dI/dV ≈ V α. Similarly, the temperature
for an additional bead. Likewise, all liquid to the right of the tunnelling dependence of the tunnelling conductance at small, fixed bias also
particle must shift to the right by the same amount. This is in contrast to must follow a power law. The exponent α depends on the inter-electron
the 3D case, in which the charge of an incoming particle is compensated interaction strength and the number of channels19. The quantization
by a swift redistribution of the itinerant electrons, which are free to move of electron motion across the wire’s width results in the wire having a
away from the incoming electron in all directions, thus causing less of a finite number of channels. The wider the wire, the larger the number of
collective disturbance of the so-called Fermi sea of electrons. channels and the smaller the electron shift necessary to accommodate
An immediate consequence of this intuitive implication of the role of an extra electron, leading to the decrease of α towards zero (the conven-
collective behaviour when confining charged particles to one dimension tional case of tunnelling into a clean conductor in higher dimensions).
is that the tunnelling rate for particles entering a 1D wire is suppressed These power-law dependences were specifically predicted for17,18,
around zero bias — the so-called zero-bias anomaly. By contrast, the and measured in, carbon nanotubes, for tunnelling both into an end
tunnelling rate into a clean, conventional 3D metal is featureless (nei- and into the bulk20–22. The experimental results20–22, notwithstanding
ther suppressed nor enhanced) at zero bias, confirming the unimpeded the difficulties in interpretation associated with the finite-size effects
creation of particles or holes close to the Fermi level. This qualitative and zero-bias anomalies in the resistive leads23–25, are considered to be a
picture of tunnelling into a 1D electron system can be quantified within clear manifestation of Luttinger liquid physics. Further confirmation of
the so-called Luttinger liquid theory, ‘bosonization’ being a practical the Luttinger liquid behaviour came from resonant tunnelling experi-
ments with gallium-arsenide-based quantum wire dots26 and nanotube
dots27, nanotube capacitance measurements28 and molybdenum selenide
B nanowires that allow variation of the number of channels available for
V electron motion29 (Fig. 1).
Despite the agreement between the tunnelling anomaly measurements
d I
and theory, sceptics may consider it to be circumstantial evidence of the
unusual behaviour of electrons associated with their confinement to one
dimension. Indeed, the presence of disorder typical for highly resistive,
conventional (non-1D) conductors also impedes the spreading of charge
of tunnelling electrons. Because both Luttinger liquids and such higher-
dimensional systems display a zero-bias anomaly23–25, perhaps the pro-
Figure 2 | Momentum-resolved tunnelling spectroscopy using two parallel
wires. Electrons or holes tunnelling (arrows) between two parallel wires a
posed effect could be confused with the zero-bias anomaly in resistive
distance d apart. The momentum boost in the tunnelling event, Δp = edB, leads. It would help to know the range of biases over which the power
is achieved by application of a magnetic field, B, perpendicular to the law for tunnelling conductance is expected to hold. An experiment on
wires (into the page in this example). The energy resolution is achieved by multiwalled carbon nanotubes produced results hardly distinguishable
controlling the interwire bias, V. from the hallmark experiments20–22, with a power law observed over a
210
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

Box 2 | Spin density waves in a Luttinger liquid

Electron fluids support waves of spin density, as well as waves of charge
density. To see this, first consider a free-fermion gas, neglecting the
interparticle interaction. Imagine that in some portion of the 1D system,
an excess of spin-up particles is created and is compensated, so as

EF↑, EF↓
x
to maintain charge neutrality, by the deficit of spin-down ones (the
up–down axis in spin space may be picked arbitrarily). Even if the total
density of particles remains unchanged, the energy of the system will
increase, as locally the chemical potentials of the spin-up and spin-
down particles deviate from the common equilibrium value. This energy
increase provides the stiffness that defines the speed of the spin mode. and a weak but long-range charge–charge interaction hardly affects
In the free-fermion gas, the speeds of mass density and spin density the spin velocity, vs, but increases the velocity of the charge mode, vρ.
modes both equal the Fermi velocity. The phases of oscillations of the Note that the presence of interactions makes the waves of charge and
local Fermi levels of the spin-up and spin-down electrons are opposite spin truly collective excitations: they propagate with different velocities
to each other and thus have no effect on the charge density (see figure, and therefore cannot be associated with a motion of a single particle
where EF↑ is the Fermi energy for spin-up electrons, EF↓ is the Fermi endowed with the two qualities. The accommodation of a tunnelled
energy for spin-down electrons and x denotes position along the wire). electron requires charge and spin displacements, which propagate along
Therefore, a spin wave does not create a perturbation of charge density, the electron fluid with unequal velocities.

suspiciously wide range of biases30. Another uncertainty comes from parallel 1D electron channels formed in a semiconductor field-effect
the limitations caused by finite-size effects in the first experiments20–22, device6–8,35. In such a device, the momentum of the tunnelling electron
which, for example, made it impossible to distinguish between the is tuned using a magnetic field, B, applied perpendicular to the wires,
conducting and insulating states of a nanotube in the limit of very low and the energy resolution is achieved by controlling the interwire bias,
temperatures. The possibility of ‘dielectrization’, that is, formation of an V (Figs 2 and 3a), yielding information about the electron spectral func-
insulating state with a gap in the electron spectrum at low energies, was tion, A(p, ε), through the interwire current, I(B, V). A complication of
considered theoretically18,31–34. The single-point tunnelling phenomena this technique in comparison with, for example, angle-resolved photo-
described above gave only the first glimpse into the behaviour of elec- emission spectroscopy36,37 is the unavoidable electrostatic interaction
trons confined to one dimension. between the wires. Because of this interaction, some effort is necessary
to extract A(p, ε) from the measurement of I(B, V) (refs 6, 7).
Spin–charge separation Ideally, spin–charge separation would manifest itself as two peaks in the
Another distinct manifestation of the correlated behaviour of electrons energy dependence of A(p, ε) at a fixed value of p. For definiteness, we con-
when confined to one dimension is the phenomenon of spin–charge centrate here on the case of p values slightly less than the Fermi momen-
separation. The electron liquid, along with the waves of electron charge tum, pF, and ε values less than zero, that is, on the probability of extracting
density, supports waves of electron spin density. These are collective a particle from a Luttinger liquid. Then, the two peaks signify the two
excitations, propagating with two different velocities (Box 2). extremes in sharing of the momentum by the elementary charge and spin
The successful observation and direct visualization of spin–charge excitations (holons and spinons, respectively38). The high-energy peak
separation has been achieved in experiments on so-called momentum- (with the largest value of |ε|) is located at ε = vρ(p − pF) and corresponds
resolved tunnelling6. In such experiments, the aim is to extract an elec- to the entire momentum being given to a holon. The low-energy peak,
tron from the wire with a given momentum, p. Such an electron would at ε = vs(p − pF), corresponds to the entire momentum being carried away
not be associated with a unique energy, ε(p), owing to the interactions by a spinon (Fig. 3). This peak actually corresponds to the lowest-energy
in the liquid, and would therefore leave behind multiple excitations (as state of the Luttinger liquid, which may be created by removing from it a
above, the same considerations apply for holes and particles and, thus, particle with momentum p; A(p, ε) must equal zero for any smaller value of
for tunnelling from and into the wire). The probability of forming a |ε|. For |ε| values slightly above the threshold, |vs(p − pF)|, a small part of the
state with energy ε if the added/extracted electron has momentum p is hole’s energy is spent on the creation of charge waves with small wavevec-
quantified by the spectral function, A(p, ε). In 1D wires, this information tors, |q| << |vs(p − pF)|/vρ, and the greater part is carried by a spinon.
can be obtained from momentum-conserving tunnelling between two Luttinger liquid theory predicts that the two described peaks in the

a c 12 Figure 3 | Probing spin–charge separation and charge fractionalization

14 in interacting 1D wires using momentum-resolved tunnelling
spectroscopy. a, Schematic structure of the double-wire geometry, made
12
10 using the cleaved-edge overgrowth technique7. The double-wire system
Magnetic field (injected momentum) (T)

10 resides at the edge of a gallium arsenide/aluminium gallium arsenide

Sp

double-well structure. Dark green, gates; blue, AlGaAs; white, GaAs; light
in

8 Char 8 green, 1D wire segments; transparent box, epitaxial confining layer on the
ge
cleaved edge. b, Schematic of circuit used to measure the spectral function
b 6 and charge fractionalization. IL, left-moving current; IR, right-moving
IL IR
VSD current; VSD, bias voltage. c, Measured spectral function, ∂I(V, B)/∂V,
6 4
indicating the spin and charge excitation branches (plotted versus injection
2 energy, eV, on the horizontal axis). A smooth background has been
d subtracted from the data. The colour scale indicates the magnitude of
4 0 ∂I(V, B)/∂V (arbitrary units). The features labelled spin and charge cross
1 at zero energy at a magnetic field of around 7 T. d, Measured spin velocities
–2
(open symbols) and charge velocities (filled symbols) for different carrier
vF /v

2
–4 densities (colours), plotted as the Fermi velocity, vF, divided by the measured
velocity, v. A lower carrier density corresponds to stronger repulsion
–6 between electrons and hence to a larger charge excitation velocity. The
0 0
0 120 –15 –10 –5 0 5 spin modes are nearly independent of the carrier density. Solid curves are
Carrier density (μm–1) Injection energy (meV) theoretical fits. (Panel reproduced from ref. 6.)

211
© 2010 Macmillan Publishers Limited. All rights reserved
INSIGHT REVIEW NATURE|Vol 464|11 March 2010

a Q and several experimentally testable predictions have already been made,

p → pF among them the survival of the power-law singularity of A(p, ε) to higher
Q/2 Q/2 energies for certain modes43 and particle–hole asymmetry in the relaxa-
tion rates10. The second of these predictions can be tested using exist-
ing6 experimental techniques. We return to the question of spin–charge
separation at higher energies below.
υpl → ∞

Charge polarization and break-up

b Q
p → pF In the discussion of momentum-resolved tunnelling, we considered an
(1 – f)Q fQ Q
idealization of an infinitely long junction between two parallel wires. In
practice, the junction typically has a length of few micrometres, which
is long on the scale of the Fermi wavelength and does not limit the use of
L momentum as a good quantum number7. Electrons with given momenta
υρ = υF υρ > υF υ ρ = υF are injected near the middle of a long quantum wire (Fig. 3b). This poses
the question of how the injected charge spreads once it is in the wire. Pre-
dictably, the answer depends on the circumstances, leading to interesting
Figure 4 | Spreading of charge on tunnelling into an interacting wire. a, An considerations related to the break-up, or ‘fractionalization’, of charges.
electron liquid in a homogeneous infinite wire in the absence of shielding First, look at the ideal case of a quantum wire in the absence of any
supports plasmons that transfer the tunnelled charge to the outer parts other conductors that may shield the electric field produced by charges
of the system; in the limit vpl/vF → ∞, the initial momentum of the charge in the wire. In this case, the dispersion relationship for the charge mode
does not affect the pattern of transient charges. b, Transient charges at a is ω(q) ≈ q√[ln(1/wq)], where w is the wire’s width and q is the wavevector
finite charge mode velocity, vρ, in the presence of free-fermion leads. The of the charge excitation, or plasmon. If the tunnelling electrons have
charge pulse splits into transmitted and reflected components (dashed) at low energies (such that their momenta are close to ±pF), then only low-
the boundary of two media with different ‘refraction indices’ for the charge energy plasmons can be excited. Such excitations have small wavevectors
waves. In the set-up shown, multiple reflections at the two boundaries result (q → 0) and, thus, diverging velocities, vpl ≈ √[ln(1/wq)] → ∞. Relative
in the entire tunnelled charge, Q, being transferred into the right-hand lead
and zero charge exiting into the left-hand lead.
to the plasmons, the velocity of a tunnelling electron, v → vF (where vF
is the Fermi velocity), is so small that its direction is inconsequential;
tunnelling of a charge Q with a well-defined momentum thus creates
electron spectral function are power-law singularities, with exponents two pulses, each of charge Q/2, running in opposite directions (Fig. 4a).
defined by the strength of the inter-electron interaction39. Singular points Although this is somewhat counterintuitive, it is no different from the
in the energy–momentum plane form one pair of lines, ε = vρ(p − pF) and dynamics of charge screening in higher dimensions: in a conducting liq-
ε = vs(p − pF), that intersect at (0, pF), and a similar pair that intersect at uid, a polarization charge develops that locally compensates the charge
(0, −pF). In the experiment (Fig. 3), the momentum of a tunnelling elec- of the tunnelling electron and spreads it towards the edges of the system.
tron is tuned using B and its maximal energy is determined using V. The An electron tunnelling into a two-dimensional electron liquid, for exam-
intersecting straight lines tracing the singularities of A(p, ε) correspond ple, would create a circularly symmetric ‘splash’ of polarization charge.
to the intersecting straight lines tracing the singularity in the differential Shielding of the long-range Coulomb forces reduces the infinite (for
conductance in the V–B plane. q → 0) plasmon velocity, vpl, to a finite velocity, vρ, of charge wave propaga-
Measurements6,7 reveal clear maxima of ∂I(V, B)/∂V along a system of tion. For a finite ratio vρ/vF, the waves of polarization moving in opposite
lines in the V–B plane, consistent with theory40. Two of the lines indeed directions away from the tunnelling point differ from each other (Fig. 4b).
form a crossing (see Fig. 3c, where the injection energy, eV, is plotted The fraction, f, of the tunnelling charge, Q, that is carried by the wave
against B), at a point that can be unambiguously identified with the tun- propagating in the direction of the incident electron motion can be found
nelling of electrons with momenta approaching pF. The velocities of the from the momentum conservation law (for pedagogical reasons, it is bet-
corresponding excitations can be extracted from the slopes of the two ter to consider the case in which Q >> e). The momentum of the tunnelling
lines at the crossing point. These velocities are in reasonable agreement charge, (Q/e)mvF, is carried away by particles of the same mass, m, moving
with theoretical estimates for the velocities of charge and spin modes in opposite directions with velocity vρ. The momentum conservation law
(Fig. 3d). Current measurements are not accurate enough for to tell takes the form fvρ − (1 − f)vρ = vF, yielding f = ½(1 + vF/vρ); in the absence of
whether ∂I(V, B)/∂V is actually divergent along the lines corresponding interactions (vρ = vF), the expected result, f = 1, is obtained.
to the spectra of the two modes. We should emphasize the distinction between the notions of charge
Further recent work exploiting a similar idea, based on tunnelling quantization and fractionalization. Nowhere in the above discussion
between a wire array and a two-dimensional electron gas, also found was a particular value of the tunnelling charge, Q, important, consistent
multiple excitation velocities consistent with theory41. Taken together, with the idea of a liquid as a description of a continuous medium. The
experiments6,7,41 provide direct confirmation of the phenomenon of quantized value of the charge of the particles forming the liquid must
spin–charge mode separation in 1D electron systems at low energies. come from a separate, microscopic consideration. (For example, the
Although there is a qualitative agreement with the Luttinger liquid quantized fractional charge of quasiparticles under the conditions of
theory for momenta approaching pF , the experiments on momentum- the fractional quantum Hall effect is defined by the special properties
conserving tunnelling have sharpened a question that is not addressed of the corresponding microscopic wavefunction44.)
within the Luttinger liquid theory, that of what happens to spin–charge This discussion of charge fractionalization is applicable as long as the
separation at higher energies. The two experiments6,7 do demonstrate polarization charges do not hit the ends or some other inhomogeneity of
that a remnant of the charge mode spectrum survives at higher bias, in the wire. In principle, a direct determination of f can be achieved from a
the form of a parabolic dispersion relationship similar to a free-particle time-resolved measurement of the transient currents in the wire or from
excitation. However, traces of the spin mode fade away at higher ener- a measurement of the current noise spectrum45. However, d.c. measure-
gies. Finding a theoretical answer to the question requires accounting ments cannot determine f. This limitation is illustrated here in the simplest
for the nonlinearity of the generic electron spectrum, which necessitates geometry of free-fermion leads attached to a Luttinger liquid of length L.
going beyond the Tomonaga–Luttinger model, in which the linearity of The contact with the leads may be modelled as a smooth spatial variation
the spectrum is a central assumption. Some suitable tools for develop- of the charge wave’s speed from vρ to vF. Being smooth on the length scale
ing a theory of a liquid of quantum particles with a generic dispersion set by the Fermi wavelength, such an inhomogeneity does not backscatter
relationship (a nonlinear Luttinger liquid) were found only recently9,42,43 individual electrons; by analogy with optics, it rather acts as an interface of
212
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

two media with different refraction indices for the charge waves (Fig. 4b). behaviour of A(p, ε) is described by a power-law function of ε(p) − ε, as
To analyse the d.c. measurement, consider the propagation of charges in the Tomonaga–Luttinger model. Unlike in that model, however, the
over times much greater than that, L/vρ, needed to traverse the Luttinger power-law behaviour at the threshold is valid at any momentum, and the
liquid. The propagation occurs in the free-fermion part of the system. As exponent of the power law depends on the momentum.
the electrons do not backscatter, the momentum conservation law can be To explain the power-law form of A(p, ε), here we allude46 to the effect
applied as was done above, but with the replacement vρ → vF. This leads to known as the Anderson orthogonality catastrophe47. For definiteness,
the conclusion that the entire tunnelled charge eventually ends up moving consider the part of A(p, ε) associated with the creation of a hole in the
in the direction of the initial electron’s momentum, regardless of the value system. The introduced hole produces a potential acting on other par-
of f within the Luttinger liquid (Fig. 4b). ticles. The threshold in A(p0, ε) corresponds to the ground-state energy
The concept of charge fractionalization provides a convenient basis for of the many-body system adjusted to that potential. The ‘old’ (pre-hole)
calculations within Luttinger liquid theory. Experiments performed at low and ‘new’ many-body ground states have zero overlap47. The overlap
injection energy, using the more sophisticated configuration of a three- with excited states is finite and scales as some power of the excitation
terminal d.c. measurement (Fig. 3b) with momentum-selective source and energy (measured relative to the new ground state). This non-zero over-
drains attached to the wire, are in excellent agreement with theory8. Such lap with excited states of the Fermi sea results in the redistribution of the
d.c. measurements can also be analysed within the framework of charge spectral weight in A(p0, ε) from a single point (ε = ε(p0)) to all energies
fractionalization, but they do not allow determination of f. above the threshold, according to a power law in ε(p0) − ε.
A natural question to ask is how the asymmetry between currents This picture, which was initially developed for weakly interacting
in the two drains (Fig. 3b) behaves at higher injection energies, once particles, has been extended into a fully consistent phenomenological
the conventional Luttinger liquid theory breaks down. Answering this theory9,43 valid at arbitrary interaction strength. The phenomenology
question may provide information about the differences between the relates the threshold exponents of a dynamic correlation function to the
kinetics of injected high-energy particles and holes. properties of the corresponding threshold energy spectrum; these two
types of characteristic can be measured independently of each other.
Nonlinear Luttinger liquid The limit of weakly interacting fermions also demonstrates the dif-
To analyse the above-mentioned experiments, aimed at measuring ference between the particles and holes. Particles move at velocities
the spectral functions of electrons and holes at higher energies and greater than vF (Fig. 5), allowing the ‘Cherenkov radiation’ of particle–
at elucidating how the injected charge propagates along the wire at hole pairs; unlike holes, energetic particles do relax, producing multiple
these higher energies, it is necessary to go beyond conventional Lut- particle–hole pairs. Although particle–hole asymmetry has not yet been
tinger liquid theory. There is also an impetus to do so from within the observed, experiments to explore such deviations from Luttinger liquid
theory. The linearization of the particles’ spectrum that is assumed in theory are underway.
the Tomonaga–Luttinger model brings an immense degeneracy into Developing the nonlinear Luttinger liquid theory of spin-½ fermions is
the energy spectrum of the excited many-body states: the energy of a of special interest. Such a theory should cover the case of strong interac-
state composed of an arbitrary number of particle–hole pairs residing tions keeping fermions ‘in place’ and thus suppressing their spin-exchange
on one branch of the single-particle spectrum depends only on the total interaction and creating an almost flat band of spinon excitations.
momentum of the pairs and is insensitive, for example, to the number of
pairs. Reintroduction of the generic nonlinear dispersion relationship of Correlated-electron solid at low density
the particles into the theory removes this degeneracy in a subtle way. In the previous sections, we discussed theory and experiment in the
Recently, the nonlinear theory was developed for spinless quantum regime where the charge density is large enough to ignore the strong
particles9–11, and since then a first step has been made in generalizing the correlations between the electron positions that develop when the elec-
dynamic response theory to spin-½ fermions with a generic dispersion tron density, n, becomes sufficiently low. These correlations emerge
relationship12,13. The new methods make it possible to move beyond because although the long-range part of the electron–electron interac-
the linear Luttinger liquid description in understanding the dynamic tion chiefly affects the value of vρ, as this depends on the electrostatic
response functions (such as the spectral function, A(p, ε)). They also energy required to create long-wavelength charge fluctuations, the
provide a basis for understanding the kinetics of interacting quantum increase of its short-range part (~e2n) relative to the single-electron
particles confined to one dimension, although the full kinetic theory
has not yet been constructed. The new theory predicts the persistence
of the power-law singularities in A(p, ε) even for momenta far from the ε
Fermi points and allows the evaluation of the corresponding exponents.
In striking contrast to the conventional Luttinger liquid theory, it also
predicts asymmetry in the relaxation rates for particles and holes.
Here we demonstrate the main idea of the new theory using the
example of weakly interacting fermions without spin. Free particles have
a parabolic dispersion relationship, ε(p) = p2/2m, and here it is assumed pF
that an equilibrium population of fermions occupies states with |p| ≤ pF. p
The focus is on evaluating A(p, ε) for the hole excitations (ε < 0). In the
absence of interactions, only one state of the system is created by extract-
ing an electron with some momentum, p0 (−pF ≤ p0 ≤ pF), in the Fermi
distribution; this state consists of a single hole with momentum p0. The
velocity of this hole, |p0|/m, is less than vF, which is the velocity of the
lowest-energy particle–hole excitations. Therefore, interaction of this Figure 5 | Effects of the finite mass of particles forming the nonlinear
hole with the particles of the Fermi sea does not lead to creation of excita- Luttinger liquid. At positive curvature of the energy–momentum function,
tions, in much the same way that a particle moving at less than the speed ε(p) (solid black line), the velocity of a hole is less than vF and therefore a
hole state is stable, unlike a particle state (the particle spectrum is shown
of light in a medium does not emit Cherenkov radiation. This means that as the dark-grey broadened line). The spectrum of holes (dashed black
the existence of the energy threshold in A(p, ε) along some line, ε(p), at lines) forms the threshold for the spectral function, A(p, ε), which is finite
a momentum |p| ≤ pF is robust with respect to the interaction (Fig. 5). At everywhere in the light-grey shaded area. Creation of a ‘deep’ hole (lower
the same time, the functional form of A(p, ε) is modified by interaction open red circle) perturbs the Fermi sea, creating low-energy particle–hole
away from its free-fermion limit, A(p, ε) ≈ δ(ε − ε(p)) (where δ denotes the pairs (open and filled red circles near the Fermi point); this process results
delta function). Remarkably, this modification is universal: the threshold in the power-law threshold singularities in A(p, ε) discussed in the text.

213
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INSIGHT REVIEW NATURE|Vol 464|11 March 2010

a (ref. 57). The periodically ordered electrons forming the Wigner crystal
B
act as a spin and isospin chain. For B > 0, the ground state reflects a com-
petition between the magnetic energy, which favours parallel spins and
parallel isospins, and the exchange energy, which favours neighbouring
b Ground-state configuration
electrons having different spin and/or isospin directions14,58.
Region III Region II Region I
Because J increases rapidly with n, in a magnetic field it is expected
that there are three different regimes for a Wigner crystal state in a car-
bon nanotube as n and B are varied. In one (region I), the magnetic
energy is dominant and all the electrons are fully spin and isospin polar-
c G (μS)
0 >10.0 ized. In another (region II), the exchange dominates the Zeeman split-
Hole number
ting but not the orbital splitting, which is larger, and only the isospin is
40 30 20 10 1 polarized. In the last (region III), the exchange energy is dominant and
the polarization is minimal for the given number of electrons. Figure 6b
B (T)

5
III II I shows the ground-state configurations for these states.
0
–4.0 –3.0 –2.0 –1.0 0 1.0
Coulomb blockade spectroscopy provides a very useful tool to study
Vg (V) this experimentally. In Coulomb blockade spectroscopy, the device acts
Figure 6 | Charges at low densities in a carbon nanotube form a 1D Wigner as a single-electron transistor, producing peaks in the dependence of
crystal. a, Although the sense of motion of electrons around the nanotube conductance on gate voltage, Vg, whenever an electron is added to the
(red and blue arrows) depends on the direction of the magnetic field, B, in dot, thereby changing the electron density. The peak spacing reflects the
an armchair tube, in a tube of general chirality electrons with both senses energy required to add each electron. Because the magnetic energy contri-
of motion can be present. The sense of motion for each charge can be bution to this energy is different for each combination of quantum num-
labelled using an isospin quantum number. b, Ground-state configurations bers, measurement of each peak’s energy shift as a function of B yields the
of charges in the regions (I–III) described in the text. c, Differential charges’ quantum numbers. Figure 6c shows the evolution of the Coulomb
conductance, G, of a nanotube as a function of gate voltage, Vg, and peaks in a magnetic field for a semiconducting carbon nanotube, which
magnetic field. The vertical stripes are the traces of the conductance peaks.
(Panel reproduced, with permission, from ref. 14.)
displays all three regimes: in region I, the parallel traces that conductance
peaks produce in the B–Vg plane indicate that each added electron has
the same spin and isospin; in region II, the alternating slopes indicate that
kinetic energy (~"n2/m, where " denotes Planck’s constant divided by isospin is polarized and that spins alternate; and in region III, the zigzag
2π and m denotes the electron mass) tends to suppress the electrons’ pattern indicates the minimum polarization state14.
ability to pass by each other. This produces a 1D Wigner-crystal-like The yellow line in Fig. 6c shows a single-parameter fit to theory58, with
structure with periodically ordered charges48–52. (Note that although the fitting parameter within the theoretically predicted range58,59. The
perfect charge ordering in one dimension is not expected, the correla- red line is a plot of the boundary between regions I and II made using
tion length can readily exceed the sample length in realistic situations, the same parameter value. The quantitative agreement with theory is
yielding a state that is much more like an electron solid than an electron satisfactory. We note that a model of non-interacting electrons predicts
liquid.) For e2n >> "n2/m, the positional exchange between neighbour- very few or no spin-polarized electrons under the same conditions14.
ing electrons becomes exponentially rare and the fluctuations of their The success of the Wigner crystal picture in accounting for the rela-
relative positions become small (the condition can be recast in the form tive ease of polarizing electron spins in nanotubes in a magnetic field
naB << 1, where aB is the effective Bohr radius for a conduction electron suggests that testing a theory49 predicting one-half the usual conduct-
in the material). The suppressed exchange decreases the spin mode ance of a ballistic channel in the spin-incoherent regime would be very
stiffness, lowering the energy cost of spin excitations. If exchange is interesting. However, this would require progress in reliably obtaining
ignored entirely, the spin system becomes disordered for any positive nearly perfect contacts to ultraclean semiconducting nanotubes.
temperature. This so-called spin-incoherent regime has been a topic of
much recent theoretical interest53. Mott insulator
Studying the properties of such a system requires low-disorder The presence of enhanced spatial correlations between the electrons
samples to avoid an insulating state where disorder pins the Wigner due to the interactions (that is, Wigner crystallization) increases the
crystal. Potential clean systems in which to study such behaviour include tendency towards pinning by inhomogeneities. When pinned, an elec-
wires made using cleaved-edge overgrowth35 and semiconducting car- tron system becomes insulating at sufficiently low temperatures. Sup-
bon nanotubes54. In particular, advances in sample preparation for car- pression of conductance observed in gallium arsenide wires made using
bon nanotubes have allowed the measurement of as-grown, suspended cleaved-edge overgrowth has been tentatively explained35 in terms of
nanotubes, minimizing the tube–substrate interaction and producing such a pinning phenomenon.
very clean and low-disorder 1D systems that may reveal Wigner crystal The atomic lattice can be another source of pinning in an interacting
physics. As discussed above, one of the hallmarks of a Wigner crystal electron system. Because of the lattice periodicity, pinning opens an
state is that the antiferromagnetic exchange energy, J, is exponentially energy gap in the electron spectrum. Commensurability between the
suppressed at low densities. This has an interesting consequence for mean electron spacing and the lattice period aids the opening of the gap.
the spin polarization in an external magnetic field. In contrast to free When electrons are on a lattice at half-filling (one conduction electron
electrons, for which full polarization occurs only if the Zeeman energy, per atom), such a situation occurs. An example illustrating this is a 1D
gμBB (where B is the magnetic field, μB is the Bohr magneton and g is lattice-site chain in which a large energy cost, U, exists for double occu-
the g factor), of an electron spin exceeds the Fermi energy, in a Wigner pancy of a site and where the hopping matrix element, t, between sites
crystal polarization occurs at exponentially smaller fields, as only the can be considered small. At half-filling, every site is occupied, an elec-
condition gμBB > 2J is required. tron–hole pair costs an energy U to create and the system is an insulator.
In addition to their spin, electrons in nanotubes have an extra isospin This is essentially the physics of Mott insulators60. However, in contrast
degree of freedom, which is an additional angular momentum acquired to 3D systems, where the formation of a Mott gap requires U to exceed
by the electrons because of their motion around the axis of the nanotube some threshold, in one dimension a gap exists for any ratio U/t > 0.
(either clockwise or anticlockwise; Fig. 6a). In an axial magnetic field, Although such physics has been studied in bulk materials61, carbon
the Zeeman effect splits the spin states55,56 and their different orbital nanotubes afford the opportunity of potentially studying 1D Mott
magnetic moments, ±μorb, split the isospin states57. The isospin splitting, physics in an individual nanostructure with readily tunable param-
2μorbB, is typically larger than the Zeeman splitting by a factor of ~5–10 eters. Indeed, there is a puzzle to be solved — nanotubes predicted to
214
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 REVIEW INSIGHT

be metallic in the single-particle description are found experimentally a

dI/dV (μS)
to be gapped. Data taken from a so-called armchair nanotube (Fig. 6a) 25 40

V (mV)
show such a gap (Fig. 7a) at zero doping. The simplest single-particle 0
20
perturbations, such as curvature or strain of an armchair nanotube, are –25 (18, 18)
0
prohibited from opening a gap by symmetry constraints62.
–5 0 5 10
Such a gap may possibly be due to the many-body effect in Mott Vg (V)
insulators. A number of works have included electron–electron inter-
actions in theories of carbon nanotubes and found that the interac-
tions should open a gap at half-filling18,31–34. The structure of a general b
U
nanotube is determined by a pair of integers (N, M), which specifies the U/N
tube’s chirality and radius, r. For N = M, an ‘armchair’ nanotube (Fig. 6a), t t
the honeycomb lattice can be mapped onto an equivalent 1D problem t⊥ t t
known as the two-leg ladder31 (Fig. 7b). When long-ranged interactions t⊥
are taken into account, theories predict an energy gap of Δ ≈ 1/r β for
metallic nanotubes, with β ranging from β = 1/(1 − g) to β = 2 (ref. 63),
depending on detailed assumptions. Here g = vF/vρ ≈ 0.2–0.3 for nano-
tubes18,33 and is a measure of the strength of the interactions therein.
Experiments show an approximate 1/r1.3 dependence15, in agreement c Gap
with the theoretical range of predicted values of the exponent. Holes Electrons
Nevertheless, even in the most robust case of an armchair nanotube it 5 30
remains possible that the observed gap originates from another single-

G (μs)
20

B (T)
2.5
particle perturbation, a twist. It is therefore beneficial to develop an 10
approach that experimentally rules out the single-particle gap-opening 0
mechanisms for nanotubes of arbitrary structure. Theory predicts that 0.5 1 1.5 2
such gaps close at some value of an applied axial magnetic field and that Vg (V)
a metallic state is recovered62,64. Interestingly, experiments performed on
Figure 7 | Electron correlations in 1D carbon nanotubes give rise to
pristine, suspended carbon nanotubes show that the gap reaches a mini- insulating gaps. a, Colour plot of differential conductance, dI/dV, as
mum, non-zero value at a critical magnetic field (~2.5 T for the device a function of source–drain bias, V, and gate voltage, Vg, for an (18, 18)
in Fig. 7c), before widening again15. The observation of a minimum nanotube. The large region of dI/dV suppression near the centre indicates
gap rules out all of the above non-interacting theories of gap creation the presence of a gap. (Data courtesy of V.V.D., M. Takekoshi, P. Kim,
in these nanotubes. J. Hone and T. Heinz.) b, Mapping of the honeycomb lattice onto an
Another indication of strongly interacting electrons in nanotubes equivalent two-leg ladder system. Brown and green distinguish which
comes from the presence of low-energy neutral electronic excitations atoms in the nanotube are mapped onto which ladder leg. The parameter
with energies less than the gap energy15. (By contrast, if the electron U is the electronic on-site interaction discussed in the main text, and t
system were non-interacting, the lowest-energy excitation would be and t⊥ are the electron hopping matrix elements. The effective on-site
interaction in the equivalent ladder is reduced by a factor N, to U/N,
that which moves an electron from the valence band to the conduction
for an (N, N) nanotube. The black arrow indicates the tube’s cylindrical
band, costing an energy equal to the gap energy.) Although the origin of axis. c, Conductance, G, as a function of magnetic field, B, and gate
these low-energy excitations is not yet fully understood, their presence voltage, showing a gap that reaches a minimum value for B ≈ 2.5 T. (Panel
is consistent with the Mott theory31–34, which predicts neutral excita- reproduced from ref. 15.)
tions of the Mott insulator with energies less than the gap energy, such
as spin excitations, and an energy scale similar to that observed. This
provides further evidence for a many-body origin of the observed gap a highly tunable means of harnessing many-body physics in the solid
in carbon nanotubes. state and developing new applications such as a pump for quantized
In future, signatures of a 1D Mott insulator around its metal–insulator fractional charge59. A number of interesting theories42,53 concerning
transition point65 may be investigated. Furthermore, the exotic spin liq- spin-incoherent and nonlinear Luttinger liquids may be investigated.
uid excitations predicted for carbon nanotubes34 could be studied in There is also significant theoretical work ahead aimed at filling the
detail. Finally, gaps could be opened even in the absence of pinning. It gaps between the limits of the Tomonaga–Luttinger model and incoher-
has been theorized for Wigner crystals48 that in wider channels the 1D ent Luttinger liquids and between the limits of ideally ordered 1D inter-
Wigner crystal will make a transition to a zigzag phase with a gapped acting systems and general disordered ones. In the case of disordered
mode, in addition to a gapless one, as its hallmark. Recent conductance Luttinger liquids, the recognized72 need for further theory is driven by
measurements of wide quantum wires66 are consistent with this picture; experiments with carbon nanotubes out of equilibrium73 and by cur-
however, the gapped mode has not yet been directly observed. rent theoretical predictions such as many-body localization74. Given
the rapid evolution of its experimental and theoretical techniques, the
Future directions field of correlated many-body 1D systems looks set for more exciting
Further experiments, including those on systems and using techniques developments in the future. ■
not discussed here, are expected to provide more insight into the nature
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308, 88–92 (2005).
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periodic potential to create an artificial Mott insulator that can provide resolved electron tunnelling.

215
© 2010 Macmillan Publishers Limited. All rights reserved
INSIGHT REVIEW NATURE|Vol 464|11 March 2010

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from one-dimensional SrCuO2. Nature Phys. 2, 397–401 (2006). acknowledges support from the US National Science Foundation (NSF) Division of
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325, 597–601 (2009). Correspondence should be addressed to M.B. (marc.bockrath@ucr.edu).

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Vol 464j11 March 2010jdoi:10.1038/nature08757

REVIEWS
Targeting early infection to prevent HIV-1
mucosal transmission
Ashley T. Haase1

Measures to prevent sexual mucosal transmission of human immunodeficiency virus (HIV)-1 are urgently needed to curb the
growth of the acquired immunodeficiency syndrome (AIDS) pandemic and ultimately bring it to an end. Studies in animal
models and acute HIV-1 infection reviewed here reveal potential viral vulnerabilities at the mucosal portal of entry in the
earliest stages of infection that might be most effectively targeted by vaccines and microbicides, thereby preventing
acquisition and averting systemic infection, CD4 T-cell depletion and pathologies that otherwise rapidly ensue.

uring more than one-quarter of a century of the HIV-1 stages of lentivirus infections. CD4 T-cell depleting and pathological

D pandemic, millions have died from AIDS, millions have

been orphaned, and more than 30 million people world-
wide are living with HIV-1 infection1. Although advances
in antiretroviral therapies and access to treatment are countering this
great and continued toll in the developed world and sub-Saharan
processes are also very quickly set in motion in the early stages of
systemic infection that then play out over the months and years in the
slow phase of infection.
SIV rhesus macaque nonhuman primate model. These conclusions
are drawn from studies in the SIV rhesus macaque nonhuman primate
Africa, the epicentre of the pandemic2, prevention of sexual mucosal (NHP) model of transmission of HIV-1 to women14, which provides a
transmission, the principal route of acquisition1, is clearly needed to window through which mucosal transmission and the first 2 weeks of
contain the continuing growth of the pandemic and ultimately eradi- infection can be viewed in vivo in ways that, for practical and ethical
cate AIDS3. reasons, are not possible in HIV-1 infection, which is only clinically
The prevention record is mixed. Circumcision has proven effective manifest after this time15. From NHP studies of the earliest stages of
in preventing 50% to 60% of female to male transmission4–6, and infection16, we have the following picture (Fig. 1) of the critical events
there are the first hints recently of vaccine and microbicide candi- following mucosal exposure to high doses of SIV: virus can cross the
dates that could prevent sexual transmission to men and women, mucosal epithelial barrier within hours17 to establish what has turned
albeit at low efficacy of about 30%7,8. However, vaccine and micro- out to be a small founder population of infected cells16,18. This founder
bicide candidates have not proved efficacious or have even enhanced population then undergoes a necessary local expansion during the first
acquisition in previous trials9,10. These disappointing failures and lim- week of infection to generate sufficient virus and infected cells to
ited successes reinforce the view of leading investigators11 that it is now disseminate and establish a self-propagating systemic infection
especially timely to reinvigorate and broaden research on the virus, the throughout the secondary lymphoid organs16.
immune response to it, and the pathogenesis of transmission and Beginning in the second week of infection, replication explodes in
infection to identify the correlates of prevention that will enable the the lymphatic tissues where virus has access to many more susceptible
design of fully protective measures against acquisition of HIV-1. target cells in close spatial proximity, compared to the dispersed low-
In this review, I provide a personal perspective and synthesis of density populations of susceptible target cells at the portal of entry.
studies on sexual mucosal transmission relevant to this goal. I largely Virus levels in blood and tissues peak near the end of this second week
of infection, before declining towards relatively stable lower levels by
focus on the results of in vivo analyses of relevant tissues, and on
4 weeks after exposure. At this time, these infected lymphatic tissues
transmission to women, because of the increasing feminization of
comprise a reservoir where virus is produced and stored and where
the pandemic in sub-Saharan Africa12, and because of insights into
proviruses are harbored in latently infected cells in SIV-infected rhesus
HIV-1 transmission to women derived from extensive studies of the
macaques19, just as in HIV-1 infected humans20. Furthermore, this
simian immunodeficiency virus (SIV) rhesus macaque nonhuman
reservoir is already the site of CD4 T-cell depletion and other patho-
primate model. I conclude from these studies that prevention strat-
logical processes that will eventually lead to progression to disease
egies should target the earliest stage of infection because of: (1) viral
(Fig. 2).
vulnerabilities at this stage at the portal of entry in the small, infected This description of transmission, early infection and ill effects of
founder populations; (2) local expansion necessary to establish sys- systemic infection (Figs 1 and 2) is derived from studies in the SIV
temic infection; and (3) the ill effects that otherwise rapidly ensue high-dose vaginal challenge NHP model in which rhesus macaques
from systemic infection. are atraumatically inoculated twice in the same day with two doses of
105 TCID50 (50% tissue culture infectious dose) and billions of viral
Fast stage of slow lentivirus infections particles16. The strengths of this model are: (1) the window (Fig. 1) it
The retrovirus genus to which HIV-1 and SIV belong was long ago provides on critical events that precede the earliest time clinical signs
named Lentivirinae13 based on the long incubation period and slow and symptoms of HIV-1 infection disease are manifest15; (2) infec-
clinical course of disease that typify infection. However, it is now tion of a high proportion of animals with a known time of exposure;
clear from the SIV rhesus macaque model that local events critical (3) access to relevant tissues in a relevant time frame, which increases
to establishing systemic infection take place very quickly in the early chances to observe directly virus–host cell interactions and critical
1
Department of Microbiology, University of Minnesota, Minnesota 55455, USA.
217
©2010 Macmillan Publishers Limited. All rights reserved
REVIEWS NATUREjVol 464j11 March 2010

Time frame Establishment

Vagina Cervix Lymphatic Activated CD4+
tissue T-cell target
Window
Crossing the reservoir
barrier
Hours
Weeks Thoracic duct Early
Immune
Local Infected 2-4 Lymph activation TReg cells
GALT
propagation founder node Suppress
Viral population
Small
vulnerabilities R0<1
Opportunities Spleen Lung
for prevention Brain Liver

Days Local Infected Massive depletion of gut lamina propria Prevent Too little Virus-specific
expansion cells CD4+ T cells too late CD8+ T cells
Virus

Sufficient Battlefield map

production Week 2
Draining
Dissemination lymph node Large numbers of
Self- SIV-specific CD8+ T cells
propagating +
Week 1 systemic
infection
Large numbers of
Establishment SIV-infected cells
Lymphatic
tissue E:T ratio <2
reservoir
Thoracic duct Lymph node
GALT
Weeks

Spleen Lung
Brain Liver

Figure 1 | Time frame, sites and major events in vaginal transmission and SIV-specific
effector CD8+ T cell
the fast phase of lentivirus infection. The SIV rhesus macaque animal
model provides a window through which to view early infection. Within SIV-infected
hours, virus in the inoculum may gain access through breaks in the mucosal target cell

epithelial barrier to susceptible target cells. The small focal infected founder Effector–target
population is initially composed mainly of infected resting CD4 T cells conjugate

lacking conventional markers of activation. The founder population

expands locally in these ‘resting’ and in activated CD4 T cells. Local Figure 2 | Ill effects of systemic infection and too little too late immune
expansion is necessary to disseminate infection to the draining lymph node, response. After dissemination and establishment of systemic infection in
and subsequently through the bloodstream to establish a self-propagating weeks 2–4 (upper left panel), peak viral replication in gut-associated and
infection in secondary lymphoid organs. Crossing the barrier, small founder other lymphatic tissues results in massive depletion of CD4 T cells in gut
populations (with the associated risk that the basic reproductive rate, R0, will lamina propria. Immune activation (upper right panel) has drawbacks in
fall below one), and local expansion are vulnerabilities for the virus in week 1 supplying activated CD4 T-cell targets for viral replication and inducing a
of infection. These vulnerabilities create opportunities for prevention Treg response that suppresses the immune response. Immune activation
targeting this stage. The male (blue) and female (pink) figures at the lower does elicit a virus-specific CD8 T-cell response that is too little and too late to
left are positioned in the time frame marked weeks, after systemic infection prevent gut CD4 T-cell depletion or clear infection. Although there are large
has been established. It is at this time that HIV-1 infection is first clinically numbers of SIV-specific CD8 T cells in, for example, lymph nodes (lower
manifest, and, hence, the need for the animal model to view sexual mucosal panel), there are also large numbers of infected cell targets so that at the
transmission and the earlier stages of infection. The larger female figure effector to target (E:T) ratio achieved (,2), infection is only partially
symbolizes the disproportionate acquisition of HIV-1 infection and focus of controlled.
the review on transmission to women.
have so far proved an inefficient way to infect animals reliably24,
events; and (4) similarities in anatomy, physiology and immunology except when facilitated by genital ulceration25. I later discuss the
of the female reproductive tract of rhesus macaques to humans, and implications and relevance of rapid dissemination from the portal
the general similarities of pathogenic SIV infection to HIV-1 infec- of entry under these conditions.
tion in CD4 T-cell depletion, pathology and AIDS14,21. Opportunities and lost opportunities. Studies in this high-dose vaginal
This animal model also has weaknesses. First, the SIV challenge challenge model support the conclusion that prevention strategies
dose exceeds the highest dose of HIV-1 to which humans are exposed should target the first week of infection both to take advantage of viral
in semen, even in the acute stage of HIV-1 infection where virus vulnerabilities and two opportunities for prevention (Fig. 1) and to avert
concentrations and transmission rates are the highest22. We thus have the ill effects of systemic infection in the second week and beyond
to assume that virus–host interactions documented in tissue analyses (Fig. 2). The first viral vulnerability and opportunity for prevention is
in the high dose challenge also occur with lower dose challenges at a related to the establishment of the small founder population, which
local level, but at such low frequencies that they would escape detec- must be sustained at a basic reproductive rate, R0, $ 1, otherwise infec-
tion in tissue samples. In further defence of the high dose challenge, tion will be aborted. Thus, early intervention measures that reduce
single genome analysis (see below) in multiple low-dose intra-rectal the growth rate to ,1 will have that desired outcome. The second
challenges that more closely approximate typical doses of HIV-1 in opportunity is preventing local expansion so that insufficient virus
semen provides evidence consistent with one of the key findings to and infected cells are produced to disseminate and establish systemic
emerge from the high dose model: that infected founder populations infection. This second opportunity for prevention may not hold for
are small as a consequence frequently of infection with a single virus rectal and oral transmission, given the evidence that dissemination
genotype23. beyond the portal of entry occurs within a few days of rectal or oral
In addition, this cell-free virus model of course cannot provide exposure, without apparent local expansion26–28.
insight into cell-associated virus transmission. An animal model of The ill effects depicted in Fig. 2 of losing those opportunities to
the latter potential mode of HIV-1 transmission would certainly be prevent systemic infection are similar in SIV and acute HIV infec-
valuable, but would have to be developed, as infected cell inocula tions, and include: massive depletion of memory CD4 T cells in the
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NATUREjVol 464j11 March 2010 REVIEWS

lamina propria of the gut mediated by direct effects of infection and Mucosal front line
apoptosis of bystander cells29–35; acute enteropathy induced by pro- Crossing the barrier. I now return to a fuller description of the
inflammatory cytokines and virotoxic effects of the virus that induce earliest steps in transmission and local infection based on direct
intestinal epithelial apoptosis36–38; and immune activation, the down- observations on cervical vaginal tissues. Virus depicted in Fig. 1
side of which includes a new supply of activated CD4 T cells to enters at anatomical sites where the mucosal barrier is most easily
support virus replication, and a T regulatory (Treg) response that breached, by mechanisms such as the microtrauma associated with
has been called premature, because the immunosuppressive effects sexual intercourse49 that provide immediate access to target cells in
that measurably dampen the SIV-specific cellular immune response the submucosa (Fig. 1). Only a single layer of columnar epithelium
precede clearance or control of viral replication39. Although immune guards the endocervix, and the transformation zone between ecto-
activation does elicit a virus-specific CD8 T-cell response, it is ‘too and endocervix is also a single layer of epithelium and a site of high cell
little and too late’ to prevent gut CD4 T-cell depletion or do more turnover that could facilitate entry50,51. These anatomical differences
than partially control infection40. and recent mapping studies of the sites where infected founder popu-
These serious ill effects on the host’s immune system in early lations were identified in vaginal transmission of SIV are consistent
infection are only the beginning of immunopathological processes with the transformation zone and endocervix as preferential sites of
set in motion at this stage that will have their greatest impact in the entry52. This hypothesis could also explain the increased acquisition of
later chronic stages of SIV and HIV-1 infection: microbial transloca- HIV-1 associated with cervical ectopy—extension of the simple
tion associated with continued damage to the gut lining that con- columnar epithelium to cover a portion of the ectocervix—observed
tributes to chronic immune activation and the continued loss of CD4 in some studies53.
T cells41; and TGF-b1 Treg cells42 that initiate a fibrotic process, which The transformation zone and endocervix, however, are not exclusive
disrupts the lymphatic tissue architecture that supports the migra- sites of entry, as SIV has been transmitted to hysterectomized
tion, growth and survival of T cells, thereby contributing both to CD4 animals54, and HIV-1 in the congenital absence of a cervix55, establish-
T-cell depletion and limiting immune reconstitution with antiretro- ing that there are target cells and independent entry mechanisms for
viral therapies43. vaginal transmission. These observations, however, do not address
Small founder population and local expansion. The evidence for a preferred sites of entry and the role of inflammation and trauma in
small, focal infected founder population of cells depicted in Fig. 1, the individual described in ref. 55; nor does the failure of diaphragms to
with local expansion as an antecedent and prerequisite for dissemi- decrease HIV-1 transmission exclude cervix as a preferred site of entry,
nation and systemic infection, is based on in vivo tissue analyses of because of the confounding effects of poor adherence in that study56.
vaginal transmission. These analyses revealed only small foci of pro- Initial target cells. In the SIV rhesus macaque model, the earliest
ductively infected cells, defined as cells with detectable SIV RNA, at 3 focal collections of infected cells are CD4 T cells with a surprising
to 4 days after inoculation16,18. Infection then expands locally before phenotype. The initial expectation from growing HIV isolates and
detection of infection in the draining lymph nodes and systemically SIV in tissue culture was that they would be macrophage-tropic and/
throughout the secondary lymphoid organs, consistent with the local or replicate in activated CD4 T cells, using the CCR5 co-receptor
expansion and staged dissemination model shown in Fig. 1. (reviewed in ref. 57). However, in vivo, it turned out that the initially
There is evidence that dendritic cells with detectable viral antigens infected cells were CD4 T cells with none of the expected markers of
reach the draining lymph nodes much earlier—18 to 24 h after expo- activation18. They were therefore called31 ‘resting’, with the implica-
sure17—but apparently the threshold for a self-propagating infection tion that they probably had some residue of co-receptor expression
is not crossed until virus and infected cells are produced locally in and prior activated state to enable them to support productive infec-
sufficient quantities to establish infection distal to the portal of entry, tion, in contrast to truly resting CD4 T cells. In the gut, a4b7 may
because the draining lymph nodes are not the site where productive facilitate entry and preferential infection of T-helper 17 CD4 T cells
infection is first detected18. Thus, dilution, trapping in cervical with this minimally activated phenotype58,59.
mucus, the physical mucosal epithelial barrier and other mechanisms Infected ‘resting’ CD4 T cells comprise about 90% of the detectably
transform exposure to a large quantity of virus in the inoculum to a productively infected cells in the initial foci and subsequent local
small founder population of infected cells that must then expand expansion at the portal of entry, and the population of infected cells
locally for a few days before systemic infection is established. that produces peak levels of virus in SIV infections18,31,60 in the
Genetic bottleneck in HIV-1 transmission. The earliest stages of lymphatic tissues in the second week of infection. Over 60% of
mucosal transmission of HIV-1 have not been directly observed, but CD4 T cells in gut with detectable SIV DNA in early infection have
we can infer parallels to SIV’s small, infected founder populations also been shown to be memory CD4 T cells with relatively low levels
from the genetic bottleneck at HIV-1 transmission. It has been known of CCR530; in early HIV-1 infection close to 90% of infected cells were
for some time that in mother-to-child and heterosexual transmission, typed as CD4 T cells and the majority had a ‘resting’ phenotype18,61.
virus isolates from the newly infected individual are genetically much Moreover, replication-competent clones derived from single genome
less diverse than viruses from the transmitter44,45. More recently, amplification of isolates in acute HIV-1 infection primarily infected
sequencing viruses in heterosexual transmission pairs and in acute CD4 T cells rather than macrophages in culture48. Thus, CD4 T cells
HIV-1 infection provided evidence that a single virus (or infected cell) are probably the principal cell type infected at the portal of entry and
initiated productive infection in close to 80% of the individuals tested, throughout the lymphatic tissue reservoir from the earliest stages of
and two to five viruses in the other 20%46,47. The implied small, infection.
infected founder populations in HIV-1 mucosal transmission again Target cell availability. One reason that this may be the case is target
indicate that the greatest opportunities for prevention are strategies cell availability. CD4 T-cell susceptible target cell populations in the
that target these initially small and genetically homogeneous foci of vagina, ecto- and endocervix in rhesus macaques60,62 and humans50,63
infection in the first week of infection. are largely spatially dispersed populations lying just beneath the epi-
However, the small size of the infected founder population does thelium, and, to a lesser extent, deeper submucosa, with some small
not imply that the transmitted virus itself is inherently any easier to focal aggregates and intraepithelial lymphocytes. There are also
contain. To the contrary, phenotypic analysis of transmitted HIV-1 macrophages and dendritic cells in the submucosa, and dendritic
revealed masking of CCR5 co-receptor binding regions, and equiva- cells within the epithelium in both species, but ‘resting’ CD4 T cells
lent or enhanced resistance to fusion inhibitors and neutralizing outnumber macrophages and dendritic cells by 4 to 5:1 and two to
antibodies compared to virus isolated from chronically infected three or more infected ‘resting’ CD4 T cells are often found clustered
individuals47,48. in the small focal clustered infected founder populations60.
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REVIEWS NATUREjVol 464j11 March 2010

These census analyses are consistent with the hypothesis that Vagina Cervix
MIP-3α
numbers and spatial proximity contribute to preferential propaga-
tion initially in this cell type60. However, target cell availability is pDCs
unlikely to be the sole determinant of the cell tropisms in early infec-
tion, because activated CD4 T cells are the other cell type most fre- Thinned/disrupted
MIP-1β
mucosal barrier
quently infected at this stage, even though there are 70 times as many Target
‘resting’ CD4 T cells and relatively more abundant CD41CCR51 cells
Pre-existing
inflammation
Interferons
Anti-viral
macrophages and dendritic cells in the vicinity. New target
cells
chemokines

Mucosal epithelium: immunity’s front line. Mucosal epithelial Fuel Restrict

defences against viral entry are conventionally portrayed as a passive infection infection

physical barrier protecting against invasion, but, in actuality, the

epithelium lining endocervix and upper female reproductive tract Local
is the active front line of the host immune system. This mucosal expansion

epithelial front line mediates innate defences against microbes under

the hormonal control of oestradiol and progesterone64–66, and func-
tions as a sentinel and signalling system with Toll-like receptors that Figure 3 | Inflammation, innate immunity, mucosal epithelial signalling
recognize and respond to pathogen-associated molecular patterns by and target cell availability at mucosal front lines. Exposure of endocervical
secreting: (1) microbicidal defensins and other antimicrobial pep- epithelium to virus, a component of virus or inoculum initiates signalling
tides; (2) secretory leukocyte protease inhibitor; (3) the microbicidal (jagged arrow) that increases expression of MIP-3a in the epithelium that
enzymes lactoferrin and lysozyme; (4) surfactant protein A; and (5) recruits pDCs. They in turn recruit, through chemokines such as MIP-1b,
complement. Mucosal epithelial signalling through chemokines and the CD4 T cells that fuel local expansion. Interferons from the pDCs and
chemokines also suppress viral replication but the balance is tipped in favour
cytokines also recruits plasmacytoid dendritic cells (pDCs)67,68 and of the virus by the cells that fuel the local expansion necessary for
other cells that mediate innate defences and inflammation, and ini- dissemination and establishment of systemic infection. Pre-existing vaginal
tiate and link innate and adaptive immunity. inflammation also facilitates infection by thinning and disrupting the
The balancing act in innate defences. The physical barrier and active multilayered lining, and providing a pool of target cells for local expansion.
mucosal front-line defences described above would be expected to
inhibit SIV and HIV entry and to provide an array of inhibitory scales in Fig. 3 are meant to convey the counterbalancing effects of the
activities against viral replication: (1) SDF-1, MIP-1a/b and innate and inflammatory response in restricting viral infection, and
RANTES to block viral entry mediated by the co-receptors CXCR4 facilitating it, through increasing target cell availability, with the
(blocked by SDF-1) and CCR5 (blocked by MIP-1a/b and greater target cell availability tipping the scales in favour of the virus.
RANTES)57; (2) MIP-3a expression in response to vaginal inocu- The influx of new target cells to fuel local expansion is also a
lation of SIV to immediately recruit interferon (IFN)-a/b producing
vulnerability that can potentially be exploited in preventing trans-
pDCs to the endocervix52; and (3) large increases in IFN-c expression
mission. Glycerol monolaurate inhibits female reproductive tract
in cervical vaginal tissues by 6 days after exposure69. These increases
epithelial signalling and may have prevented local expansion to
in inhibitory chemokines immediately after exposure and the first
thereby protect animals exposed to high doses of SIV52.
few days of infection and unknown contributions of innate defences
Inflammation and mucosal transmission. Innate defences and assoc-
cited in the preceding section presumably provide a cumulative level
iated inflammation generally facilitate transmission of SIV and HIV-1
of protection that accounts in part for the small infected founder
populations in SIV infection despite exposure to a large dose of virus. by compromising the integrity of the mucosal barrier and increasing
This innate immune response might also explain restricted cellular target cell availability. In the model illustrated in Fig. 3, pre-existing
tropisms in vivo, for example protection of IFN-producing pDCs inflammation is shown in association with thinned and disrupted
from infection. In HIV-1 transmission, similar defence mechanisms vaginal epithelium. This provides immediate access to large numbers
probably also contribute to the generally low probability of vaginal of target cells in the underlying submucosa, and thus the association of
transmission of HIV-1 of ,1 in 100 to 1,000, depending on the viral initial vaginal SIV infections at such a site52. The seemingly paradoxical
load of the transmitting partner70,71. enhancing effects of imiquimod and CpG ODN74 applied vaginally on
Paradoxically, these innate antiviral and inflammatory defence SIV transmission may also be explained by the effects on mucosal
mechanisms in SIV and HIV-1 infections at the same time may integrity and target cell availability of inflammation induced by these
facilitate transmission, by increasing target cell availability, and by Toll-like receptor agonists.
creating conditions for highly efficient cell-to-cell spread of infection Pre-existing genital infections and ulcers may facilitate systemic
where HIV-1 replication has been shown to become less sensitive to infection not only by effects on the mucosal barrier and target cell
inhibition by interferon72. Mapping local expansion52 revealed availability, but also by providing a haematogeneous route for imme-
growth by accretion of newly infected cells around foci of infected diate systemic spread. For example, SIV-infected and other cells
founder cells, and spread of infection along tracts of infiltrating inoculated vaginally were quickly disseminated throughout the
inflammatory cells. These findings led to the model shown in Fig. 3: lymphatic tissues in the setting of experimentally induced genital
SIV overcomes the problem of replication in the low density and ulceration25.
dispersed populations of CD4 T cells at the portal of entry through Co-existing genital infections also increase HIV-1 acquisition
exploiting the innate immune and inflammatory response that brings through macro- or micro-ulcerations compromising the integrity of
in large numbers of target cells to create a generally favourable environ- the mucosal barrier and the increased availability of target cells asso-
ment to fuel expansion wherever the initial focus is established, and to ciated with these infections75,76. These conditions relieve the genetic
generate conditions where SIV has been shown to propagate most bottleneck described above, such that multiple genetic variants rather
efficiently by direct cell-to-cell spread or at close range73. than one or two are transmitted to individuals with underlying genital
The mechanism inducing the influx of target cells fits nicely with the infections77.
concept of mucosal epithelium functioning as a sentinel and signalling Target cell availability may also explain the failure of acyclovir treat-
system65. In this case, exposure to virus or other components of the ment of herpes simplex virus 2 (HSV-2) infection to reduce the assoc-
inoculum stimulates increased expression of MIP-3a in the endocer- iated acquisition of HIV-1, despite decreased shedding and healing of
vical epithelium, attracting pDCs beneath the epithelium, which in HSV-2 ulcers78,79. This result, in line with the generally disappointing
turn recruit CD4 T cells through MIP-1b and other chemokines52. The results in reducing HIV-1 acquisition by treating sexually transmitted
220
©2010 Macmillan Publishers Limited. All rights reserved
NATUREjVol 464j11 March 2010 REVIEWS

infections75,76, can now be attributed to persistence of enriched target about immunological memory. It may be that a vaccine would have
cell populations after ulcer healing80. to induce and maintain a population of effectors at the portal of entry
Seminal contributions to transmission. Even without pre-existing to be up to the challenge. This is more likely to be the case for rectal
inflammation, exposure of the mucosal epithelium to semen initiates and oral transmission where infection is also even more quickly
signalling to orchestrate the necessary changes in the female re- disseminated26–28.
productive tract for tolerance of allogeneic sperm, implantation Consistent with the concept of a protective mucosal population of
and development of the fetus81,82 that may also facilitate transmis- immune effectors, vaccine-induced high-avidity CTLs have been
sion. Exposure of cervical vaginal epithelium to semen elicits shown to delay simian-human immunodeficiency virus (SHIV) dis-
increases in chemokines such as MIP-3a83 (similar to the effect of semination from mucosa following rectal challenge86, and prior
exposure to the viral inoculum in experimental SIV infection52) and infection of rhesus macaques with an attenuated SHIV has been
pro-inflammatory cytokines (GM-CSF, IL-1, IL-6 and IL-8) that shown to provide protection against vaginal challenge associated
recruit neutrophils, dendritic cells, macrophages and lymphocytes, with SIV-specific CTLs in cervical vaginal tissues87. Furthermore, a
which accumulate beneath the cervical and uterine epithelium. Thus, persistent prior infection with rhesus cytomegalovirus expressing
a microenvironment conducive to transmission is created by the SIV antigens that elicited and maintained virus-specific effector
recruited cells, the immunosuppressive effects of TGF-b and pros- memory CD4 and CD8 T cells at extra-lymphoid sites has recently
taglandin E in semen, other transmission-enhancing factors in semen been shown to provide partial protection against repeated intra-
such as the amyloid fibres derived from prostatic acid phosphatase84, rectal challenge88.
alterations in mucosal integrity effected by neutrophils migrating More generally, measures that target the establishment and expan-
through the epithelium, and increased target cell availability sion of small founder populations in the earliest stages of infection
mediated by mucosal epithelial signalling. are most likely to be effective in prevention (Fig. 1). Thus, pharmaco-
logical interventions with antiretroviral therapies within the window
Mucosal cellular immune response in which the infected founder population has just been established
Too little and too late. I now turn from early infection and virus can prevent or increase control and moderate the pathological con-
interactions with innate defences to the adaptive immune response. sequences of infection89. Microbicides that block binding and co-
Even though this response is not detectable until the end of the receptor-mediated entry and reverse transcription have been shown
second week of infection40, its timing, location and magnitude are to protect against SHIV and SIV vaginal and rectal challenges in the
still instructive for designing effective preventive measures. rhesus macaque model90–92. Similarly, moderating the growth in
The cellular immune response that has so far been characterized target cells available to fuel the local expansion on which the virus
follows antigen expansion and peak replication, and therefore has depends can also prevent vaginal transmission52.
been described40 as ‘‘too late and too little’’, because it is too late to
clear virus locally or prevent systemic spread, and insufficient in Challenges and gaps in knowledge
magnitude to prevent the massive CD4 T-cell depletion in gut and
Studies described herein reveal both challenges in preventing mucosal
other pathological consequences of early infection. Nonetheless, the
HIV-1 transmission and opportunities to do so, particularly with
robustness of the immune response in specific tissue compartments
strategies targeting early infection. Designing and assessing the effec-
is a determinant of the extent of control of viral replication and thus
tiveness of countermeasures would be informed and enabled by
rate of disease progression.
addressing the following six issues.
Tissue-compartment-specific partial control. The changing rela-
tionships over time in numbers and locations of immune effectors— First, discovering ways to generate a resident or rapidly responding
virus-specific CD8 T cells—and their infected targets in infected tissues mucosal population to eliminate the small founder population at the
can be visualized and quantified by combining in situ tetramer staining portal of entry that avoids the immunosuppressive and target cell
of the effectors with in situ hybridization to detect viral RNA1 cells85. availability problems inherent in the immune activation necessary for
This analysis, illustrated in Fig. 2, provides images of the spatial pro- that response.
ximity and conjugates of effectors and targets, and a direct way to Second, identifying correlates of prevention in the recent Thai
determine the in vivo effector/target ratio. It is immediately apparent trial7 of a two-component vaccine in which neither component alone
in this infected lymph node that there are not only large numbers of had previously shown efficacy.
effectors but also large numbers of targets, so the effector/target ratio is Third, monitoring occult infections in trials of vaccines and micro-
,2. Such a low effector/target ratio correlates with poor control of viral bicides. Occult infections are defined as HIV-1-exposed individuals
replication—measured as reduction from peak viral load. By contrast, who remain seronegative but who have detectable cellular immune
at high effector/target ratios $50–100:1 achieved at 3 weeks after responses to HIV-1 and DNA in CD4 T cells93; or rhesus macaques
vaginal exposure in cervical vaginal tissues, there was a commensurate repeatedly challenged vaginally or rectally with low or high doses of
,100-fold reduction in viral load. SIV94–96 that develop transient viraemia with low to undetectable
This analysis tells us that the extent of control by SIV-specific CD8 antibodies or cellular immune response, and have detectable provirus
T cells is tissue-compartment-specific and related to the relative in vaginal and lymphoid tissues94,96. They may later be manifest as
numbers of both effectors and targets. The opportunities for preven- typical pathogenic systemic infections, have been documented after
tion in the earliest stages of infection tell us that both timing and apparent protection with a microbicide52, and thus represent a poten-
location may be critical determinants of outcome. When infected tial public health issue and point to the need for long-term follow up in
founder populations are small and focal, and have not as yet testing vaccine and microbicide candidates.
expanded sufficiently to have disseminated and established systemic Fourth, defining mechanism(s) by which circumcision does or
infection, a relatively small number of effectors at the mucosal site of does not protect4–6, and the lack of circumcision facilitates male
entry might be at the right place at the right time to be ‘enough and transmission.
soon enough’ to clear infection85. Fifth, developing and investigating the following in NHP models:
Targeting early infection. The ‘soon enough’ part of this concept (1) cell-free virus transmission under conditions that more closely
implies that a very rapid local recall response and local expansion of approximate human exposures; (2) cell-associated viral transmis-
virus-specific cytotoxic T lymphocytes (CTLs) might eradicate founder sion; and (3) each route of mucosal transmission—vaginal, rectal,
populations before local expansion spreads infection. Whether the oral and penile—which will probably differ, based on their distinct
few days available in vaginal transmission would be sufficient or not anatomy and functions, both in local propagation and routes and
is unclear from experience with conventional vaccines and ideas speed of systemic spread.
221
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REVIEWS NATUREjVol 464j11 March 2010

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6234–6246 (2009). Acknowledgements I thank J. V. Carlis, R. P. Johnson, Q. Li, J. D. Lifson,
74. Wang, Y. et al. The toll-like receptor 7 (TLR7) agonist, imiquimod and the TLR9 D. Masopust, S. Pambuccian, P. J. Southern, J. Estes, D. Douek, H. W. Virgin and
agonist, CpG ODN, induce antiviral cytokines and chemokines but do not prevent B. D. Walker for discussions. Errors of commission are mine as are errors of
vaginal transmission of simian immunodeficiency virus when applied omission, with apologies to the authors of work that I could not cite because of
intravaginally to rhesus macaques. J. Virol. 79, 14355–14370 (2005). space limitations and exclusive focus on tissue analyses. I thank C. O’Neill and
75. Glavin, S. R. & Cohen, M. S. The role of sexually transmitted diseases in HIV T. Leonard for help with the manuscript and figures. Work from my laboratory cited
transmission. Nature Rev. Microbiol. 2, 33–42 (2004). in the review was supported by grants from the National Institutes of Health (AI
76. Kaul, R. et al. The genital tract immune milieu: an important determinant of HIV 38565, AI 48484, AI 71976) and the International AIDS Vaccine Initiative.
susceptibility and secondary transmission. J. Reprod. Immunol. 77, 32–40 (2008).
77. Haaland, R. E. et al. Inflammatory genital infections mitigate a severe genetic Author Information Reprints and permissions information is available at
bottleneck in heterosexual transmission of subtype A and C HIV-1. PLoS Pathog. 5, www.nature.com/reprints. The author declares no competing financial interests.
1–13 (2009). Correspondence should be addressed to the author (haase001@umn.edu).

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 Vol 464j11 March 2010jdoi:10.1038/nature08898

PERSPECTIVES
Immunology and the elusive AIDS vaccine
Herbert W. Virgin1 & Bruce D. Walker2
Developing a human immunodeficiency virus (HIV) vaccine is critical to end the global acquired immunodeficiency syndrome
(AIDS) epidemic, but many question whether this goal is achievable. Natural immunity is not protective, and despite
immunogenicity of HIV vaccine candidates, human trials have exclusively yielded disappointing results. Nevertheless, there
is an indication that success may be possible, but this will be dependent on understanding the antiviral immune response in
unprecedented depth to identify and engineer the types of immunity required. Here we outline fundamental immunological
questions that need to be answered to develop a protective HIV vaccine, and the immediate need to harness a much broader
scientific community to achieve this goal.

nti-HIV drugs have extended the lives of millions of Gaps in knowledge

A infected people, but the epidemic continues its course,

with high rates of new infections in regions least able to
cope medically, socially and financially. For example, in
some particularly hard hit areas infection rates in young women soar
from less than 1% at age 15 to in excess of 50% by their mid-twenties.
Given the failure of empirical HIV vaccine approaches, understanding
the nature of the immune response needed for protection is the essential
missing ingredient. We still lack fundamental knowledge regarding the
nature, quality and quantity of immune responses that should be
induced, the ideal antigens to include, how to overcome the tremendous
A vaccine will be required to ultimately conquer AIDS, but many sequence variability engendered by the error-prone HIV reverse tran-
scientists believe that this is probably decades away, if even possible. scriptase, or even whether preventive vaccine strategies should focus on
Thus far, HIV vaccine trials in humans have resulted in either no or protection from infection or protection from disease progression.
relatively unimpressive protection despite measureable immuno- Classically, immunization has depended on programming memory
genicity of administered HIV antigens1–4. Indeed, natural immunity T and B cells to remember encounter with antigen. The current
itself is far from protective, inducing both humoral and cellular vaccine failures indicate that this is not sufficient, and that immuni-
immune responses but not leading to spontaneous clearance or pro- zation strategies need now to be engineered with close attention to the
tecting against HIV superinfection5. Therefore, simply generating an type and differentiation state of the immune response generated,
immune response similar to what is generated in natural infection is the efficacy of vaccine-induced effector mechanisms against specific
unlikely to immunize effectively against HIV. This conclusion sup- vulnerable steps in the pathogenesis of HIV infection, the capacity of
ports the view that our fundamental approach to HIV vaccination the induced response to maintain activity at relevant body surfaces, the
needs to be re-examined. influence of the virome and microbiome on vaccine responses, the
We believe that the development of an effective HIV vaccine is an fitness cost of mutations forced on HIV by specific immune responses,
achievable goal, and that there are many ‘knowable unknowns’ that the capacity of vaccine-generated immune responses to avoid exhaus-
can be addressed immediately to advance this effort. The goal of this tion, and the capacity of the different parts of the immune response
Perspective is to outline progress from studies of HIV and the closely generated by vaccine to synergize with one another at the relevant sites.
related AIDS-inducing monkey retrovirus simian immunodeficiency Because HIV initially establishes infection in a very small number of
virus (SIV), together with insights derived from studies of other cells6, appropriate pre-existing immunity has the potential to prevent
viruses, showing that some level of control of lentiviruses can be infection, clear infection, or limit replication so that the set point of
achieved. This suggests that there are immune mechanisms that, if viraemia is low enough during chronic infection to prevent progres-
induced by a vaccine, may prevent HIV infection or at least limit the sion to AIDS and inhibit spread from one person to another. A better
level of viraemia in infected people. In addition to the critical value in understanding of the molecular mechanisms responsible for immune
continuing and expanding current efforts to test vaccine candidates control of acute and chronic viral infections, and immune interactions
in humans, we emphasize the importance of a long-term plan to with HIV at various stages of disease (Fig. 1 and ref. 6) will be needed to
enhance understanding of basic immunological mechanisms that direct effective vaccine strategies to meet this desperate global need.
may be able to prevent amplification and systemic spread from the
limited initial nidus of HIV infection (see the accompanying paper6). Signals that vaccination may work
We need better integration of novel concepts and information on There are, finally, weak signals from a human vaccine trial, and more
new immune system genes and mechanisms derived from work in robust findings from efforts to vaccinate against SIV, indicating that a
model systems into vaccine science. These advances need to be measure of protective immunity to HIV or SIV can be induced. A recent
rapidly translated into the HIV field to optimize chances for effective phase III trial combining an HIV envelope protein immunogen with a
vaccination against AIDS. Achieving this goal will be greatly facili- recombinant canarypox-HIV vector suggested an encouraging (albeit
tated by engagement of scientists from diverse but currently isolated insufficient) 30% reduction in acquisition1. In monkeys, induction of
disciplines, who, by providing new insights, can accelerate progress virus-specific CD8 T-cell responses does not prevent infection, but if
towards an effective vaccine. strong and broad enough, can curtail SIV replication after homologous
1
Washington University School of Medicine and Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research, Campus Box 8118, 660 South Euclid
Avenue, Saint Louis, Missouri 63110, USA. 2Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Howard Hughes
Medical Institute, 149 13th Street, Charlestown, Massachusetts 02129, USA.

224
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NATUREjVol 464j11 March 2010 PERSPECTIVES

Figure 1 | HIV pathogenesis and outstanding

What form(s) of HIV spreads? questions related to vaccination. Shown are the
Free HIV? Cell-associated HIV? steps in HIV pathogenesis and ‘knowable
HIV virus unknowns’ that, when defined, may alter how we
Is HIV pathogenesis or immunity
different at different mucosal sites?
approach vaccination against HIV. APC, antigen-
presenting cell.

Does compression of the quasispecies

associated with transmission have
CD4 T cell Dendritic cell
implications for vaccines?
Does HIV productively infect and/or
impair the function of APCs?
Can HIV latency be
Do innate iimmune mechanisms How are APCs optimally licensed prevented or eliminated?
c acquisition?
affect for induction of immunity?

Replicate in Viraemia
de
How does HIV evade HI be cleared by
Can HIV
mucosa nity once
immunity o infection
?
vaccine immunity?
i established?
is estab
t b

Blood

What determines HIV fitness?

ess?
For transmission?
Secondary infection ty?
For evasion of immunity?
lymphoid organs For establishment of set point?
Are specific viral genotypes
associated with elite controllers?
CD4 T-cell depletion
Lymph
node

AIDS

or heterologous virus challenge7,8. Use of simian cytomegalovirus vaccination (Fig. 2). For example, about one in 300 people maintain
(CMV) as a vector to continuously deliver SIV antigens limits systemic extremely low HIV loads without treatment, some for more than 30
infection in some animals after intrarectal challenge with SIV9. These years14, and HIV-2 pathogenesis is similar to HIV-1 pathogenesis and
and similar studies show that exposure to lentivirus antigens can lead to yet causes disease far less frequently15. Similarly, SIV can cause high-
resistance to or control of infection and provide controlled models to level viraemia, infect and deplete CD4 cells, and yet not cause AIDS16.
address substantial gaps in our knowledge of what immune mechan- Together these data indicate that there are as-yet-unidentified viral or
isms are responsible for the observed effects. host factors that attenuate lentivirus pathogenesis. Identification of
Encouragement afforded by these vaccine results is enhanced by these may define the type of immune responses needed to reach the
studies of natural transmission that indicate that induction of even goal of vaccinating to prevent disease progression and spread within
modest protective immunity at mucosal surfaces may tip the balance the population (Fig. 2). A key step in this direction would be iden-
towards less efficient spread of HIV. Only one-third of infants born to tification of novel molecules involved in control of chronic virus
infected mothers acquire infection; sexual transmission occurs after infection in model systems, and rapid translation of such new findings
between 1 out of 100 and 1 out of 1,000 exposures (although the risk for detailed analysis during HIV and SIV infection.
may be far higher when the transmitting partner is acutely infected)10;
exposure to infected blood products does not consistently lead to Fight using all the tools immunity offers
infection11; commercial sex workers may remain uninfected despite Current evidence overwhelmingly argues for vaccines that simulta-
extensive exposure12; and most infections are initiated by a single neously elicit effective CD4, CD8 and B-cell responses; however, an
virus13. These data indicate that the induction of even modest pro- integrated understanding of the relationships between innate and
tective immunity at mucosal surfaces before infection may be effec- adaptive immune responses to HIV antigens, and how this might
tive at inhibiting spread of HIV. Indeed, this may be the mechanism lead to enhanced immunization, is yet to be achieved (Fig. 3).
of apparent protection in the recent Thai vaccine trial, which limited Furthermore, although it is clear that innate immune mechanisms
HIV acquisition but had no effect on set-point viral load once contribute to control of HIV17, it is not clear whether it is possible to
systemic infection was established. Detailed studies of the earliest harness this response via vaccination. Indeed, the HIV vaccine field
events in transmission in animal models and humans are needed to has been largely divided into B-cell approaches and CD8 T-cell
define the mechanisms that explain resistance to infection in the vast approaches. There is little evidence that any vaccine achieves protec-
majority of HIV exposures. tion through antibody alone, or ever prevents initial infection
entirely; the same holds for T-cell responses. Antibody functions
Détente teaches us how to go to war optimally in the setting of simultaneous CD4 T-cell responses that
Not all HIV infections lead to AIDS, indicating that it is possible for provide help; CD4 and CD8 responses are probably needed to limit
immunity to establish a very long-term détente with lentiviruses; or eliminate HIV replication at the initial site of infection through
the mechanisms involved might provide insight into the goals for innate and adaptive mechanisms. Similarly, T-cell vaccines largely
225
©2010 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES NATUREjVol 464j11 March 2010

HIV: Immune people unavailable, on SIV replication25, indicating that previous associations between
Define correlates elite controllers limit progression
of immunity SIV: Protection from acquisition and CD4 T-cell activation and increased viraemia are correlative only20.
lowering of set point demonstrated,
mechanisms unknown
Furthermore, CD4 T cells can provide direct antiviral effects24, and
the presence of strong virus-specific CD4 T-cell responses distinguishes
HIV: Uncertain
Choose
se desired
SIV: Neutralizing antibody,
non-pathogenic HIV-2 infection from HIV-1 infection15. Possible sup-
immune responses
es
CD8 T cells, CD4 T cells port for a role for immune CD4 T cells in vaccination may be found in
the Thai HIV vaccine trial results, in which decreased HIV acquisition
Vaccine goal
was observed with a vaccine that induces HIV-specific CD4 T cells1.
Prevent infection? Vaccine strategy Vaccine New data, A much greater emphasis on understanding the antiviral effector
Limit set point? development assess correlates,
Prevent progression?
Vector?
Adjuvant? and trial revise approach
properties of CD4 T cells is needed, and the role of these cells in control
Prevent spread? Dose? of HIV at mucosal sites and vaccination must be one of the highest
Frequency?
priorities for future studies.

Choose antigen HIV: Highly variable B cells and antibody come back into focus
targets rapid mutation
Animal challenge studies indicate that a pre-existing neutralizing
antibody response can prevent AIDS virus infection. Notably, a signifi-
Define genome HIV: Well described,
moving target as
cant minority of HIV infections generate heterogeneous mixtures of
and proteome epidemic expands polyclonal high-affinity broadly neutralizing antibodies to multiple
Figure 2 | Approach to vaccination and special challenges of HIV different envelope epitopes26, giving hope that properly formulated
vaccination. Shown are the steps in development of a vaccine strategy for vaccines might generate protective responses. However, it is unknown
HIV. In blue are the key issues corresponding to each step that influence our whether vaccine-induced elicitation of such antibodies can counter
capacity to immunize against HIV. enormous and growing HIV quasispecies diversity and prevent initial
infection on exposure. So far, vaccine-elicited antibody responses have
focused on CD8 T-cell responses7,8 are unlikely to succeed without not been protective and the form of the virus (free versus intracellular)
antibody and HIV-specific CD4 T-cell responses9,18. Even if we knew that spreads is unknown (Fig. 1) so that epitopes exposed during
what we wanted to induce in a vaccine, the science of engineering natural transmission are undefined. Moreover, the envelope glyco-
specific immune responses to exceed the capacity of the natural protein is very difficult to produce in its folded trimeric form; this
immune response is in its infancy. A key step in this important may be required to provide an effective immunogen. Furthermore,
direction will be to define relationships and potential synergies because HIV spread between cells may occur by means of multiple
between CD4 T-cell, CD8 T-cell, antibody and B-cell responses mechanisms, including virological synapse-mediated cell adhesion
during vaccination and in situations in which long-term viral control coupled to viral endocytosis, it is not clear that antibody will be able
is achieved in infected people. to inhibit all forms of viral spread in vivo27.
As only a tiny subset of an infected person’s HIV quasispecies
The case for re-embracing CD4 T cells transmits6, the relationship between the diversity of sequences of
the surface glycoprotein gp120 worldwide and appropriate vaccine
There is much controversy concerning the role of HIV-specific CD4
epitopes is unknown, but it is possible that much of this diversity is
T cells in control versus promotion of HIV replication. HIV repli-
irrelevant to immunogen design28,29. Recent results from large popu-
cates best in vitro in activated CD4 T cells, there is evidence for
lation screening studies have revealed the presence of novel broadly
selective infection of HIV-specific CD4 T cells by HIV19, and a cor-
neutralizing antibodies in some infected people, and provide important
relation between SIV recrudescence and CD4 T-cell activation after
new avenues for antigen design through targeting these epitopes that
depletion of CD8 T cells has been reported20. Such observations are subdominant or entirely unrecognized in the majority of infected
support concerns that induction of a large population of HIV- persons (for example, see refs 28, 30). Reasonable levels of one of the
immune CD4 T cells might enhance rather than control HIV infec- few available broadly neutralizing monoclonal antibodies blocks low-
tion. This view is countered by overwhelming evidence that CD4 T dose intravaginal challenge with SIV using the HIV gp120 (SHIV)31,32,
cells are essential for antiviral responses in vivo including CD8 T-cell and encouraging recent studies show that complete protection from
responses and effective B-cell responses in every system in which it SIV challenge has been achieved by adeno-associated virus (AAV)-
has been studied (for example, see refs 21–23 and references therein). mediated continuous endogenous expression of immunoadhesins
For example, CD8 T cells do not efficiently access mucosal sites to derived from neutralizing antibodies33.
combat viral infection without CD4 T-cell help18, and the outcome of Even if protective B-cell epitopes are found, the mechanisms
retroviral infection depends critically on speed and extent of virus- responsible for antibody action in vivo need to be much better under-
specific CD4 T-cell responses24. The basic rules of immunology there- stood to foster induction of a polyfunctional humoral response
fore indicate that it will be impossible to generate long-lived, stable, (Fig. 3)30,34,35,. Meeting this need is complicated by the lack of sur-
high-level vaccine-induced immunity to HIV without inducing a rogate in vitro assays, other than virus neutralization, for protection.
protective CD4 T-cell response, but most HIV vaccine strategies have We may be missing epitopes that generate antibodies that protect
not specifically focused on inducing these responses. through antibody-dependent cell-mediated cytotoxicity, complement
Current data strongly suggest that HIV-specific CD4 T cells are fixation, or the capacity to block infection without neutralizing, as
beneficial rather than detrimental. The initial burst of HIV and SIV observed for other viruses (see refs 36–39 and references therein).
replication during acute infection does not occur in HIV- or SIV- Antibodies may also be important through interaction with Fc recep-
specific CD4 T cells6, and whereas infection of these cells could con- tors on granulocytes, macrophages, dendritic cells, or B cells to alter
ceivably add fuel to this early fire, a strong effector-memory CD4 T-cell antigen presentation, tropism, or replication in vivo36. Furthermore,
response is associated with diminished, not enhanced, SIV replication B cells are more than simply factories for making antibody, as B cells
after intrarectal challenge9. Moreover, although HIV preferentially unable to make antiviral antibody exert T-cell-dependent control of
infects HIV-specific CD4 cells, the overall magnitude of this increase chronic virus infection in mice40. Efficient antibody-mediated control
is small, and the great majority of HIV-specific CD4 T cells are not of Friend leukaemia retrovirus requires CD4 and CD8 T-cell responses
infected in vivo, even in the presence of high-level viraemia19. Blockade and is regulated by specific major histocompatibility complex (MHC)
of CD4 T-cell activation after CD8 T-cell depletion using anti- alleles37,41. This may be particularly relevant to AIDS as specific MHC
interleukin (IL)-15 antibody in SIV-infected macaques has no effect alleles are associated with more effective immune control of HIV
226
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NATUREjVol 464j11 March 2010 PERSPECTIVES

Can anybody prevent infection? Figure 3 | Immune responses to HIV and

Can the envelope be expressed as Is neutralization the only relevant
outstanding questions related to vaccination.
an antigen in authentic form? assay for protective antibody Shown are the steps in the immune response to
responses to HIV? HIV. For each component of the immune
How do we generate broadly response, colour-matched ‘knowable unknowns’
Can the initial conditions of vaccination: cross-protective antibodies? are specified which, when defined, may alter how
Enhance mucosal response? Antibody we approach vaccination against HIV.
Enhance memory? Would a strong antibody response
Deal with HIV diversity? improve the efficacy of T cells?
Generate effector cells present constantly? B cell How do we optimize antibody
Prevent T-cell or B-cell exhaustion?
responses at mucosal surfaces
Generate relevant effector mechansims?
where they are needed?
Essential help Can the immune system clear
established infection by targeting
replication and latently infected cells?
APC CD4 Target
T cell cell Are there undiscovered immune
effector functions relevant to HIV?
CD4?
C CD8? Antibody? Innate immunity?
Essential help
Is it possible to target T cell and antibody
Can APCs be altered epitopes that extract a high cost in viral
or targeted to optimize fitness when they mutate?
effective T-cell responses? CD8
T cell Can the most potent antiviral CD8 T-cell
responses be defined?
Are T-cell mechanisms operative
to control set point relevant correlates of Would pre-exisiting immune CD4 T cells
protection for vaccine development? help or hurt immune control of HIV infection?

How can we generate the optimal CD4 helper

response for protective antibody and CD8 responses?
How do we get immune T cells to the Are there undiscovered mechanisms
mucosal surfaces where they are needed? of T-cell antigen recognition?

infection. Unexplored interactions between B cells, antibody res- the first critical hours of infection in the intestine or genitourinary
ponses and T cells are knowable unknowns that are probably critical tract (Fig. 1 and ref. 6). A major opportunity to engineer vaccine
to HIV vaccine strategies (Fig. 3). responses comes from studies in mice and non-human primates
A major future challenge will be converting information on pro- showing that the immune system is dampened during persistent
tective epitopes and antibody-mediated mechanisms of protection virus infection, generating ‘exhausted’ T cells to prevent tissue
into protective vaccine antigens. Antibodies to HIV often target damage (reviewed in ref. 22). The key observation for vaccine design
accessible gp120 epitopes that can mutate without fitness cost rather is that manipulation of the mechanisms responsible for exhaustion at
than subdominant epitopes that may be more conserved and thus the time of first antigen exposure can fundamentally alter vaccine
more targetable. A fundamental question is how to generate CD4 responses.
T-helper cell activity for vaccine antibody responses to potentially Exhausted cells express downregulatory receptors, lack multiple
protective subdominant epitopes. One approach to generating effector functions, and show weak proliferative responses. Exhaus-
protective responses to subdominant epitopes is inoculation tion is best studied in T cells, but may also affect B cells50,51. For T cells,
with viral vectors that express blocking antibody-like molecules33. exhaustion is mediated by factors including FOXP3-positive regula-
Alternatively, the HIV glycoprotein may have to be engineered to tory T (Treg) cells, inhibitory receptors such as PD1 and CTLA4 and
optimize responses to cross-protective subdominant epitopes. others22,48, and cytokines such as IL-10 (refs 52, 53) or transforming
growth factor-b54. In mice infected with lymphocytic choriomenin-
CD8 T cells are important yet insufficient gitis virus (LCMV) recent data show that IL-21, perhaps made by
The failure of the first T-cell HIV vaccine in humans2,3 indicates that CD4 T cells, prevents exhaustion55–57. Blockade of IL-10 or PD1
more needs to be understood about the breadth, specificity and quality increases control of chronic LCMV and or SIV infection52,58,59. This
of protective CD8 responses to HIV to optimize vaccine design is relevant to vaccine strategies as control of chronic LCMV infection
(Fig. 3). Targeting of Gag epitopes is associated with lower viral loads is enhanced by either blockade of IL-10 during therapeutic DNA
than targeting Env, a finding that needs to be evaluated in SIV models vaccination52 or blockade of PD1 during vaccinia vector vaccina-
to assess whether this should inform vaccine design42. Some SIV- and tion60. These data support linking studies of immunoregulatory
HIV-specific T cells are surprisingly ineffective at killing virus-infected mechanisms operating during chronic infection to vaccination
targets43,44, and the relevance of killing to the protective activity of CD8 efforts. IL-10 and PD1 inhibit CD8 responses by independent
T cells in vivo remains unknown. Immunoregulatory networks that mechanisms; simultaneous blockade of both may be advantageous53.
exhaust adaptive CD4 and CD8 responses during chronic infection New data show that administration of the ‘immunosuppressive’ drug
probably contribute to CD8 ineffectiveness45–48. Studies using assays rapamycin counterintuitively enhances memory CD8 T-cell differ-
that more closely replicate the in vivo situation in which viral antigens entiation61, indicating that pharmacological manipulations at the time
are processed and presented by primary cells rather than being pro- of vaccination may enhance differentiation of induced T or B cells.
vided as synthetic peptides at supra-physiological doses are urgently Administration of antigen in specific contexts such as via dendritic
needed, along with high-throughput single-cell analysis of T-cell phe- cells may induce immune responses that can enhance chronic
notypes and gene expression, to define differences between effective immune control of HIV62. This striking finding indicates that similar
and ineffective T-cell responses49. approaches to vaccination might induce immune responses that
either eliminate the initial nidus of HIV infection or limit replication
Tuning the immune system to fight AIDS and thus control set point viraemia. Similarly, one might argue to
To vaccinate against HIV we need to induce B and T cells that have block induction of Treg cells during vaccination. However, counter-
and retain very specific differentiated properties allowing them to intuitively, elimination of Treg cells can blunt the mucosal immune
control replication of established infection and to function during response and control of vaginal infection with herpes simplex virus, a
227
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PERSPECTIVES NATUREjVol 464j11 March 2010

finding potentially related to altering the balance between mucosal of correlates of HIV immunity, are yet to be defined. Translocated
and lymphatic responses63. This provides a cautionary note as one intestinal products may not be the entire cause of systemic immune
may not be able to translate all mechanisms of chronic control of HIV activation. It has been presumed that standard diagnostic tests rule
to the vaccination effort. Another major issue is how to use adjuvants, out a role for pathogens in HIV- and SIV-related enteropathy, but
which may, in addition to enhancing the overall immunogenicity of this is not tenable as metagenomic analyses reveal many novel enteric
an antigen, be used to direct and optimize the differentiation of cells viruses in humans and primates (for example, see refs 73, 74) whose
responding to vaccine antigens. Here again are knowable unknowns: role in HIV/AIDS has not been assessed. Furthermore, monkeys
it is important to determine how to engineer augmented vaccine harbour widely variant populations of intestinal bacteria that may
responses via manipulation of immunoregulatory pathways during influence progression to AIDS and systemic vaccine responses75. The
vaccination (Fig. 3). virome and microbiome of the host have profound effects on the
nature of the mucosal and systemic immune response to new patho-
Battling HIV in mucosal tissues gens, including the basal level of activation of innate immune cells and
HIV delivers a near-lethal hit to the host immune system by killing a the development of T-cell subtypes in the intestinal mucosa22,69,76.
huge proportion of T cells in the gastrointestinal tract (ref. 6 and Future studies of immune cell function in HIV-infected individuals
Fig. 1), and persistent viraemia may feed off of systemic immune and of HIV vaccine responses need to be designed while bearing in
activation associated with mucosal damage during HIV infection. mind the potential role of the microbiome and virome in determining
Mucosal events may contribute to HIV and SIV progression, vaccine responses.
variation in vaccine responses, and variations in measurements of
surrogate markers of protection. Continuous activation of mucosal Exploiting evolution in a vaccine trap
HIV-immune cells may be required for protective vaccine responses HIV sequences have tremendous diversity; circulating HIV isolates
given the speed of mucosal HIV pathogenesis6. The immune effector exhibit up to 35% sequence variation in gp120 (ref. 77) and tremendous
mechanisms that target HIV in the mucosa are unknown, and rela- variability is generated in an individual after HIV passes through the
tionships between mucosal and systemic immunity are poorly under- transmission bottleneck6,13,78. The HIV quasispecies probably has
stood, leading to uncertainty over the benefits of mucosal versus pathogenetic properties independent of the capacity to mutate to evade
systemic vaccination. Tissue-specific immune responses have a key immunity. For example, poliovirus studies show that a quasispecies can
role in control of viral infections22,64,65, and thus differences among confer pathogenetic properties such as neurotropism and virulence
the vagina, penis, intestine and oropharynx might determine vaccine during viral pathogenesis79.
efficacy. Furthermore, emerging evidence from humans and mice The tremendous variability of HIV also exposes some weaknesses.
suggests that specific mechanisms maintain local immunity in skin In simultaneously infected adult identical twins, the earliest CD8
and genital tissues64,65. As studies of HIV or SIV immunity typically epitope escape mutations arose at the same nucleotide in both indi-
analyse peripheral blood cells, we may be working from an inaccurate viduals, indicating that there are constraints on HIV evolution, and
view of immunity to HIV and SIV. An increased focus on the role of that some escape pathways are detrimental to viral fitness80. Popula-
mucosal events in HIV vaccination is therefore needed. tion studies show predictable pathways to escape within epitopes
Although appropriate emphasis has been placed on local res- presented by specific HLA alleles81,82. These mutations, particularly
ponses, there is nevertheless convincing evidence of effective induc- in Gag, can impair viral fitness83,84. It is perhaps not surprising that
tion of mucosal responses to enteric murine norovirus infection and CD8 T cells targeting epitopes in Gag are associated with lower viral
human papilloma virus after subcutaneous vaccination66,67, for load than CD8 T cells targeting the more mutable gp120 (ref. 42).
mucosal protection against SIV after subcutaneous vaccination with Part of the role of HLA B*57 in people able to control HIV replication
a CMV-based vector9, and for generation of substantial mucosal to undetectable levels without medication (‘elite controllers’) seems
responses after systemic immunization in mice and macaques68. to be generation of fitness-impairing epitope mutations that cannot
Thus, systemic vaccination can elicit effective mucosal immunity, easily be compensated83,85. Such CD8 T-cell-induced mutations
and systemic immunoglobulin-G (IgG) responses, rather than associated with lower viral load can revert after transmission, pro-
mucosal IgA responses, may be a reasonable vaccine goal. If lympho- viding in vivo evidence of impaired fitness86,87. The ability to define
cytes with specific differentiated properties are essential for mucosal the fitness landscape of HIV now exists, and this information could
immunity to HIV they must be better defined to optimize systemic have a significant impact on vaccine development.
induction of mucosal immunity. The mucosal immune responses Careful observation of HIV evolution under selective pressure in
associated with continuous antigen and potential adjuvant effects69 vivo also has the potential to provide key new insights into the nature
of live vaccine vectors such as cytomegalovirus9 or live attenuated of potent immune responses. Clear signatures of selection have been
SIV70, and with the immunization scheme used in the Thai vaccine identified that are not accounted for by known CD4 or CD8 T-cell, or
trial, need to be defined. B-cell, epitopes82; known epitopes do not explain all CD4 and CD8
One of the key sources of data for immune correlates of HIV T-cell responses observed88. CD8 T-cell control of chronic infection
immunity that might be converted into vaccine strategies is the nature with herpesviruses and polyomaviruses can involve non-classical
of immunity to HIV during chronic infection (Fig. 2). Mucosal- MHC molecules, and similar responses have been reported in
damage-associated translocation of intestinal contents, or immune- humans22. Thus, there are additional epitopes, and also potentially
suppression-associated intestinal infection, may lead to systemic other mechanisms of innate or adaptive immune control, that await
immune activation that fosters HIV disease progression16,71,. Further- discovery.
more, SIV-infected non-progressors show fewer signs of intestinal These findings define a weakness in HIV’s most potent defence, the
barrier dysfunction than progressors16. Studies in mice deficient for capacity to mutate, and indicate the potential for effective immuni-
innate immune genes also show that alterations in mucosal barrier zation via an ‘evolutionary trap’, driving HIV into a fitness dead end
function result in systemic activation of adaptive immunity72. by targeting epitopes in fitness-critical regions of viral proteins.
Mucosal damage is therefore a new factor to be considered with Experiments in both HIV and in animal models in which immune
regards to immune effects of HIV infection, and might well alter pressure on viruses can be easily controlled will be needed to develop
measurements of HIV-immune cells or antibody and thus contribute the methods for evolutionary trap vaccination. This will require
to false conclusions about the effects of HIV on immunity, and vice integration of computational biology, evolutionary biology and
versa, during chronic infection. high-throughput sequencing to determine the sequence space within
The antigens, pathogens, or microbial products that induce sys- which a ‘fit’ virus must reside. Full-length genomes will have to be
temic immune activation, and may therefore complicate assessment analysed to define escape mutations and linkages between these
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NATUREjVol 464j11 March 2010 PERSPECTIVES

mutations and intragenic and extragenic suppressor mutations that additional animal models in which mice are adapted to HIV98, or
restore fitness. Studies have already shown that immune-induced HIV is adapted to additional primates99.
mutations in gp120 are more frequent yet less likely to confer a fitness
cost to HIV84 than those in Gag that impair fitness83,84. However, even Tough choices ahead
then compensatory mutations can sometimes rapidly restore viral There is hope for HIV vaccination. There are many knowable
fitness89. Combining an evolutionary trap with mosaic vaccines based unknowns (Figs 1 and 3) that can guide this effort, and there is much
on computationally derived sequences representing maximal coverage scientific knowledge yet to be applied. However, there are also major
of HIV diversity is an attractive approach90. scientific challenges that must be met to optimize chances of generating
an effective vaccine. Although a successful vaccine might be around the
Weapons deployed by vaccination against HIV corner in the next human trial, instead the path to an effective vaccine
In the end, inducing immune responses to HIV will be effective only if might be many years long and require novel discoveries and insights
potent effector mechanisms are deployed against the virus. Because into the functioning of the immune system. Thus, the scientific issues
viral integration and establishment of a pool of latently infected cells addressed in this Perspective are best considered in the broader context
occurs very rapidly after transmission (Fig. 1 and ref. 6), these res- of how to structure the research effort to ensure success. Because many
ponses will have to be present continually or arise extremely rapidly to of the basic immunological issues that need to be solved to conquer
prevent establishment of chronic infection. Unfortunately, we still do AIDS are similar to those for hepatitis C, hepatitis B, malaria, tuber-
not know what an effective immune response to HIV includes, and culosis and cancer, enhancing basic understanding of HIV immunity
therefore are compromised in our quest for a vaccine (Figs 1–3). will have broad impacts. Calls for a greater focus on basic science to
Recent focus has been on inducing polyfunctional T-cell responses facilitate HIV vaccine research were expressed 16 years ago101. We, and
as a surrogate for effective responses, but which functions are truly many others102, make the same call today, as the kind of effort com-
relevant remains unknown. Significant effort needs to be directed to mensurate with the problem is far from being realized.
defining vaccine-relevant effector mechanisms that can eliminate the So what are the barriers, in addition to the complexity of the
initial nidus of HIV infection, prevent systemic spread from an initial immunology, to rapid progress in basic science relevant to HIV vac-
localized mucosal infection, or block the establishment of latency. The cination? Most important among these barriers, and most controversial
prevention of latency might enhance vaccination by preventing the because it has important practical, philosophical, political, scientific
virus from accessing a quiescent state of infection invisible to the and financial implications, is the separation of most HIV-related
immune system (see below). In this area, particular emphasis needs research from research in other areas of immunology and virology.
to be placed on defining novel mechanisms as currently known Considering the many issues that need to be solved to give us the best
effector responses do not consistently correlate with control of HIV. long-term chance of effective HIV vaccination (for example, Figs 1 and
Clues to potential novel immune effector mechanisms come from 3), it becomes clear that not all of the answers can efficiently come from
analysing the complex cellular factors that promote or restrict HIV studies of HIV. The history of research on HIV immunology is filled
replication91,92. HIV has evolved mechanisms to thwart many host with paradigm shifts based on studies of viruses and species commonly
proteins that limit viral replication. The importance of defining the defined as not AIDS related. Examples include T-cell exhaustion, Treg
relationship between immune evasion strategies, host restriction and cells, PD1, CTLA4, inhibitory cytokines, and many others. Many were
immunity is shown by the recent demonstration that Rfv3, which first defined in mice by studying LCMV, a virus unrelated to HIV, and
determines the effectiveness of antibody against Friend retrovirus many have taken years to translate from murine to human studies.
in mice, is encoded by Apobec3, which encodes a protein that is a Studies of other human pathogens are also highly relevant. For
homologue of human APOBEC3G and APOBEC3F proteins targeted example, systems-based studies of human vaccines that do work, such
by HIV vif 93–95. The task we face is defining mechanisms that are not as the yellow fever virus vaccine, provide new genes and pathways,
effectively blocked by HIV and which can be deployed by vaccine- some entirely unexpected, that may contribute to highly functional
generated immune cells. Cytotoxic killing of an infected cell requires CD8 T-cell and B-cell vaccine responses49. Studies of cytomegalovirus
recognition that occurs optimally only after HIV is integrated into led to the development of an HIV vaccine approach effective against
the genome and proteins are expressed. Breaking the cycle of replica- mucosal challenge9. There is no debate that HIV vaccine research has
tion and latency may therefore require different or additional benefitted significantly from research in other areas, and vice versa. The
mechanisms, in particular those that block early events in HIV patho- question is how best to move forward to realize the great potential
genesis (Fig. 1 and ref. 6). offered by integrating currently isolated efforts.
One potential weapon that might be used against HIV would be We need, as a scientific community, to break down barriers
prevention or regulation of latency. It is possible that the capacity of between the AIDS effort and other relevant areas of science to access
HIV to enter an immunologically silent latent state in which the previously untapped human and technological resources for HIV
provirus is present but antigenic proteins are not expressed early after vaccination. But this needs to be done in context, such that those
infection of the mucosal surface could be responsible for vaccine with expertise from other fields are able to effectively tap into the
failures or inefficient vaccination. Thus, immune responses that pre- wealth of current knowledge on HIV pathogenesis. It is not enough to
vent the establishment of latency might enhance vaccine effective- simply funnel more funding to current AIDS research efforts. The key
ness. Latent reservoirs of HIV and SIV are incompletely defined96, point here is that there is no lack of enthusiasm among scientists
and this knowledge is critical to the vaccine effort. Reactivation of outside the HIV field to get involved in HIV/AIDS related research;
HIV from latency is regulated by multiple overlapping factors includ- rather there is merely a lack of a way in. The study section system and
ing chromatinization of the provirus, methylation, T-cell activation, foundation efforts should be reformed with this principle in mind.
NFkB and cytokines including tumour-necrosis factor-a97–99. Studies in multiple systems, especially those that define novel
Notably, the immune system can regulate the nature of latency in cells mechanisms and pathways related to chronic viral infection, should
infected with herpesviruses100, and thus the nature of latency is not be linked to ongoing HIV efforts and pursued with urgency propor-
necessarily cell intrinsic—a potentially important clue that needs to be tionate to the severity of the global AIDS crisis and under the AIDS
exploited. Latency in macrophages or other cells that may have a role umbrella.
in persistent infection is less well understood than in T cells. Therefore, We propose that AIDS-related research be redefined to promote
it is not known whether vaccine-induced effector mechanisms should the integrated study of all mechanisms that may facilitate under-
target HIV in macrophages or dendritic cells, in addition to T cells. A standing HIV pathogenesis and immunity. The HIV/AIDS problem
further profound advantage of defining immune and cell-intrinsic is too severe to allow the relevant science to be restricted by policies
control of latency in more detail could be the development of that separate disciplines that could have a real impact on this deadly
229
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PERSPECTIVES NATUREjVol 464j11 March 2010

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chronic enterocolitis. PLoS Pathog. 4, e20 (2008). (H.W.V. and B.D.W.), the Bill and Melinda Gates Foundation (B.D.W.), and a gift
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bacteria. Cell 139, 485–498 (2009).
Author Contributions H.W.V. and B.D.W. contributed equally to all aspects of the
77. Korber, B. T., Letvin, N. L. & Haynes, B. F. T-cell vaccine strategies for human
planning and writing of this review.
immunodeficiency virus, the virus with a thousand faces. J. Virol. 83, 8300–8314
(2009). Author Information Reprints and permissions information is available at
78. Keele, B. F. et al. Identification and characterization of transmitted and early www.nature.com/reprints. The authors declare no competing financial interests.
founder virus envelopes in primary HIV-1 infection. Proc. Natl Acad. Sci. USA 105, Correspondence should be addressed to H.W.V. (virgin@wustl.edu) or B.D.W.
7552–7557 (2008). (bwalker@partners.org).

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 Vol 464 | 11 March 2010 | doi:10.1038/nature08784

ARTICLES
Retroviral intasome assembly and
inhibition of DNA strand transfer
Stephen Hare1*, Saumya Shree Gupta1*{, Eugene Valkov1{, Alan Engelman2 & Peter Cherepanov1

Integrase is an essential retroviral enzyme that binds both termini of linear viral DNA and inserts them into a host cell
chromosome. The structure of full-length retroviral integrase, either separately or in complex with DNA, has been lacking.
Furthermore, although clinically useful inhibitors of HIV integrase have been developed, their mechanism of action remains
speculative. Here we present a crystal structure of full-length integrase from the prototype foamy virus in complex with its
cognate DNA. The structure shows the organization of the retroviral intasome comprising an integrase tetramer tightly
associated with a pair of viral DNA ends. All three canonical integrase structural domains are involved in extensive
protein–DNA and protein–protein interactions. The binding of strand-transfer inhibitors displaces the reactive viral DNA
end from the active site, disarming the viral nucleoprotein complex. Our findings define the structural basis of retroviral DNA
integration, and will allow modelling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

Retroviral integrase (IN) recognizes and acts on the termini of the Crystallization of the PFV intasome
linear double-stranded DNA molecule that is produced by reverse Most characterized INs predominantly promote the insertion of one
transcription1,2. Initially, in a reaction termed 39-processing, IN viral DNA end into one strand of a target DNA duplex in vitro. In
removes two or three nucleotides from one or both viral DNA ends contrast, we recently reported that recombinant PFV IN catalyses
to expose the 39 hydroxyl groups of the invariant CA dinucleotides. efficient concerted insertion of two PFV DNA ends into target
Next, after import of the viral DNA into the nucleus, IN inserts both DNA17. Here, we obtained soluble and fully functional PFV intasome
39 ends of the viral DNA into opposing strands of cellular genomic preparations using recombinant PFV IN and double-stranded oligo-
DNA. Mechanistically and structurally, IN belongs to a diverse family nucleotides that mimic the viral DNA ends (Supplementary Fig. 1).
of polynucleotidyl transferases3, which notably includes RNaseH4 To bypass the initial catalytic step, 39-processing, the IN–DNA com-
and the transposases from Tn5 (ref. 5) and eukaryotic mobile ele- plexes were assembled using ‘pre-processed’ oligonucleotides with
ment Mos1 (ref. 6) (reviewed in refs 7, 8). The reactions catalysed by recessed termini, which model the viral DNA ends before their inser-
these enzymes proceed by SN2-type nucleophilic substitution, tion into host chromosomal DNA. The IN–DNA complexes were
assisted by divalent metal cofactors4,9. In retroviral IN, a pair of remarkably stable and did not dissociate or lose activity, even after
divalent metal cations (Mg21 or Mn21) are thought to be coordi- prolonged incubations under high ionic strength conditions (Sup-
nated by three carboxylates of the invariant D,D-35-E motif within plementary Fig. 1 and data not shown).
the catalytic core domain (CCD). To function, IN also requires its
After extensive crystallization trials we identified a crystal form of
amino-terminal domain (NTD), a three-helical bundle stabilized by
full-length wild-type PFV IN in complex with a 19-base-pair (bp)
binding a Zn atom, and a carboxy-terminal domain (CTD) that
donor DNA that diffracted X-rays to 2.9 Å resolution, enabling us to
adopts an SH3-like fold10,11. In vivo, IN acts within a large nucleo-
determine the three-dimensional structure of the intasome (Sup-
protein complex that contains viral DNA and several virus- and host-
plementary Table 1). The asymmetric unit contained a single IN
cell-derived components. The minimal functional complex involving
dimer with a tightly associated viral DNA molecule, and a pair of
viral DNA and IN, referred to here as the intasome, can be assembled
symmetry-related IN dimers formed an oblong tetramer (130 3 65 3
in vitro from purified components12.
70 Å; Fig. 1a and Supplementary Movie 1). The IN dimer–dimer
Despite its acute importance for antiretroviral drug discovery and
interface is stabilized by intermolecular NTD–CCD interactions, as
decades of rigorous research7,13, the complete structure of IN, either as a
separate protein or in the context of the functional intasome, is lacking. previously observed in partial structures of lentiviral INs18,19. The
Accordingly, the structural organization of the enzyme active site, which overall shape of the tetramer is reminiscent of the low-resolution
is believed to adopt its functional state only after viral DNA binding, is envelope obtained by a recent negative-stain electron microscopy
unknown. Because clinically useful HIV-1 IN strand-transfer inhibi- study of HIV-1 IN complexes20. Nonetheless, its architecture is markedly
tors14,15 (InSTIs) preferentially bind to and inhibit the intasome com- different from all previously reported models.
plex as compared to free IN16, the mechanism of drug action is poorly
understood. We have now obtained diffracting crystals of the full-length Overall architecture of the PFV intasome
IN from the prototype foamy virus (PFV) in complex with its cognate The inner subunits of the tetramer (coloured green and blue in Fig. 1)
viral DNA. The availability of these crystals enabled us to determine are responsible for all contacts involved in tetramerization and viral
the long-sought structure of the retroviral intasome and explain the DNA binding. The CCDs of the outer subunits (gold in Fig. 1) seem
mechanism of strand-transfer inhibitor action. to provide supporting function, their NTDs and CTDs not resolved
1
Division of Medicine, Imperial College London, St-Mary’s Campus, Norfolk Place, London W2 1PG, UK. 2Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute,
44 Binney Street, Boston, Massachusetts 02115, USA. {Present addresses: Hannover Biomedical Research School, D-30625 Hannover, Germany (S.S.G.); School of Chemistry and
Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia (E.V.).
*These authors contributed equally to this work.

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©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

90º
a b
CCD

CCD

CCD NTD Linkers:

CCD–CTD

NTD–CCD

CTD
CTD
CTD

NTD
NTD CCD

NED

CCD

Figure 1 | Architecture of the PFV intasome. a, Views along (left) and Glu 221 active-site residues are red sticks; Zn atoms are grey spheres.
perpendicular to (right) the crystallographic two-fold axis. The inner Locations of the canonical IN domains (NTD, CCD and CTD) are indicated.
subunits of the IN tetramer, engaged with viral DNA, are blue and green; b, Inner (green) IN chain with domains and linkers indicated. The
outer IN chains are yellow. The reactive and non-transferred DNA strands orientation is the same as in the right panel of a.
are magenta and orange, respectively. Side chains of Asp 128, Asp 185 and

in the electron density maps. Previous retroviral IN CCD structures considerably from the ideal B form. Each CTD makes contact with
showed a conserved dimeric interface7, and this interface is retained the phosphodiester backbone of both viral DNA molecules, essentially
between the inner and outer IN subunits of the intasome. Partial crosslinking the structure. Notably, the NED and NTD of each cata-
structures of INs from HIV-1 (ref. 21), simian immunodeficiency22 lytic subunit interact with the viral DNA molecule engaged at the active
and Rous sarcoma23 viruses showed considerable variability of the site of the opposing CCD (Fig. 1a). The NED interacts with the phos-
CCD–CTD linker region. Within the PFV intasome, the CCD–CTD phodiester backbone, whereas the other elements (NTD, CTD, CCD,
linker adopts an extended conformation for most of its length, track- NTD–CCD and CCD–CTD linkers) further contribute to interactions
ing parallel to the NTD–CCD linker from the same subunit (Fig. 1b). with DNA bases. These sequence-specific interactions include the
The interdomain linkers truss both halves of the intasome together, main-chain carbonyl group of Gly 218, which forms a hydrogen bond
and the structure is further stabilized by a pair of CTDs interacting with guanine 4 of the non-transferred strand (Fig. 2a). The protein–
with both inner CCDs (Fig. 1a), in addition to an extensive network DNA interactions extend into the beginning of the CCD a4 helix,
of protein–DNA interactions (see later). Thus, in contrast to previous which packs into the minor groove at the end of the viral DNA duplex
IN tetramer models based on two domain structures in which the (Fig. 2a and Supplementary Fig. 3). The side chain of Arg 222 is
dimer–dimer interface appeared highly flexible18,19, the overall con- involved in hydrogen bonding with T5 and C6 bases of the non-
formation of the assembled intasome is well constrained. Homology transferred strand (Fig. 2a). Another set of sequence-specific contacts
modelling suggests that the notably shorter interdomain linkers in involves residues from the NTD–CCD and CCD–CTD linkers extend-
HIV-1 IN (Supplementary Fig. 2) can extend sufficiently to allow a ing from the opposing IN dimer. The side chain of Arg 313 intercalates
similar overall architecture and topology in the HIV-1 intasome the minor groove of viral DNA, stacking its guanidinium group against
(data not shown). A further small domain, which we refer to as the adenosine 12 of the reactive strand and forming a hydrogen bond with
NTD extension domain (NED), precedes the PFV IN NTD (Fig. 1b). cytosine 11 (Fig. 2b). Nearby, the side chain of Asn 106 interacts with
On the basis of amino acid sequence comparisons and secondary thymine 8 of the non-transferred strand. These interactions cause
structure predictions, NEDs are present in other spumaviral and notable widening of the minor groove of the viral DNA (Fig. 2b and
possibly gammaretroviral INs (data not shown). Supplementary Fig. 3b). The overall extent of the protein–DNA inter-
action agrees well with the ,16 bp IN footprint in functional HIV-1
IN–DNA interactions within the PFV intasome intasomes12.
In total, almost 10,000 Å2 of molecular surface is buried within IN– At the reactive viral DNA terminus, the base pair involving the
DNA interfaces of the intasome. The protein–DNA interactions cytosine of the invariant CA dinucleotide (C16 and A17 of the reactive
involve amino acid residues from each domain of the inner IN sub- strand; Supplementary Fig. 3a) is distorted by a buckle of ,30u,
units, their interdomain linkers, and 17 nucleotides from each viral whereas the terminal adenine is completely isolated from its com-
DNA end (Supplementary Figs 2 and 3). Thus, as observed in the case plement on the non-transferred strand (Fig. 2a). The active-site loop
of Tn5 transposase5, the canonical retroviral IN domains (NTD, CCD (PFV IN residues 211–220) is directly involved in separating the viral
and CTD) do not have discrete functions; each contributes to extensive DNA strands, acting as a plough with highly conserved residues
protein–protein and protein–DNA contacts within the functional Pro 214 and Gln 215 (corresponding to HIV-1 Pro 145 and Gln 146,
complex. The most intimate protein–DNA interactions are found see Supplementary Fig. 2 for PFV/HIV-1 structure-based alignment)
within the terminal six nucleotides, where the viral DNA deviates forming its share. In particular, Gln 215 displaces thymine 3 of the
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ARTICLES NATURE | Vol 464 | 11 March 2010

a T2 would be expected to destabilize the scissile phosphodiester group in

D128 target DNA. Superposition of the Ca atoms of the active-site Asp and
D185 Glu residues showed notable conservation between the metal and
P214 DNA substrate binding modes of the Tn5 synaptic complex27 and
A17 PFV intasome (Supplementary Fig. 5). Furthermore, the positions
T3 E221
of the metal ions are nearly identical to those of Cd21 and Zn21
Q215
cations observed in structures of the avian sarcoma virus IN CCD28.
G218 C16
Of note, soaking crystals in MgCl2 or MnCl2 did not change the
G4 organization of the intasome active site (Supplementary Fig. 6).
A15 Hence, the positioning of the 39 end of viral DNA is independent of
T5 bound divalent metal ions.
α4
R222

Model for target DNA binding

C6 G14
The active sites of the inner IN subunits, engaged with the 39 termini of
b the viral DNA, are located deep within the dimer–dimer interface.
Therefore, the only mode of host chromosomal DNA (target DNA)
N106 binding that would not require marked rearrangement of the inta-
some or severe DNA bending is along the cleft between IN dimers
C11 R313 G9 (Fig. 4). This target DNA binding mode could not have been predicted
based on previous partial IN structures, and starkly differs from what
C10 we recently proposed18. Modelling B-form DNA within the cleft
G10 results in near perfect alignment of the active sites with opposing
R69
T9 target DNA phosphodiester bonds separated by 4 bp, the known
A11 spacing of concerted PFV integration17. It is easy to see how muta-
A12 tions within the a2 helix of the CCD, described in ref. 29, would
T8
prevent target DNA binding (Fig. 4). We tentatively speculate that
NTD the NTDs and/or the CTDs of the outer IN subunits, disordered in our
structures, could be involved in target DNA capture30. However, this
Figure 2 | Sequence-specific protein–DNA interactions. a, View of the IN target-binding model requires verification using mutagenesis or crys-
active site with bound DNA. Amino-acid side chains discussed in text, DNA tallographic approaches.
bases and the main-chain carbonyl of Gly 218 are shown as sticks.
b, Interactions of the same DNA molecule with the NTD and NTD–CCD and
Mechanism for strand-transfer inhibitor action
CCD–CTD linkers from the opposing (blue) IN subunit. Note the widening
of the minor groove owing to insertion of Arg 313 and Asn 106 side chains. We have previously shown that PFV IN is sensitive to HIV-1 InSTIs17.
The Arg 69 side chain packs into the major groove, forming hydrogen bonds These compounds are thought to engage metal ion cofactors in the
with guanine 10 of the non-transferred strand. IN active site by interactions with uniquely positioned oxygen atoms
of the pharmacophore31. The role of the remaining common InSTI
non-transferred strand, which turns away from the interior of the feature, a fluorobenzyl group, is enigmatic. Intasome structure
DNA duplex (Fig. 2a and Supplementary Fig. 3b). The 59-overhang refinement using diffraction data collected on crystals soaked in
of the non-transferred strand is threaded between the CCD and inter- the presence of Mg21 and the clinical InSTI MK0518 (also known
domain linkers, where it forms extensive contacts with the active-site as raltegravir)14 or GS9137 (elvitegravir)15 showed strong further
loop and the CTD from the same IN chain (Supplementary Fig. 3c). electron density within the active sites of the inner IN subunits.
The involvement of the CCD a4 helix and the active-site loop in Structures of MK0518 or GS9137 with pairs of Mg atoms could be
intimate interactions with the viral DNA end agrees well with results easily fitted into the maps and refined to 2.85 and 3.15 Å resolution,
of chemical and photo crosslinking of functional HIV-1 IN–DNA respectively (Fig. 3b, c, Supplementary Fig. 4d, f and Supplementary
complexes24–26. Table 1). Moreover, soaking crystals in the presence of the drugs and
Mn21 produced similar results, with manganese atoms and drug
The active site of the functional intasome molecules refining at almost precisely the same positions
The reactive 39 termini of the donor DNA molecules are positioned (Supplementary Fig. 4b, c, e, g). Concordantly, InSTIs inhibited both
within close proximity of the Asp 128, Asp 185 and Glu 221 active- the Mg21- and Mn21-dependent activities of the PFV intasome
site carboxylates (Fig. 2a). Although the crystals could be grown in (Supplementary Fig. 1d).
the presence of MgCl2, which considerably improved their diffrac- On the basis of the structures, the two InSTIs seem to have very
tion limit (Supplementary Table 1), data resolution did not allow similar modes of binding and action, involving an induced fit mech-
unambiguous visualization of Mg21 cations in the active site. anism. Their metal chelating oxygen atoms orient towards the metal
Fortuitously, similar to other retroviral INs1, PFV IN can efficiently cofactors of the active site, whereas their halobenzyl groups fit within
use Mn21, a more electron-rich element, as a metal ion cofactor a tight pocket created by displacement of the 39 adenosine (A17).
(Supplementary Fig. 1d). A difference electron density map calculated Within it, the drugs make intimate Van der Waals interactions with
using diffraction data collected on crystals soaked in the presence of the bases of the invariant CA dinucleotide, guanine 4 from the non-
MnCl2 revealed two strong positive peaks (9.4 and 12.4s) within the transferred strand, and conserved residues Pro 214 and Gln 215
active sites of the inner IN subunits. This result confirmed the (Fig. 3b, c). In addition, the isopropyl and methyl-oxadiazole groups
expected two-metal binding mode of retroviral INs and revealed the of MK0518 are involved in hydrophobic and stacking interactions
positions of metal ion cofactors within the assembled active site, which with the side chains of Pro 214 and Tyr 212, respectively (Fig. 3b),
could be refined at full occupancy (Fig. 3a, see also Supplementary further stabilizing this drug in the active site. Through its quinolone
Fig. 4a for metal atom omit and final electron density maps). On the base and isopropyl group, GS9137 interacts with Pro 214 (Fig. 3c).
basis of the current model for two-metal active-site catalysis4, metal Crucially, this mode of drug binding results in displacement of the
atom B, coordinated by the carboxylates of Asp 128 and Glu 221, is in reactive 39 viral DNA end from the active site (Fig. 3b, c), which can
place to activate the 39 hydroxyl group of the pre-processed viral DNA only result in deactivation of the intasome. Thus, after binding of
for strand transfer, whereas metal A, bound by Asp 128 and Asp 185, MK0518, the reactive 39 hydroxyl group moves away from the active
234
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

a b c

Q215 Q215 Q215

P214 Y212 A17 P214 Y212 P214 Y212
A17 G4 A17 G4
G4

3′OH
S217
S217 3′OH S217 3′OH
E221 E221 E221
B A D185 D185 D185
D128 D128 D128
N224 β1 N224 β1 N224 β1
α4 α4 α4

Y212 Y212 Y212

P214 P214 P214

3′OH MK0518 GS9137

3′OH
3′OH
B
A

Figure 3 | PFV IN active site in committed and drug-bound states. yellow, C; blue, N; red, O; grey, F; green, Cl. The complex is shown as a
a–c, Views without drug (a) and with MK0518 (b) or GS9137 (c) bound. solvent accessible surface in bottom panels, coloured by atoms (light grey, C;
Protein and DNA in top panels are cartoons, with A17, DNA bases and the red, O; blue, N). Grey spheres are Mn21 (a, labelled A and B) or Mg21
side chains of indicated amino acids as sticks. Drug atoms are coloured: (b, c) ions.

site by more than 6 Å, compared to its positions in the Mg21- or Furthermore, mutations of HIV-1 IN residue Tyr 143, which, on the
Mn21-containing, or apo crystals. basis of our structure, is expected to interact with the methyl-
Because the core contact points consisting of invariant nucleotide oxadiazole group of MK0518 (Fig. 3b and Supplementary Fig. 2),
bases and amino acid residues are conserved in HIV-1, the mode of are known to confer resistance to this drug33. Common InSTI resist-
InSTI binding and action are unlikely to significantly differ. The ance pathways involve mutations of HIV-1 IN Gln 148 or Asn 155
extensive contacts with the viral DNA end observed in our structures (ref. 33), which correspond to PFV IN residues Ser 217 and Asn 224,
determine why the InSTIs preferentially interact with and inhibit the respectively (Supplementary Fig. 2). Mutations at these positions are
DNA-bound form of HIV-1 IN16. Moreover, the induced fit caused likely to interfere with coordination of metal cofactors by the active-
by displacement of the 39 adenosine by the halobenzyl groups of these site carboxylates, as proposed recently34. Conceivably, a slight shift in
compounds explains why the deletion of this base markedly increased metal ion cofactor positions might suffice to abrogate drug binding,
InSTI on- and off-rates for binding to HIV-1 IN–DNA complexes32. which relies on its spatially constrained metal chelating groups, albeit
at a steep price of impaired viral replication fitness due to detuning of
the IN active site structure.
Our findings will allow the generation of reliable HIV-1 IN and
InSTI pharmacophore models, which will be invaluable for the
development of next-generation strand-transfer inhibitors. Their
α2 design should take advantage of the most conserved elements of
the IN active site determined here, such as the bases of the invariant
α2 CA dinucleotide, positions of the metal co-factors and the main-
90º
chain atoms of the protein.

METHODS SUMMARY
Protein–DNA complexes were assembled using full-length wild-type PFV IN17
α2
and synthetic double-stranded DNA that modelled the viral U5 end
(59-TACAAAATTCCATGACA-39/59-ATTGTCATGGAATTTTGTA-39) by dia-
lysis, reducing the NaCl concentration from 500 to 200 mM. A typical result of
complex assembly is shown in Supplementary Fig. 1a. The intasome was crystal-
lized by vapour diffusion in hanging drops with a reservoir solution containing
Figure 4 | Predicted target DNA binding orientation. Cartoon 1.35 M ammonium sulphate, 25% (v/v) glycerol, 4.8% (v/v) 1,6-hexanediol and
representation of the intasome as in Fig. 1a with active-site side chains shown 50 mM 2-(N-morpholino) ethanesulphonic acid (MES), pH 6.5. The crystals
as red sticks and a2 helices, known to contribute to target DNA binding29, in were soaked in the presence of MK0518, GS9137, Mg21 and/or Mn21, as
cyan. The DNA molecule modelled in black and grey shows the most likely required. Diffraction data were obtained at the beamlines I02 and I04 of the
orientation for target DNA binding. Note that the CTD, juxtaposed to the Diamond Light Source (Oxford, UK), and the structure was solved by molecular
target DNA in this model, is known to possess sequence non-specific DNA replacement. Crystallographic and refinement statistics for the seven resulting
binding activity35. models are summarized in Supplementary Table 1. The structures, refined to
235
©2010 Macmillan Publishers Limited. All rights reserved
ARTICLES NATURE | Vol 464 | 11 March 2010

2.85–3.25 Å resolution, included PFV IN residues 10–374 and 116–278 in chains 22. Chen, Z. et al. X-ray structure of simian immunodeficiency virus integrase
A and B, respectively, without gaps, and both complete strands of the pre- containing the core and C-terminal domain (residues 50–293)—an initial glance
processed donor DNA molecule (17 and 19 nucleotides in chains C and D, of the viral DNA binding platform. J. Mol. Biol. 296, 521–533 (2000).
23. Yang, Z. N., Mueser, T. C., Bushman, F. D. & Hyde, C. C. Crystal structure of an
respectively). The models had good geometry with 92.2–96.4 and not more than
active two-domain derivative of Rous sarcoma virus integrase. J. Mol. Biol. 296,
0.4% of amino acid residues in most preferred and disallowed regions of the 535–548 (2000).
Ramachandran plot, respectively. 24. Alian, A. et al. Catalytically-active complex of HIV-1 integrase with a viral DNA
substrate binds anti-integrase drugs. Proc. Natl Acad. Sci. USA 106, 8192–8197
Full Methods and any associated references are available in the online version of (2009).
the paper at www.nature.com/nature. 25. Esposito, D. & Craigie, R. Sequence specificity of viral end DNA binding by HIV-1
integrase reveals critical regions for protein-DNA interaction. EMBO J. 17,
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Published online 31 January 2010. 26. Jenkins, T. M., Esposito, D., Engelman, A. & Craigie, R. Critical contacts between
1. Craigie, R. in Mobile DNA II (eds Craig, N. L., Craigie, R., Gellert, M. & Lambowitz, HIV-1 integrase and viral DNA identified by structure-based analysis and photo-
A. M.) 613–630 (ASM Press, 2002). crosslinking. EMBO J. 16, 6849–6859 (1997).
2. Lewinski, M. K. & Bushman, F. D. Retroviral DNA integration - mechanism and 27. Steiniger-White, M., Rayment, I. & Reznikoff, W. S. Structure/function insights
consequences. Adv. Genet. 55, 147–181 (2005). into Tn5 transposition. Curr. Opin. Struct. Biol. 14, 50–57 (2004).
3. Dyda, F. et al. Crystal structure of the catalytic domain of HIV-1 integrase: 28. Bujacz, G. et al. Binding of different divalent cations to the active site of avian
similarity to other polynucleotidyl transferases. Science 266, 1981–1986 (1994). sarcoma virus integrase and their effects on enzymatic activity. J. Biol. Chem. 272,
4. Nowotny, M., Gaidamakov, S. A., Crouch, R. J. & Yang, W. Crystal structures of 18161–18168 (1997).
RNase H bound to an RNA/DNA hybrid: substrate specificity and metal- 29. Nowak, M. G., Sudol, M., Lee, N. E., Konsavage, W. M. Jr & Katzman, M. Identifying
dependent catalysis. Cell 121, 1005–1016 (2005). amino acid residues that contribute to the cellular-DNA binding site on retroviral
5. Davies, D. R., Goryshin, I. Y., Reznikoff, W. S. & Rayment, I. Three-dimensional integrase. Virology 389, 141–148 (2009).
structure of the Tn5 synaptic complex transposition intermediate. Science 289, 30. Miller, M. D., Bor, Y. C. & Bushman, F. Target DNA capture by HIV-1 integration
77–85 (2000). complexes. Curr. Biol. 5, 1047–1056 (1995).
31. Grobler, J. A. et al. Diketo acid inhibitor mechanism and HIV-1 integrase:
6. Richardson, J. M., Colloms, S. D., Finnegan, D. J. & Walkinshaw, M. D. Molecular
architecture of the Mos1 paired-end complex: the structural basis of DNA implications for metal binding in the active site of phosphotransferase enzymes.
Proc. Natl Acad. Sci. USA 99, 6661–6666 (2002).
transposition in a eukaryote. Cell 138, 1096–1108 (2009).
32. Langley, D. R. et al. The terminal (catalytic) adenosine of the HIV LTR controls the
7. Jaskolski, M., Alexandratos, J. N., Bujacz, G. & Wlodawer, A. Piecing together the
kinetics of binding and dissociation of HIV integrase strand transfer inhibitors.
structure of retroviral integrase, an important target in AIDS therapy. FEBS J. 276,
Biochemistry 47, 13481–13488 (2008).
2926–2946 (2009).
33. Buzón, M. J. et al. The HIV-1 integrase genotype strongly predicts raltegravir
8. Nowotny, M. Retroviral integrase superfamily: the structural perspective. EMBO
susceptibility but not viral fitness of primary virus isolates. AIDS 24, 17–25 (2010).
Rep. 10, 144–151 (2009).
34. Grobler, J. A., Stillmock, K., Miller, M. D. & Hazuda, D. J. Mechanism by which the
9. Engelman, A., Mizuuchi, K. & Craigie, R. HIV-1 DNA integration: mechanism of
HIV integrase active-site mutation N155H confers resistance to raltegravir.
viral DNA cleavage and DNA strand transfer. Cell 67, 1211–1221 (1991).
Antivir. Ther. 13 (suppl. 3), A41 (2008).
10. Cai, M. et al. Solution structure of the N-terminal zinc binding domain of HIV-1
35. Engelman, A., Hickman, A. B. & Craigie, R. The core and carboxyl-terminal
integrase. Nature Struct. Biol. 4, 567–577 (1997).
domains of the integrase protein of human immunodeficiency virus type 1 each
11. Eijkelenboom, A. P. et al. The DNA-binding domain of HIV-1 integrase has an SH3-
contribute to nonspecific DNA binding. J. Virol. 68, 5911–5917 (1994).
like fold. Nature Struct. Biol. 2, 807–810 (1995).
12. Li, M., Mizuuchi, M., Burke, T. R. Jr & Craigie, R. Retroviral DNA integration: Supplementary Information is linked to the online version of the paper at
reaction pathway and critical intermediates. EMBO J. 25, 1295–1304 (2006). www.nature.com/nature.
13. Marchand, C., Maddali, K., Métifiot, M. & Pommier, Y. HIV-1 IN inhibitors: 2010
update and perspectives. Curr. Top. Med. Chem. 9, 1016–1037 (2009). Acknowledgements We thank F. Dyda for critical reading of the manuscript,
14. Summa, V. et al. Discovery of raltegravir, a potent, selective orally bioavailable R. Clayton and M. Cummings for the generous gift of InSTIs and helpful
HIV-integrase inhibitor for the treatment of HIV-AIDS infection. J. Med. Chem. 51, discussions, T. Sorensen and the staff of the I02 and I04 beamlines of the Diamond
5843–5855 (2008). Light Source for assistance with X-ray data collection. P.C. and co-workers are
15. Sato, M. et al. Novel HIV-1 integrase inhibitors derived from quinolone antibiotics. funded by the UK Medical Research Council, and A.E. by the US National Institutes
J. Med. Chem. 49, 1506–1508 (2006). of Health.
16. Espeseth, A. S. et al. HIV-1 integrase inhibitors that compete with the target DNA Author Contributions E.V. and P.C. carried out initial trials with truncated PFV IN
substrate define a unique strand transfer conformation for integrase. Proc. Natl constructs; S.S.G. and P.C. obtained full-length PFV IN–DNA complexes, carried
Acad. Sci. USA 97, 11244–11249 (2000). out crystallization screening and optimization; S.H. soaked and prepared crystals
17. Valkov, E. et al. Functional and structural characterization of the integrase from for data collection; S.H. and P.C. collected diffraction data and solved the
the prototype foamy virus. Nucleic Acids Res. 37, 243–255 (2009). structures; S.H. refined the final models; S.H. and S.S.G. carried out gel-filtration
18. Hare, S. et al. Structural basis for functional tetramerization of lentiviral integrase. and activity assays; P.C., S.H. and A.E. wrote the paper.
PLoS Pathog. 5, e1000515 (2009).
19. Wang, J. Y., Ling, H., Yang, W. & Craigie, R. Structure of a two-domain fragment of Author Information Atomic coordinates and structure factors have been
HIV-1 integrase: implications for domain organization in the intact protein. EMBO deposited with the Protein Data Bank under accession codes 3L2Q (Apo), 3L2R
J. 20, 7333–7343 (2001). (Mg), 3L2S (Mn), 3L2T (Mg/MK0518), 3L2U (Mg/GS9137), 3L2V (Mn/MK0518)
20. Michel, F. et al. Structural basis for HIV-1 DNA integration in the human genome, and 3L2W (Mn/GS9137) structures. Raw diffraction images are available on
role of the LEDGF/P75 cofactor. EMBO J. 28, 980–991 (2009). request. Reprints and permissions information is available at www.nature.com/
21. Chen, J. C. et al. Crystal structure of the HIV-1 integrase catalytic core and reprints. The authors declare no competing financial interests. Correspondence
C-terminal domains: a model for viral DNA binding. Proc. Natl Acad. Sci. USA 97, and requests for materials should be addressed to P.C.
8233–8238 (2000). (p.cherepanov@imperial.ac.uk).

236
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08784

METHODS replacement. The solution was found in Phaser39 in space group P41212 using
Preparation and analyses of the PFV intasome. Full-length PFV IN (corres- PFV IN CCD (Protein Data Bank (PDB) accession 3dlr)17, HIV-1 IN CTD
ponding to residues 752–1143 of PFV POL) was produced in Escherichia coli (residues 223–266 of chain A from PDB accession 1ex4)21 and a generic 7-bp
strain PC2 (ref. 36), transformed with pSSH6P-PFV-INFL17 and purified as B-form DNA duplex as search models (Supplementary Fig. 7a). The initial phases,
previously described17. The protein was stored in aliquots at 280 uC in 0.5 M modified with prime-and-switch mode of RESOLVE40 (Supplementary Fig. 7b)
NaCl, 5 mM dithiothreitol (DTT), 10% glycerol, 50 mM Tris-HCl, pH 7.4. Ion- were used as input for Buccaneer41, which built most of the protein part of the
exchange HPLC-purified oligonucleotides were purchased from Eurogentec. structure, including the NTD, NTD–CCD and CCD–CTD linkers. The models
Protein–DNA complexes were prepared by dialyses of mixtures containing were improved by iterative manual building in Coot42, simulated annealing in
120 mM PFV IN, 50 mM synthetic DNA duplex, 500 mM NaCl, and 50 mM Phenix43 and maximum likelihood restrained positional refinement in Refmac44.
BisTris propane-HCl, pH 7.45, against excess 200 mM NaCl, 2 mM DTT, Non-crystallographic symmetry restraints for CCD regions similar in chains A
25 mM ZnCl2, 20 mM BisTris propane-HCl, pH 7.45, for 18–24 h at 18 uC. and B (residues 121–200, 241–257 and 263–273) were applied throughout.
Dialysed material was supplemented with an extra 120 or 800 mM NaCl (0.32 Translation, liberation and screw (TLS) refinement45 was included in the final
or 1 M NaCl final), incubated for 1 h on ice and analysed by size-exclusion stages. Geometry of the final structures was analysed using Molprobity46.
chromatography using a Superdex 200 HR 10/30 column, attached to an Examples of electron density and omit maps are given in Supplementary Figs 4
ÄKTA Purifier system (GE Healthcare). The column was operated in 0.32 or and 7. Supplementary Table 1 lists unit cell parameters and crystallographic
1 M NaCl supplemented with 20 mM BisTris propane-HCl, pH 7.45, at statistics. Coordinates and restraints files for the drug molecules were created
1 ml min21, 20 uC. Strand-transfer assays with size-exclusion chromatography- using the PRODRG2 server47. Figures were prepared using PyMOL (DeLano
purified intasome were carried out using established buffer conditions17. A Scientific) and ESPript48, molecular surface areas were calculated using
typical reaction contained 300 ng supercoiled pGEM9 target DNA, 12 mA280 nm Areaimol38,49 and protein–DNA contacts were analysed with help of NUCPLOT50.
units (,30 nM) intasome, 125 mM NaCl, 5 mM MgCl2 (or MnCl2), 10 mM 36. Cherepanov, P. LEDGF/p75 interacts with divergent lentiviral integrases and
DTT, 4 mM ZnCl2, 25 mM BisTris propane-HCl, pH 7.45, in a final volume of modulates their enzymatic activity in vitro. Nucleic Acids Res. 35, 113–124 (2007).
40 ml. The reaction conditions were modified as required. After incubation at 37. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and
37 uC for 30–60 min, the products were deproteinized, separated in 1.5% (w/v) image plate data. Joint CCP4 and ESF-EAMCB Newsletter on Protein Crystallography
agarose gels and visualized by staining with ethidium bromide, as previously no. 26 (1992).
described17. 38. Collaborative Computational Project, Number 4. The CCP4 suite: programs for
Crystallization and structure determination. More than 30 DNA constructs protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
were tested in initial crystallization trials with full-length wild-type and several 39. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40,
658–674 (2007).
mutant PFV IN proteins in ,40,000 initial sparse matrix conditions. Although
40. Terwilliger, T. C. SOLVE and RESOLVE: automated structure solution and density
several crystal forms could be identified and optimized, only one, obtained using modification. Methods Enzymol. 374, 22–37 (2003).
a 19-bp mimic of pre-processed U5 end of PFV DNA (59-TACAAAA 41. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing
TTCCATGACA-39/59-ATTGTCATGGAATTTTGTA-39) and wild-type full- protein chains. Acta Crystallogr. D 62, 1002–1011 (2006).
length IN, diffracted X-rays to a resolution better than 6 Å. For crystallization, 42. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
the protein–DNA complex assembled by dialysis and supplemented with a Crystallogr. D 60, 2126–2132 (2004).
further 120 mM NaCl (320 mM NaCl final) was concentrated to 10–14 mg ml21 43. Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods
using Amicon Ultra-4 centrifugal devices (Millipore). Intasome preparations Mol. Biol. 426, 419–435 (2008).
were stable for extended periods of time when stored on ice. Crystals were grown 44. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular
by vapour diffusion in hanging drops at 18 uC by mixing 1 ml IN–DNA complex structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255
(1997).
and 1 ml of reservoir solution containing 1.35 M ammonium sulphate, 25 mM
45. Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to model
MgCl2, 25% (v/v) glycerol, 4.8% (v/v) 1,6-hexanediol, 50 mM MES-NaOH, anisotropic displacements in macromolecular refinement. Acta Crystallogr. D 57,
pH 6.5. MgCl2 was omitted to obtain apo crystals used in Mn21 and InSTI/ 122–133 (2001).
Mn21 soaking experiments. Crystals typically appeared within 24–48 h and 46. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for
grew to a size of ,100 3 100 3 75 mm within 7–10 days. To obtain drug-bound proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).
forms of the complex, intasome crystals were incubated in stabilizing solution 47. Schüttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput
(reservoir plus 200 mM NaCl and 7 mM DTT), supplemented with 1 mM crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363
MK0518 or GS9137 for 60 h at 18 uC. To visualize metal atoms in the active sites, (2004).
crystals grown without MgCl2 were soaked in the presence of 25 mM MnCl2 in 48. Gouet, P., Robert, X. & Courcelle, E. ESPript/ENDscript: Extracting and rendering
sequence and 3D information from atomic structures of proteins. Nucleic Acids
the absence or presence of 1 mM MK0518 or GS9137.
Res. 31, 3320–3323 (2003).
The crystals were frozen by rapid immersion in liquid nitrogen, and diffrac- 49. Lee, B. & Richards, F. M. The interpretation of protein structures: estimation of
tion data, acquired on I02 and I04 beamlines of the Diamond Light Source static accessibility. J. Mol. Biol. 55, 379–400 (1971).
(Oxford, UK), were processed using Mosflm37 and SCALA38. Availability of 50. Luscombe, N. M., Laskowski, R. A. & Thornton, J. M. NUCPLOT: a program to
the PFV IN CCD structure17 and the high solvent content of the crystal form generate schematic diagrams of protein-nucleic acid interactions. Nucleic Acids
(68.5%) made it possible to determine the intasome structure by molecular Res. 25, 4940–4945 (1997).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08852

ARTICLES
Somatic sex identity is cell autonomous in
the chicken
D. Zhao1*, D. McBride1*, S. Nandi1, H. A. McQueen3, M. J. McGrew1, P. M. Hocking2, P. D. Lewis4, H. M. Sang1
& M. Clinton1
In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of
a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has
yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring
phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining
mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and
indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we
transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female
chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the
donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was
transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a
marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in
birds, sexual differentiation is substantively cell autonomous.

Sexual development in vertebrates is thought to be governed by DMRT1 in male embryos has a ‘feminizing’ effect on the developing
general principles defined in the early to mid-twentieth century1,2. testis15.
These principles state that the sexual phenotype of individuals is In an attempt to clarify the nature of the sex-determining mech-
dependent on the gonad: male and female somatic cells and tissues anism in birds, we have investigated the composition of a number of
are initially sexually indifferent and sexual dimorphism is imposed by gynandromorph chickens. These birds are rare, naturally occurring
the type of gonad that develops. Although these principles have been phenomena in which one side of the animal appears male and the
challenged, most notably by work on songbird neural development3–6 other female16. We investigated these birds with the expectation that
and marsupial development7,8, these observations are generally con- this condition resulted from a sex-chromosome aneuploidy on one
sidered as exceptions. In the currently accepted model, gonadal differ- side of the bird, and that our analysis would provide evidence regard-
entiation is triggered in sexually indifferent embryos by the transient ing the nature of the avian sex-determining mechanism. Contrary to
action of a sex-determining gene. In mammals, the sex-determining expectations, our analysis established that the gynandromorphs were
gene is known to be the testis-determining Sry gene carried by the in fact male:female chimaeras, and that the gynandromorphic pheno-
male-specific Y chromosome9. Although all vertebrates are thought type was due to ZZ (male) and ZW (female) somatic cells responding
to conform to this general model, with the exception of Sry in mam- in different ways to the same profile of gonadal hormones. These
mals and Dmy in medaka10,11, no other vertebrate sex-determining observations led to a series of transcriptome screens and embryonic
genes have been confirmed. transplantation studies showing that male and female avian cells
In terms of morphology, birds seem to conform to the mammalian possessed an inherent sex identity. Our studies demonstrate that in
pattern: male and female embryos are sexually indistinguishable until chickens, gonadal development and the sexual phenotype are largely
around days 5–6 of incubation (Hamburger and Hamilton12 (H&H) cell autonomous and not principally dependent on sex hormones.
stage 28/29) when the action of a sex-determining gene is thought to
initiate testis or ovary development13. However, in birds, not only is Gynandromorph birds are mixed-sex chimaeras
the identity of the putative sex-determining gene unknown, the We obtained three adult lateral gynandromorph birds (designated
nature of the sex-determining mechanism has not been established. G1, G2 and G3) which we maintained and observed over a period of
Current theories of sex determination in birds include the presence of 24 months. These birds occur naturally and it has been suggested that
an ovary-determining gene on the female-specific W chromosome, this condition results from the loss of a single sex chromosome at the
and a dosage mechanism based on the number of Z chromosomes two-cell stage17. All three birds were ISA brown commercial hybrids
(females have one Z and one W sex chromosome whereas males have with sex-linked plumage colour. ISA brown males are heterozygous
two Z sex chromosomes)14. Currently, the best candidate for a testis- for the dominant silver and recessive gold genes (Ss) and so have
determining gene in birds is DMRT1 (doublesex and mab-3-related white plumage; females possess only the gold gene (s-) and have
transcription factor 1). Expression of DMRT1 is restricted to the brown plumage. The birds displayed a marked bilateral asymmetry,
gonads and it has recently been shown that repressing levels of where one side of the animal appeared phenotypically female and the
1
Division of Developmental Biology and 2Division of Genetics and Genomics, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin,
Midlothian EH25 9PS, UK. 3Institute of Cell Biology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JR, UK. 4Animal and Poultry Science Department, University of
KwaZulu-Natal, Pietermaritzburg, South Africa.
*These authors contributed equally to this work.
237
©2010 Macmillan Publishers Limited. All rights reserved
ARTICLES NATURE | Vol 464 | 11 March 2010

other side phenotypically male (Supplementary Fig. 1). Figure 1 a b 100

shows a picture of G1 where the right side of the bird is female in

Mean percentage
80

of cell type
coloration (brown) and has a small wattle and small leg spur. In
60 ZZ cells
contrast, the left side is male coloured (predominantly white), has ZW cells
40
a large wattle and a large leg spur, a heavier leg structure and an
obviously greater mass of breast muscle, typical of a cockerel. Post 20
mortem, whole tissues from both sides were weighed and measured ZZ/ZW 0
Sk. Wa. BM Sk. Wa. BM
and samples of all tissues were taken for later analysis. The measure- ‘Female side’ ‘Male side’
ments performed on individual tissues from both sides of all three
animals supported the observation that these animals were, at least Figure 2 | Male and female cells in gynandromorph birds. a, FISH analysis
of sex chromosomes in gynandromorph blood cells. Shown are interphase
phenotypically, half male and half female. On the side that appeared
nuclei prepared from cultured blood cells from gynandromorph G1
male, tissues were larger and heavier and bones were longer and hybridized according to standard FISH procedures with probes specific to
denser than corresponding tissues and bones from the side with a both the W and Z chromosome (XhoI repeat on W chromosome, and Z
female appearance (Supplementary Table 1). Fluorescent in situ chromosome bacterial artificial chromosome (BAC) containing the VLDL
hybridization (FISH) analysis using Z and W chromosome probes receptor gene identified by screening the HGMP chicken BAC library).
was performed on preparations of blood cells from all three animals Erythrocytes were hybridized with probes for Z chromosome (green) and W
and on multiple preparations of cultured skin cells from both sides chromosome (red). Cells contain either two Z chromosomes or one Z and
of birds G2 and G3. Whereas an autosomal probe demonstrated a one W chromosome. b, Mean relative proportions of ZZ and ZW cells in
tissues from male and female sides of gynandromorph birds. The average
diploid chromosome constitution for G1 blood cells, the sex chro-
percentage of ZW and ZZ cells (Supplementary Table 2) in three tissues from
mosome probes demonstrated that approximately half of gynand- the phenotypically female side and from the phenotypically male side of
romorph G1 cells were female (ZW) and half were male (ZZ) three gynandromorph birds is shown. Tissues from the sides that appear
(Fig. 2a). Similar FISH analyses of blood and primary fibroblast female contain more ZW (female) than ZZ (male) cells, whereas tissues from
cultures from birds G2 and G3 demonstrated that all three animals the sides that appear male are composed predominantly of ZZ cells. BM,
were composed of a mixture of normal diploid male and female cells breast muscle; Sk., skin; Wa., wattle.
(Supplementary Fig. 2 and Supplementary Table 2). Although a
recent analysis of a gynandromorph zebra finch3 demonstrated that composition of a variety of tissues from both sides of the individual
both Z-chromosome and W-chromosome containing cells were pre- birds. Southern analysis using sex chromosome probes on genomic
sent, the possibility remained that such animals were composed of a DNA extracted from multiple tissues revealed that none of the tissues
mixture of ZW and Z0 cells. Here we show that gynandromorph from either side was composed exclusively of either ZZ- or ZW-
birds are genuine male:female chimaeras and provide an explanation containing cells; that is, all tissues examined comprised a mixture of
for a phenomenon that has been debated for centuries18. both female and male cells (examples shown in Supplementary Fig. 3).
We next investigated whether the apparent bilateral asymmetry Multiple Southern analyses were performed on separate DNA samples
reflected the distribution of ZZ and ZW cells by examining the cellular extracted from different regions of skin, from wattle and from breast
muscle from both sides of all three birds, to quantify the relative
proportions of male and female cells. Phosphorimager analyses com-
paring the hybridization signal obtained from DNA from gynandro-
morphic tissues with that obtained from known amounts of male and
Right Left
female DNA produced a measure of the relative proportion of male
and female cells in each tissue. Figure 2b shows the mean proportion
of female and male cells in skin, wattle and breast muscle from the
‘male’ side and ‘female’ side of all three birds. It is clear that tissues
from the side that appeared female contained more ZW (female) than
ZZ (male) cells, whereas tissues from the side that appeared male were
composed predominantly of ZZ cells (Supplementary Table 2). Our
data establishing the presence of both ZZ- and ZW-containing cells
indicate that it is highly unlikely that these birds arise as a consequence
of mutation at the two-cell stage of development, and would support
the hypothesis that gynandromorphs arise as a result of failure of
extrusion of a polar body during meiosis and subsequent fertilization
of both a Z- and W-bearing female pronucleus19.
The development of gonads in the gynandromorph birds was of
obvious interest (Supplementary Fig. 4). The type of gonad present
did not correspond to the external appearance but rather reflected the
cellular composition of the individual organs. The gonads differed
for each gynandromorph: G1 contained a testis-like gonad on the left
side, G2 contained an ovary-like gonad on the left side, and G3
contained a swollen testis-like structure on the left side (in contrast
to G1 and G2, G3 appeared female on the left side and male on the
right). The G1 testis-like gonad was composed primarily of sperm-
containing seminiferous tubules, whereas the G2 ovary-like gonad
was composed predominantly of large and small follicles. The gonad
from G3 comprised a mixture of empty tubules and small follicular-
like structures (ovo-testis). Southern analyses demonstrated that the
Figure 1 | Image of gynandromorph bird (G1). ISA brown bird where the morphological appearance of the gonads conformed to the cellular
right side has female characteristics and left side has male characteristics composition in that the structures that appeared to be testis and
(white colour and larger wattle, breast musculature and spur). ovary were composed principally of ZZ- and ZW-containing cells,
238
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

respectively, whereas the ‘ovo-testis’ comprised a mixture of ZZ- and transcripts are unknown, these findings not only supported the
ZW-containing cells. concept that the tissue phenotype was not dependent on gonadal
Although the findings from our gynandromorph analyses are products, but also reinforced the suggestion that the phenotype
uninformative in terms of elucidating the avian sex-determining was defined by inherent differences between the male and female cells.
mechanism, they do lead to the conclusion that the classical dogma
of sex differentiation, where the phenotype is mainly determined by Chimaeras confirm cell-autonomous sexual differentiation
gonadal hormonal secretions, does not apply to birds. These results To test the hypothesis that the male and female cellular composition
strongly indicate that the avian phenotype is dependent on the nature defines phenotype, we generated embryos containing chimaeric
of the cells comprising the individual tissue rather than being gonads comprised of a mixture of male and female cells. Gonadal
imposed by the type of gonad formed: both sides of these animals chimaeras were generated by transplantation of sections of presump-
are exposed to an identical profile of gonadal products yet each side tive mesoderm from green fluorescent protein (GFP)-expressing
responds differently to these stimuli. For example, although it is well embryos23 at developmental stage 12 (day 2) to replace the equivalent
established that growth of the wattle is sensitive to testosterone20, it is tissue of non-GFP embryos at the same stage of development (Fig. 4a).
clear from Fig. 1 that a major determinant in wattle size is the cellular Donor tissue was transplanted only to the left side of recipient
composition of the tissue, and therefore cellular identity and gonadal embryos as only the left ovary develops fully in the chick. Trans-
hormones both have a significant role in establishing the sexual planted embryos were returned to the incubator and allowed to
phenotype of this tissue. Our analyses led us to conclude that male develop until stage 35 (day 9). By stage 35, donor cells were incor-
and female chicken somatic cells may have a cell-autonomous sex porated into tissues on the left side of the embryo in the region
identity. between the fore and hind limbs (Supplementary Fig. 6). In addition
to the gonads, donor cells were observed in a variety of tissues includ-
Sex differences precede gonadal hormone influences ing skin, muscle, mesonephros, Wolffian duct and Müllerian duct. A
To investigate whether differences exist between male and female minimum of four donor:host chimaeric gonads were generated for
cells independently of any possible gonadal influences we compared each of the four possible combinations: male:male, female:female,
the transcriptomes of male and female embryos at developmental male:female and female:male. For each of these donor:host com-
stages before the formation of the gonads (data not shown). These binations, chimaeras were generated with different levels of donor
analyses identified both messenger RNAs and microRNAs (miRNAs) contribution—ranging from examples where the contribution of
that were expressed in a sexually dimorphic fashion throughout the donor cells was limited to isolated individual cells dispersed through-
embryos at stages before the formation of the gonadal precursor (the out the host gonad, to instances where areas of the host gonad were
genital ridge; H&H stage 21) and well before the generally accepted almost exclusively composed of donor cells. Gonad:mesonephros
point of sex determination in the chicken (that is, around day 5/6 of pairs were collected at stage 35 and longitudinal frozen sections
incubation). Screening for mRNAs expressed exclusively in male or were prepared for confocal microscopy. GFP expression was used to
in female embryos led to the identification of an mRNA encoded by a estimate the extent of donor contribution to the individual chimaeric
W chromosome gene that was expressed ubiquitously in females. gonads and immunohistochemistry (IHC) analysis was performed
This gene was designated FAF for female-associated factor and with antibodies for both anti-Müllerian hormone (AMH) and aro-
sequences were deposited in the EMBL/GenBank databases (acces- matase. AMH is a marker of functionally ‘male’ cells24 (expressed by
sion numbers AJ606294–AJ606297). Whole-mount in situ hybridiza- precursor Sertoli cells of the sex cords) whereas aromatase is a marker
tion analysis of embryos at stages before genital ridge formation of functionally ‘female’ cells25 (expressed by cells in the female medul-
showed that FAF mRNA is expressed throughout the female embryo lary region). At stage 35 of development, the male gonad is composed
as early as 18 h of incubation (H&H stage 4) (Fig. 3a). We also of a thin layer of cortex tissue surrounding a medullary region which
identified a ubiquitously expressed miRNA that is present at levels contains the developing sex cords (expressing AMH) separated by
approximately tenfold higher in males than in females throughout interstitial connective tissue. In contrast, the female left ovary com-
development, including at stages before the expected point of sex prises a greatly thickened cortex surrounding a smaller less-structured
determination (Fig. 3b and Supplementary Fig. 5). This miRNA is medullary region (expressing aromatase). Figure 4b shows the normal
encoded on the Z chromosome and the sequence has not previously expression of AMH and aromatase in stage 35 male and female
been reported in any other species (Gallus gallus mir-2954, accession gonads, respectively. It is clear that the testis is composed almost
number AM691163). These observations are in agreement with other exclusively of medullary tissue and that AMH is expressed in distinct
studies that have identified sexually dimorphic gene expression in the cord-like structures within this tissue. In contrast, the developing
brain preceding morphological differentiation of the gonads, in both ovary comprises a definitive cortex enclosing a reduced medulla and
chicken and mouse21,22. Although the functions of these particular aromatase is expressed in cells throughout the medulla. Examples of

a H&H stage 4 H&H stage 14 H&H stage 20 b m f m f

f
miRNA

m
U6

m
f m f Stage 14 Stage 20

Figure 3 | Sexually dimorphic expression in early chick embryos. the W-chromosome repeat gene Wpkci (also called HINTW)34,35 and
a, Expression of FAF in male and female embryos before development of transcribed in the opposite orientation. f, female; m, male. b, Expression of
genital ridge/gonads. Whole-mount ISH showing expression of FAF novel chicken miRNA (Gallus gallus mir-2954). Expression in whole
(purple) in embryos at 18 h, 48 h and 72 h of development (H&H stages 4 embryos at 48 h (H&H 14) and 72 h (H&H 20) of development is shown.
(original magnification, 340), 14 (320) and 20 (310), respectively). FAF is This miRNA is clearly expressed in a sexually dimorphic fashion at stages
clearly expressed throughout the female embryos at all developmental stages before the sexual differentiation of the gonads. This miRNA matches
and is not expressed in male embryos. FAF is not expressed in extra- sequence present in chicken Z-chromosome BAC clones AC192757 and
embryonic tissues of the female. The FAF transcript is encoded by the AC187119. U6 RNA was used as a loading control.
genomic DNA complementary to the intergenic regions between copies of
239
©2010 Macmillan Publishers Limited. All rights reserved
ARTICLES NATURE | Vol 464 | 11 March 2010

a c m GFP AMH GFP/AMH

g g
Transplant m m
t

Donor Host md

b m GFP AROM GFP/AROM

d GFP AMH GFP/AMH m GFP AROM GFP/AROM

GFP AROM GFP/AROM m GFP AMH GFP/AMH

t

Figure 4 | Expression of male and female markers in chimaeric gonads. mesonephros; o, ovary; t, testis. d, Retention of female donor phenotype in
a, Generation of chimaeras. Left: schematic illustrating transplantation of mixed-sex chimaeras. IHC showing expression of AMH (red in top row) and
presumptive mesoderm from GFP-expressing embryo to non-GFP embryo aromatase (red in bottom row) in neighbouring sections from the gonad of a
at day 2. Right: image of mesonephros and gonads from chimaeric embryo at female:male (donor:host) chimaera. Donor contribution is illustrated by
day 9 showing donor contribution to left gonad (g), mesonephros (m) and GFP (green) expression. The right column shows a merged image of the
Müllerian duct (md). Original magnification, 320. b, Expression of female images in left and middle columns. Regions containing a significant host
and male markers in embryonic gonads. Expression of aromatase (AROM) contribution (defined by the bottom bracket) formed sex-cord-like
in ovary and anti-Müllerian hormone (AMH) in testis at day 9 is shown by structures and expressed AMH. Female donor cells were not incorporated
IHC. Original magnification, 3400. c, Integration of GFP-expressing donor into AMH-expressing sex cords, as shown by the lack of GFP and AMH co-
cells into host gonads. Panels in the first column show a low-magnification localization. Regions composed primarily of female donor cells (defined by
view of sections through host gonads and illustrate the extent of donor cell top bracket) behaved as ovarian-like tissue and expressed aromatase, as
contribution. Panels to the right show higher-magnification views of shown by co-localization of GFP and aromatase (yellow/orange). Scale bars
highlighted areas. Using IHC, donor cells are marked by GFP (green) in c and d indicate 100 mm. IHC was performed following standard
whereas expression of AMH and aromatase are shown in red. The fourth procedures. Primary antibodies were (1:100) goat anti-human AMH (Santa
column is a merged image of the images from the second and third columns. Cruz Biotechnology), (1:200) mouse anti-human cytochrome P450
In same-sex chimaeras, GFP-expressing donor cells co-localize with AMH- aromatase (AbD Serotec) and (1:250) rabbit anti-GFP conjugated to Alexa
expressing and aromatase-expressing cells in host testis and ovary, Fluor 488 (Invitrogen). Secondary antibodies were conjugated to Alexa
respectively (yellow/orange in the fourth column). In mixed-sex chimaeras, Fluor 594 (Invitrogen).
GFP-expressing donor cells do not co-localize with AMH or aromatase. m,

all four donor:host combinations of chimaeric gonads are shown in This is further supported by a striking example where the degree of
Fig. 4c and Supplementary Fig. 7. It is clear that GFP expression did the female donor contribution was sufficient to effectively generate
not affect the ability of donor cells to contribute to host tissues and to an ‘ovo-testis’ in the host embryo (Fig. 4d). This mixed-sex chimaera
function normally: in each case of same-sex chimaeras, either male or contained a gonad with an anterior portion composed almost exclu-
female donor cells were integrated into all somatic compartments of sively of female cells. Whereas the posterior portion contained testis-
the respective host testis and ovary (cortex, sex cords and interstitial like medulla with AMH-expressing sex cords, the region composed of
tissue). Moreover, when integrated into the appropriate ‘functional’ female cells did not form sex cords and did not express AMH. Most
compartment, donor male cells expressed AMH and donor female surprisingly, the female cells in this region expressed aromatase. This
cells expressed aromatase. In contrast, in mixed-sex chimaeras the demonstrates that although female cells in a male embryo can correctly
donor cells did not integrate into the ‘functional’ structures of the interpret gonadal location and differentiation signals, they respond in
host gonad: female donor cells in host testis medulla were not a cell-autonomous manner characteristic of a female genotype (and
recruited into the AMH-expressing sex cords and were restricted to express aromatase). Our findings are in contrast with those from
the interstitial tissue, whereas male donor cells in host ovary were mammalian mixed-sex chimaeras, where XX cells can become func-
excluded from the aromatase-expressing structures. In mixed-sex chi- tional Sertoli cells and XY cells can become functional granulosa
maeras the inability of donor cells to form functional host structures cells26,27.
was evident regardless of the relative contribution of male and female These studies demonstrate that avian somatic cells possess a cell-
cells (Supplementary Fig. 7). The fact that female chicken cells in an autonomous sex identity. Our results support and extend previous
environment and location that induces testicular development cannot findings3 that showed that differences between male and female zebra
be recruited into the functionally ‘male’ Sertoli cell compartment, and finch brains were a result of endogenous genetic differences in the
male cells in an ovary-inducing environment are excluded from a brain cells themselves. Our analysis of lateral gynandromorph birds,
functionally ‘female’ compartment, strongly supports the suggestion showing that they are male:female chimaeras, and our experimental
that chicken somatic cells possess a cell-autonomous sexual identity. generation of embryos with mixed-sex chimaeric gonads, together
240
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

Mammalian model
Sry Testis containing 100 units ml21 penicillin and 100 mg ml21 streptomycin (PBS-Pen/
Male
phenotype Strep). A strip of presumptive mesoderm flanking presumptive somites 21–23
Hormones
was removed and stored in CO2-independent medium (Invitrogen) containing
Genital Indifferent Hormones 10% FBS and Pen/Strep. Host eggs were windowed as above and kept moist by
ridge gonad Female the addition of PBS-Pen/Strep. To help visualize somites, sterile India ink (20%
Ovary phenotype
in PBS-Pen/Strep) was injected under the host embryos. Using a microneedle,
Chicken model
the vitelline membrane and a flap of ectoderm were folded back from the under-
Male Male
lying mesoderm. A strip of presumptive mesoderm was then removed from the
soma phenotype host embryo taking care to leave the endoderm intact. The GFP-donor tissue was
Testis
es then inserted into the host site and the ectodermal flap replaced. Two millilitres
Genital Hormon
of albumen was then withdrawn from the host eggs using a hypodermic syringe.
ridge DMRT1(?) Hormo
nes Transplanted eggs were tightly sealed with tape and incubated at 37 uC in a
Ovary humidified incubator. All other methods are standard.
Female Female
soma phenotype
Full Methods and any associated references are available in the online version of
Figure 5 | A novel mechanism of sex determination in the chicken. A sexual the paper at www.nature.com/nature.
identity is genetically imposed on the male and female chicken soma at
fertilization and is the major factor in determining the adult sexual Received 10 August 2009; accepted 18 January 2010.
phenotype. At the appropriate stage in development, the sexually-dimorphic
transcripts underlying the male/female identity trigger expression in the 1. Lillie, F. R. Sex-determination and sex-differentiation in mammals. Proc. Natl Acad.
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3. Agate, R. J. et al. Neural, not gonadal, origin of brain sex differences in a
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28. McQueen, H. A. et al. Dosage compensation in birds. Curr. Biol. 11, 253–257 Supplementary Information is linked to the online version of the paper at
(2001). www.nature.com/nature.
29. Melamed, E. & Arnold, A. P. Regional differences in dosage compensation on the
chicken Z-chromosome. Genome Biol. 8, R202 (2007). Acknowledgements This work was supported by DEFRA and BBSRC (BB/
30. Ellegren, H. et al. Faced with inequality: chicken do not have a general dosage E015425/1). We thank G. Miele, G. Robertson, S. Wilson, A. Sherman,
compensation of sex-linked genes. BMC Biol. 5, 40 (2007). M. Hutchison, F. Thomson and R. Mitchell for technical support and for provision of
31. McQueen, H. A. & Clinton, M. Avian sex chromosomes: dosage compensation fertilized eggs and embryos. We also thank T. Cannon for donation of
matters. Chrom. Res. 17, 687–697 (2009). gynandromorph bird G1, and R. Field and N. Russell for photography.
32. O, W.-S., Short, R. V., Renfree, M. B. & Shaw, G. Primary genetic control of somatic Author Contributions D.Z. and D.M. performed transplantation studies,
sexual differentiation in a mammal. Nature 331, 716–717 (1988). transcriptome screens, Southern analyses and general molecular biology. S.N.
33. Harry, J. L., Koopman, P., Brennan, F. E., Graves, J. A. & Renfree, M. B. Widespread performed immunostaining, H.A.M. performed FISH analyses and P.M.H.
expression of the testis-determining gene SRY in a marsupial. Nature Genet. 11, performed dissections and post-mortem measurements. M.J.M. performed ISH
347–349 (1995). and suggested transplantation strategy and P.D.L. obtained gynandromorph birds.
34. Hori, T., Asakawa, S., Itoh, Y., Shimizu, N. & Mizuno, S. Wpkci, encoding an altered Overall project was conceived by M.C. and H.M.S. M.C. carried out day-to-day
form of PKCI, is conserved widely on the avian W chromosome and expressed in supervision and wrote the manuscript. All authors edited the manuscript.
early female embryos: implications of its role in female sex determination. Mol.
Biol. Cell 11, 3645–3660 (2000). Author Information Reprints and permissions information is available at
35. Smith, C. A., Roeszler, K. N. & Sinclair, A. H. Genetic evidence against a role for www.nature.com/reprints. The authors declare no competing financial interests.
W-linked histidine triad nucleotide binding protein (HINTW) in avian sex Correspondence and requests for materials should be addressed to M.C.
determination. Int. J. Dev. Biol. 53, 59–67 (2009). (Michael.clinton@roslin.ed.ac.uk).

242
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08852

METHODS carried out as described previously39. Digoxigenin-labelled probes were prepared

Embryo transplantation. Generation of chimaeric embryos:GFP embryos23 and from linearized plasmid clones using a Roche DIG RNA labelling kit to incor-
ISA brown embryos at H&H stage 11/12 (13–15 somites) were used as donor and porate digoxigenin-11-UTP by in vitro transcription with SP6 and T7 RNA
host, respectively. The blunt end of donor eggs was pierced to create an air hole polymerases.
and a ‘window’ cut on the midline. The embryos were removed and pinned on a RNA preparation. Total RNA was extracted from pools of male and female chick
3% agarose surface containing 0.5% India ink. Embryos were kept moist by the embryos and tissues using RNA-Bee (AMS Biotechnology) according to the
addition of phosphate buffered saline (PBS) containing 100 units ml21 penicillin manufacturer’s instructions.
and 100 mg ml21 streptomycin (PBS-Pen/Strep). A strip of lateral plate meso- Differential display. RNA expression profiles in male and female embryos were
derm flanking presumptive somites 21–23 was removed and stored in CO2- compared by differential display reverse transcription PCR (DDRT–PCR).
independent medium (Invitrogen) containing 10% FBS and Pen/Strep. Host Embryos were sexed38 and pools of RNA from male and female embryos generated.
eggs were windowed as above and kept moist by the addition of PBS-Pen/ DDRT–PCR was performed as described previously40.
Strep. To help visualize somites, sterile India ink (20% in PBS-Pen/Strep) was miRNA library construction. Low-molecular-mass RNAs (,40 nucleotides
injected under the host embryos. Using a microneedle, the vitelline membrane long) were isolated from total RNA by the use of a flashPAGE fractionator
and a flap of ectoderm were folded back from the underlying mesoderm. A strip (Ambion). MicroRNA libraries were constructed essentially as described previ-
of lateral plate mesoderm was then removed from the host embryo, taking care to ously41,42.
leave the endoderm intact. The GFP-donor tissue was then inserted into the host MicroRNA northern analysis. Five micrograms of total RNA was separated by
site and the ectodermal flap replaced. Two millilitres of albumen was then electrophoresis through a 15% TBE/urea polyacrylamide gel (Bio-Rad) before
withdrawn from the host eggs using a hypodermic syringe. Transplanted eggs transfer to Hybond-N1 membrane (GE Healthcare). Locked nucleic acid (LNA)
were tightly sealed with tape and incubated at 37 uC in a humidified incubator. oligonucleotides antisense to the mature miRNA were end-labelled (mirVana
Immunostaining. Immunohistochemistry was carried out as described previ- Probe and Marker kit, Ambion) with 32P-dATP (Perkin-Elmer) and hybridized
ously36. Briefly, tissues were fixed in 4% paraformaldehyde for 2 h at 4 uC, to membranes containing miRNAs. Hybridization was carried out overnight in
equilibrated in 15% sucrose then embedded in 15% sucrose plus 7.5% gelatin ULTRAhyb-oligo (Invitrogen) at 42 uC and membranes washed at 63 uC in 0.13
in PBS, pH 7.2. Sections, 15 mm thick, mounted on Superfrost slides (Menzel) SSC/0.1% SDS43 (22 uC below the estimated melting temperature of the LNA,
were washed for 30 min in PBS at 37 uC and blocked in PBS containing 10% 85 uC).
donkey serum, 1% BSA, 0.3% Triton X-100 and 0.05% Tween 20 for 2 h at 22–24 Southern analysis. High-molecular-mass genomic DNA was extracted from
uC. Incubation with primary antibodies was carried out overnight at 4 uC, fol- tissues of embryonic and adult male and female chickens by standard phenol-
lowed by washing in PBS containing 0.3% Triton X-100 and 0.05% Tween 20, chloroform procedures43. DNA was digested with restriction endonucleases,
and then incubation with secondary antibodies for 2 h at room temperature. subjected to electrophoresis on a 1% TBE gel and transferred to Hybond-N
After washing, the sections were treated with Hoechst solution (10 mg ml21) for membrane. Probes labelled with 32P-dCTP were hybridized by standard proce-
5 min to stain nuclei. dures and signal was recorded on high-sensitivity film (Kodak) and by phos-
Fluorescent in situ hybridization (FISH). FISH analysis of metaphase or inter- phorimager analysis.
phase preparations of chicken cells was performed by standard procedures37.
BAC clones containing the VLDL receptor, aldolase B, CHRN or SCII genes were 36. Stern, C. D. In Essential Developmental Biology: A Practical Approach (eds Stern, C.
identified by screening the HGMP chicken BAC library and used to identify Z D. & Holland, P. W. H.) 193–212 (IRL, 1993).
chromosomes. A probe for the W chromosome was prepared by polymerase 37. McQueen, H. A. et al. CpG islands of chicken are concentrated on
chain reaction (PCR) amplification of a portion of the XhoI repeat region from microchromosomes. Nature Genet. 12, 321–324 (1996).
the W chromosome38. After gel purification, the probe was labelled by incor- 38. Clinton, M., Haines, L., Belloir, B. & Mcbride, D. Sexing chick embryos: a rapid and
poration of either biotin-16-dUTP (Roche) or digoxigenin-11-dUTP (Roche) simple protocol. Br. Poult. Sci. 42, 134–138 (2001).
during a further round of PCR. BAC DNA was prepared using Qiagen plasmid 39. Henrique, D. et al. Expression of a Delta homologue in prospective neurons in the
columns following recommendations for low-copy plasmid purification. Biotin- chick. Nature 375, 787–790 (1995).
40. Clinton, M., Miele, G., Nandi, S. & McBride, D. In Differential Display Methods and
16-dUTP and digoxigenin-11-dUTP were incorporated into BAC DNA by nick
Protocols 2nd edn (eds Liang, P., Meade, J. D. & Pardee, A. B.) 157–178 (Humana,
translation and labelled probes were concentrated by precipitation in the pres- 2006).
ence of 5 mg of salmon sperm DNA as a carrier and 2 mg of sonicated chicken 41. Lau, N. C. et al. An abundant class of tiny RNAs with probable regulatory roles in
genomic DNA as competitor. The pellet was resuspended in 15 ml of hybridiza- Caenorhabditis elegans. Science 294, 858–862 (2001).
tion mix, denatured and pre-annealed for 15 min at 37 uC to block repetitive 42. Lee, R. C. & Ambros, V. An extensive class of small RNAs in Caenorhabditis elegans.
sequences. Science 294, 862–864 (2001).
Whole-mount in situ hybridization. Chicken embryos and isolated gonads were 43. Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual (Cold Spring
fixed in 4% paraformaldehyde for 1 h and whole-mount in situ hybridization was Harbor Laboratory Press, 2001).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08779

ARTICLES
Systems survey of endocytosis by
multiparametric image analysis
Claudio Collinet1, Martin Stöter2, Charles R. Bradshaw1, Nikolay Samusik1, Jochen C. Rink{, Denise Kenski{,
Bianca Habermann1, Frank Buchholz1, Robert Henschel3, Matthias S. Mueller3, Wolfgang E. Nagel3, Eugenio Fava2,
Yannis Kalaidzidis1,4 & Marino Zerial1

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and
integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design
principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF)
and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal
microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel
components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such
as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-b and Notch regulate the endocytic system, and identified
new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further
showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly
but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

Endocytosis is a fundamental process supporting many functions platform, on the basis of the custom-designed image analysis software
such as nutrient uptake, intracellular signalling1, morphogenesis MotionTracking12. First, images were segmented to determine mor-
during development2 and defence against pathogens3. Dysfunctions phological and positional descriptors for each endosome (Fig. 1b).
of the endocytic system lead to severe metabolic4, infectious5 and Second, a total set of 62 parameters were extracted, 4 serving as quality
neurodegenerative diseases6. Receptors are internalized by both control to reject sub-optimal images and 58 describing defined bio-
Clathrin-dependent and -independent mechanisms into early endo- logical properties of the endocytic system, such as total internal cargo,
somes and, from here, follow different intracellular routes. For number, size (mean apparent area) and position of endosomes
example, transferrin (TF) is recycled back to the plasma membrane, (Fig. 1c, Supplementary Table 1 and Supplementary Information
whereas epidermal growth factor (EGF) is routed to late endosomes/ for details). The latter were processed to suppress plate-to-plate
lysosomes for degradation. Despite a great deal of molecular random variations, normalized (see Supplementary Information)
insights7–9 our understanding of the endocytic machinery remains and the 46 most robust parameters (see Supplementary Information
fragmentary. Furthermore, the design principles of the endocytic and Supplementary Table 1) were combined into multiparametric
pathway, its integration in the overall cellular system and high-order profiles (Fig. 1d) describing quantitatively the endocytic phenotypes.
control are still largely unknown. Addressing these problems requires In preparation for the genomic screen, we validated the quality and
methods capable of seizing the complexity at the system level (that is, reproducibility of assay and QMPIA. First, we verified that the deple-
measuring many key parameters). tion of established components of the endocytic machinery, such as
Here, we designed a new analytical approach aimed at phenotypi- Clathrin (CLTC), Dynamin-2 (DNM2), EGF receptor (EGFR),
cally profiling genes with respect to endocytosis by genome-wide Transferrin receptor (TFRC), early endosome antigen 1 (EEA1)
RNAi screening through the quantitative assessment of many system and others by RNAi (Supplementary Figs 1 and 2), yielded the
parameters. expected alterations in phenotypic profiles. For example, silencing
of TFRC reduced the values of number of endosomes, total internal
Multiparametric profiling of the endocytic system cargo and mean cargo load per endosome for TF but not EGF (Fig. 1d
We visualized endocytosis of fluorescent TF and EGF in HeLa cells and Supplementary Fig. 1e). Conversely, EGFR downregulation per-
before we could apply our platform to more disease-relevant systems. turbed the parameters of EGF but not of TF (Supplementary Fig. 1e).
After small interfering RNA (siRNA) transfection, cells internalized Second, we validated the use of a single confocal section as representa-
the two cargo markers simultaneously for 10 min, were fixed, stained tive of the three-dimensional endosomal population of the entire cell
with 49,6-diamidino-2-phenylindole (DAPI)/SYTO42 blue for nuclei (Supplementary Fig. 3). Third, we validated the reproducibility of the
and cytoplasm detection, and imaged (12 images per well) by triple- observed phenotypic profiles using the same and different siRNAs
colour high-resolution automated confocal microscopy (Fig. 1a). targeting the same gene for a set of 78 genes and 468 siRNA (6
Instead of performing a phenotypic evaluation on the basis of single siRNAs per gene). We found that profiles of the same siRNAs were
or few parameters as commonly done in previous screens10,11, we highly reproducible (Fig. 2a) (mean Pearson correlation coefficient
developed a quantitative multiparametric image analysis (QMPIA) 0.6 6 0.02 s.e.m.), compared with other monoparametric screens13
1
Max Planck Institute for Molecular Cell Biology and Genetics, 2High-Throughput Technology Development Studio, MPI-CBG, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
3
Center for Information Services and High Performance Computing (ZIH), Dresden University of Technology, D-01062 Dresden, Germany. 4Belozersky Institute of Physico-Chemical
Biology, Moscow State University, 119899, Moscow, Russia. {Present addresses: University of Utah School of Medicine, 401 MREB, 20 North 1900 East, Salt Lake City, Utah 84132-
3401, USA (J.C.R.); Sirna Therapeutics Inc., San Francisco, California 94158, USA (D.K.).

243
©2010 Macmillan Publishers Limited. All rights reserved
ARTICLES NATURE | Vol 464 | 11 March 2010

a a C12orf4 runs
EGF Synthetic EGF TF Other
TF 6.0
DAPI
4.0

Normalized z-values
2.0

0.0

–2.0
20 μm 20 μm
–4.0
b
–6.0

P1
P2

P8
P3
P4
P5
P6
P7

P9
P1

P 11

P2 8

P37
P10

P1 2
P13
P14
P1 5
P16
P17

P20
P23

P3 0
P31

P38
P2 6
P28
P39

P32
P3 3
P34
P35
P3 6

P4 9
P40
P 41

P43
P4 2

P 44

P5 7
P 50
P5 2
P55
P5 6
P57
P4
5

8
{

{
{
{
{
{
{
{
{
{
{
{

{
{
{
{
G

G
G

G
G
G

G
G

10
2

8
3
1

1
6
Parameter

c Multi-parametric analysis b C12orf4 all siRNAs

EGF TF Other
Endosome parameter groups/cargo Other endocytic parameters 6.0
G1. Number of endosomes (1). G9. Background intensity (2).
G2. Cargo uptake (2). G10. Colocalization (2).

Normalized z-values
4.0
G3. Endosome area (3).
G4. Vesicle elongation (3). Quality control parameters
G5. Endosomal cargo concentration (3). - Number of nuclei. 2.0
G6. Endosomal cargo content (3). - Nuclear size.
G7. Endosome distance from nucleus (3). - Nucleus intensity. 0.0
G8. Endosome clustering (9). - Focus score.

d EGF TF Other
–2.0

30 Control –4.0
Normalized z-value

20 Internalized TFRC
cargo Cargo load –6.0
Number Cargo density
10

P1
P2

P4

P8
P3

P5
P6
P7

P9
P1
P10
P 11
P1 2
P13
P14
P1 5
P16

P20

P39
P17

P3 6

P 38
P4 9
P2 8

P23
P2 6
P28

P3 0
P31
P32
P3 3
P34
P35

P37

P40
P41

P43

P4 5

P 50
P4 2

P44

P57

P5 2
P55
P5 6
P57
8
{

{
{
{
{
{
{
{
{
{
{
{

{
{
{
{
0

G
G

G
G
G

10
2

8
3
1

1
–10 Parameter
–20 Figure 2 | Reproducibility of multiparametric profiles by the same siRNA
–30 and different siRNAs/gene. a, Reproducibility of multiparametric profiles
–40 of a single siRNA silencing the C12orf4 gene. The same siRNA (ID: 28470)
was transfected in seven independent experiments (runs) and
P3
P1
P2

P5

P7

P9

P10
P11

P14

P16

P28

P23
P4

P6

P 36
P37
P38
P 49

P41
P 42

P57

P 56
P57
P8

P1

P12
P13

P 15

P17

P 28

P32

P35
P20

P 26

P39
P 30
P31

P 33
P34

P40

P43
P44

P50

P55
P 45

P 52

8
{
{
{
{
{

{
{
{
{
{
{
{
{
{
{
{

multiparametric profiles (differently coloured for each experiment) were

G
G

G
G
G

G
G

G
1
2

10
3

9
4

8
8

Parameter calculated and plotted as described in Fig. 1. b, Reproducibility of

Figure 1 | Multiparametric image analysis. a, Example of three-colour high- multiparametric profiles of different siRNAs targeting the same gene
resolution images collected with the automated spinning disk confocal (C12orf4). C12orf4 was silenced with six different siRNAs (IDs: 133085,
OPERA microscope (left). Nuclei and cytoplasm are pseudo-coloured in blue, 133086, 133087, 28285, 28378, 28470) in the same experiment and
Alexa-488-labelled EGF is pseudo-coloured in red and Alexa-647-labelled TF corresponding multiparametric profiles were calculated and plotted as
is pseudo-coloured in green. The synthetic image (right) was obtained after described in Fig. 1.
background intensity subtraction and modelling of endosomal structures
(see Supplementary Information). b, Close-ups show the model structure of probabilistic approach to infer gene-specific phenotypes by ‘averaging’
individual endosomes. c, List of quantitative parameters divided into groups the profiles of different siRNAs targeting the same gene. We defined the
(G1–G10). Ten groups of parameters (number of parameters in each group ‘gene profile’ as the most probable profile calculated as mode of the a
indicated in brackets) were used to measure endosomal features and four
posteriori joint probability distribution considering all individual
parameters as image quality control. See Supplementary Table 1 and
Supplementary Information for further description on the QMPIA
siRNA profiles for a given gene. Notably, here every siRNA targeting
parameters. d, Example of QMPIA profile (TFRC; see pictures in a given gene contributes, to a different extent, to the determination of
Supplementary Fig. 1c). The 46 most specific parameters (listed in gene profiles (see Supplementary Fig. 4), circumventing the need of
Supplementary Table 1 and Supplementary Information) are aligned on the similarity thresholds to define supporting siRNAs (2 of 3, 3 of 3, and
x-axis and normalized z-values are plotted on the y-axis. Parameter groups so on)18. Gene profiles instead of individual siRNA profiles were sub-
(G1–10) and parameter numbers (P1–P58) are indicated corresponding to sequently used for phenotypic clustering and gene classification.
the nomenclature in c and to the enumeration in Supplementary Table 1, As test, we screened 1,000 genes including 35 known endocytic
respectively. Black continuous lines separate the parameter groups and grey regulators (Supplementary Table 2) and 92 kinases identified in a
dashed lines separate the parameters. The horizontal bars indicate the
endocytic markers which the parameters refer to. Key parameters affected in
previous screen19 (Supplementary Table 3) using seven siRNAs per
the TFRC profile are indicated by arrows. gene. On the basis of the QMPIA, 31 of the 35 endocytic regulator
genes (,90%) and 62 out of 92 kinase genes presented a x2 probability
(see Supplementary Information), indicating that the experimental (1.0 2 P) $ 0.95 corresponding to a statistically significant enrich-
noise is not increased by the higher complexity of the assay. In contrast, ment (P 5 1.1 3 10211 and P 5 5 3 10212, respectively) in the list of
different siRNAs targeting the same gene frequently yielded different scores. Interestingly, only 7 out of the 35 endocytic regulators would
profiles (Fig. 2b) (mean Pearson correlation coefficient 0.047 6 0.01 have scored considering only two parameters (EGF and TF total
s.e.m.), consistent with high incidence of RNAi off-target13–17. As we internal cargo). Altogether, these data indicate that the QMPIA has
excluded variations in protein depletion and silencing of alternatively the advantage of higher comprehensiveness and specificity over
spliced transcripts (data not shown), the major cause of incoherence monoparametric read-outs in the identification of genes regulating
between profiles of different siRNAs is most probably off-target effects. endocytic trafficking.

Gene phenotypic profiling Genome-wide survey of EGF and TF endocytosis

Assuming that each siRNA profile has both ‘on-target’ and ‘off- To improve accuracy and coverage in the survey, we screened directly
target’ phenotypic components, we exploited the QMPIA to suppress in the primary assay three genomic libraries, two siRNA and one
as much as possible the latter contaminant. For this, we developed a endoribonuclease-prepared siRNA (esiRNA)15, corresponding to
244
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

7–8 si/esiRNAs per gene for a total of 161,492 si/esiRNAs (see providing both strong and mild, but specific, phenotypes. A list of
Methods). This approach presents two major advantages. First, the 4,609 genes, 3,804 with a ‘strong’ and 805 with a ‘mild’ phenotype
high number of si/esiRNAs analysed by QMPIA reduces the impact was compiled (Supplementary Table 4; images and profiles available
of false negatives owing to disagreement between siRNAs. Second, at http://gwsdisplayer.mpi-cbg.de). Considering both the design of the
reproducibility of phenotypes is assessed already in the primary assay and the integration of endocytosis with many cellular processes,
screen for all genes (not just the hits). We acquired 12 images per such a large number was expected because, besides the ‘core’ endocytic
sample to obtain statistically significant data, accounting for a total of machinery, many genes have a direct or indirect role in endocytosis. For
,2.5 3 106 images. Quality controls for transfection efficiency, comparison with the common practices in the field, we estimated that
QMPIA and toxicity (see Methods) were included in each plate (see screening a single library (2–3 siRNAs per gene) with a typical
Supplementary Fig. 5). Image analysis and gene profile determination two-parameter assay and validating the hits with an independent set
required ,4.5 3 106 CPU hours of calculation on a 2,584-cores com- of si/esiRNAs (4–5) would yield only 336 genes (data not shown),
puter cluster. indicating that the large number of genes in the list results from (1) a
broader window of detection (many parameters) and (2) high number
Phenotypic clustering and gene scoring of si/esiRNAs screened rather than permissive criteria for selection.
As our aim was not to identify a few hit genes but rather to conduct a To estimate the reproducibility of the hits, we re-screened twice the
systems analysis, we exploited the multidimensionality of gene profiles. kinome-phosphatome (9,330 siRNAs, 1,459 genes), including a large
First, we clustered all genes of the human genome using mean-shift fraction of the gene hits (235). We observed 84% reproducibility for
clustering20 (see Supplementary Information). We identified 14 pheno- ‘strong’ and 72.5% for ‘mild’ phenotypes. Nevertheless, to assign a
typic cluster groups (Fig. 3) stable over a wide range of the algorithm better probabilistic value to the scores, the genes in the list will be re-
parameters, indicating that the endocytic system is under constraints screened (the web database will be updated). The silencing efficacy of
and changes between a discrete set of possible states in response to the libraries was assessed by the manufacturers by quantitative RT–PCR
perturbations. Second, genes were scored on the basis of two criteria, (qRT–PCR) on a set of siRNAs and we independently confirmed it by
phenotype amplitude (probability of x2) and presence of specific western blotting (see Methods and Supplementary Fig. 7).
phenotypic traits (defined as the cluster group profiles; ‘pheno-score’; The largest portion of scored genes (,34%, 1,593; P-value of
Supplementary Information), as shown in Supplementary Fig. 6, enrichment P 5 7 3 10234) encoded for components of metabolic

a 0.4
EGF TF Other b 0.4 EGF TF Other i 0.4
EGF TF Other j Parameters list
Cluster 1 Cluster 3
Normalized z-value

Normalized z-value
Normalized z-value

0.3 0.3 0.3 Cluster 14

Cluster 2 Cluster 4 Endosome parameter groups/cargo
0.2 0.2 0.2
0.1 0.1 0.1
G1. Number of endosomes (1).
0.0
G2. Cargo uptake (2).
0.0 0.0
G3. Endosome area (3).
–0.1 –0.1 –0.1
G4. Vesicle elongation (3).
–0.2 –0.2 –0.2
G5. Endosomal cargo concentration (3).
–0.3 –0.3 –0.3
G6. Endosomal cargo content (3).
–0.4 –0.4 –0.4
G7. Endosome distance from nucleus (3).
G1
G2
G3
G4
G5

G7
G1
G1

G6

G2

G4

G9
G2
G3
G4
G5

G7
G1

G10

G3

G5

G7
G1

G6

G2

G4

G9

G6
G2
G3
G4
G5

G7
G1

G3

G5

G7

G10
G6

G2

G4

G9

G6
G3

G5

G7
G6

G10

Parameter group Parameter group Parameter group G8. Endosome clustering (9).
c EGF TF Other d EGF TF Other Other endocytic parameters
0.4 0.4 G9. Background intensity (2).
Normalized z-value

Cluster 5 Cluster 7
Normalized z-value

0.3 Cluster 6 0.3 G10. Colocalization (2).

Cluster 8
k
0.2 0.2
0.1 0.1
0.0 0.0
–0.1 –0.1 Cluster Description Hits Total
–0.2 –0.2 group size
–0.3 –0.3
–0.4 –0.4 1 Selective up-regulation of TF endocytosis 932 5,258
G1
G1

G2
G3
G4
G5

G7
G1
G2
G3
G4
G5

G7
G1

G6

G2

G4

G9
G6

G2

G4

G9

G3

G5

G7
G3

G5

G7

G6

G10
G6

G10

Parameter group Parameter group 2 Selective down-regulation of TF endocytosis 88 586

e EGF TF Other f EGF TF Other Specific effect on subcellular localization: endosomes appear clustered in
0.4 0.4 3 801 2,990
Cluster 9 the cell centre
Normalized z-value

Normalized z-value

0.3 0.3 Cluster 11

0.2
Cluster 10
0.2 Specific effect on subcellular localization: endosomes appear dispersed in
0.1 0.1 4 260 1,505
the cell periphery
0.0 0.0
–0.1 –0.1 Opposite effects on EGF and TF endocytosis: EGF endocytosis is increased
5 224 1,158
–0.2 –0.2 and TF endocytosis is decreased
–0.3 –0.3 Opposite effects on EGF and TF endocytosis: EGF endocytosis is
–0.4 –0.4 6 143 670
decreased and TF endocytosis is increased
G1
G2
G3
G4
G5

G7
G1

G10
G6

G2

G4

G9
G3

G5

G7
G6

G1
G2
G3
G4
G5

G7
G1

G10
G6

G2

G4
G3

G5

G7
G9
G6

Parameter group Parameter group 7 Effects on endocytosis of both markers: increased EGF and TF endocytosis 137 631
g 0.4 EGF TF Other h 0.4 EGF TF Other 8 Effects on endocytosis of both markers: decreased EGF and TF endocytosis 799 2,256
Normalized z-value

Normalized z-value

0.3 Cluster 12 0.3 Cluster 13 9 Selective up-regulation of EGF endocytosis 178 695
0.2 0.2
0.1 0.1 10 Selective down-regulation of EGF endocytosis 324 1,291
0.0 0.0 Selective up-regulation of EGF endocytosis with accumulation of
–0.1 –0.1 11 271 1,012
endosomes in cell centre
–0.2 –0.2
–0.3 –0.3 12 Reduced TF endocytosis with endosomes accumulated in the cell centre 204 955
–0.4 –0.4 13 Selective increase in EGF endosomes number and elongation 38 140
G1

G10
G2
G3
G4
G5

G7
G1
G6

G2

G4

G9

G1
G3

G5

G7

G10
G2
G3
G4
G5

G7
G1
G6

G6

G2

G4

G9
G3

G5

G7
G6

Parameter group Parameter group 14 Increase in elongation of TF endosomes with mild increase of TF endocytosis 37 176

Figure 3 | Phenotypic cluster groups profiles. a–i, Mean-shift genomic and cluster group 8 (blue) present opposite effects on endocytosis of both
clustering of the gene profiles (see text and Supplementary Information) EGF and TF (both increased for cluster group 7, both decreased for cluster
yielded 14 cluster groups. Multiparametric profiles of the cluster groups are group 8). e, Cluster group 9 (black) and cluster group 10 (blue) present
plotted as in Fig. 1. The amplitude of the phenotype vectors was normalized selective and opposite effects on EGF endocytosis: increased and decreased,
to 1. a, Cluster group 1 (black) and cluster group 2 (blue) present selective respectively. f, Cluster group 11 presents increased EGF endocytosis and
and opposite effects, that is, increased vs decreased internal TF, respectively. endosome accumulation in the perinuclear region. g, Cluster group 12
b, Cluster group 3 (black) and cluster group 4 (blue) present specific and presents reduced TF endocytosis and endosomes accumulated in the cell
opposite effects on the subcellular localization of endosomes (‘distance from centre. h, Cluster group 13 presents a selective increase in EGF endosomes
nucleus’); that is, endosomes are either clustered in the perinuclear region number and their elongation. i, Cluster group 14 presents mainly an increase
(cluster group 3) or dispersed in the periphery (cluster group 4). c, Cluster in TF endosomes elongation. j, List of parameter-groups compiled as in
group 5 (black) and cluster group 6 (blue) is a pair of opposite clusters Fig. 1c. k, Table providing a summary of the most representative phenotypes
presenting each opposite effects with respect to EGF and TF endocytosis: in each group (‘Description’). The number of hits and the total size of each
increased EGF endocytosis with concomitantly decreased TF endocytosis group are also indicated.
(cluster group 5) and vice-versa (cluster group 6). d, Cluster group 7 (black)
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ARTICLES NATURE | Vol 464 | 11 March 2010

and signalling pathways; 10% (468, P 5 1 3 102214) could bioinfor- the possibility that the reduction in total internal cargo was due to
matically be assigned to the ‘core’ endocytic machinery, embracing downregulation of surface receptors (Supplementary Fig. 9), we sug-
established (CLTC, DNM2, EEA1) and predicted genes on the basis of gest a new role of these pathways in the regulation of endocytic
trafficking-relevant functional domains, such as VPS9 (8/10; uptake. We found for the Notch pathway NOTCH4, PSENEN and
P 5 6 3 1024), TBC (13/38; P 5 0.18), FYVE-finger (9/27; PSEN2, MAML1 and RBPJ and for the TGF-b pathway several genes
P 5 0.27)21, PX (14/41; P 5 0.17) or BAR (4/15; P 5 0.59)22 domains. encoding for activin receptors (ACVR1B, ACVR2A, ACVR2B and
Some genes (17%; 798; P 5 1 3 10236) were ‘unknown’ owing to ACVRL1).
absence of curated annotations. We found several novel potential In conclusion, the cluster analysis assigns new functions to meta-
endocytic regulators such as four TBC domain-containing proteins bolic and signalling pathways and further predicts functions for new
(MGC16169 (also known as TBCK), TBC1D9, TBC1D9B, genes in endocytosis.
TBC1D21), two FYVE-finger proteins (MTMR4 and WDFY1), and
a protein with a VPS9 domain (RINL). Notably, the list was enriched Differential regulation of TF and EGF trafficking
in genes implicated in different human diseases (Kyoto Encyclopedia To reveal general design principles of the endocytic system we con-
of Genes and Genomes database; see Supplementary Information) sidered all ,160,000 knockdown conditions as independent per-
such as different cancers (for example, glioma (P 5 1.26 3 1028), turbed states of the system and determined correlations between
colorectal (P 5 5.53 3 1026), pancreatic cancer (P 5 8.92 3 1026) parameters. A first interesting aspect emerging from such analysis
etc.), neurodegenerative and metabolic disorders (for example, was a high divergence in EGF and TF endocytosis. Despite the good
dentatorubral-pallidoluysian atrophy; P 5 7.8 3 1023), Huntington correlation between the size of EGF- and TF-positive endosomes
disease (P 5 0.026) and type II diabetes (P 5 3.7 3 1023). (Fig. 4a) and their intracellular localization (Fig. 4b), we found an
unexpected low correlation between the levels of total internal cargo
Regulation by metabolic and signalling pathways (Fig. 4c) of the two markers. These results strongly indicate that
The cluster analysis provided insights into how endocytosis is inte- although the molecular regulators of endosome size and distance
grated with other cellular functions and made predictions concerning from nucleus are essentially shared by EGF and TF, the machineries
the function of uncharacterized genes. Many cluster groups were controlling internalization and sorting are largely distinct. We
anti-correlated (Fig. 3a–e and Supplementary Fig. 8a, b), reflecting exclude a major contribution of alternative non-clathrin-dependent
positive and negative regulation on the endocytic system. For entry pathways29 under our experimental conditions, because deple-
example, ablation of genes in cluster groups 1 and 2 increased and tion of CLTC almost completely blocked the uptake of both cargo
decreased the amount of internalized TF, respectively (Fig. 3a and markers (Supplementary Fig. 1a). One possible interpretation is that
Supplementary Fig. 8a). Interestingly, genes of metabolic pathways EGF and TF may preferentially be internalized into endocytic vesicles
were highly enriched in cluster group 1, most probably reflecting a subjected to different regulation. For example, depletion of AP-2
compensatory response to a metabolic deficit by generally increasing complex specifically reduces TF but not EGF internalization
endocytosis of nutrients. (Supplementary Fig. 10)30, whereas knockdown of other genes such
Another example of anti-correlated phenotypes is provided by as PLEKHB2, CCDC33 and RCHY1 specifically downregulated EGF
cluster groups 3 and 4 (Fig. 3b and Supplementary Fig. 8b), where internalization.
the endosomes were localized either closer to or more distant from Interestingly, silencing of certain genes resulted in the dispersal of
the nuclei, respectively. We found enrichment (P 5 0.022 in cluster EGF into small, peripheral endosomes (Fig. 4e), a pattern reminiscent
group 3 and P 5 2.3 3 1023 in cluster group 4) of components of the of APPL1-endosomes31. These endosomes are largely distinct from
actin and tubulin cytoskeleton (for example, RAC1, ROCK2, canonical EEA1-positive early endosomes31 and act as signalling plat-
CAPZB, microtubule and actin motors KIF20A and MYH9). forms32. Knockdown of ZFYVE20, ANKFY1, PIK3R1, PIK3R2 along
Interestingly, in cluster group 4 we found genes involved in cytokin- with novel genes such as CCDC128 (also known as KLRAQ1), C12orf4,
esis such as CEP55 (ref. 23) and KIF20A (ref. 24), strengthening the MGC16169 (also known as TBCK) specifically affected EEA1-positive
link with endocytosis25. endosomes (Fig. 4d–e) and increased the colocalization of EGF with
Cluster groups 8 and 10 contained several known endocytic com- APPL1 (Fig. 4h–i). Conversely, knockdown of INPP5B and RABEP1
ponents. Cluster group 8 (decreased internal TF and EGF; Fig. 3d) specifically affected APPL1-positive endosomes (Fig. 4d–f) and con-
contained many genes encoding components required for endocytic comitantly decreased their colocalization with EGF (Fig. 4h–i).
uptake, such as CLTC, DNM2 and phosphatidylinositol kinases/ Altogether, we identified new components that regulate the sorting
phosphatases (INPP4A, INPP5B, PIB5PA, INPPL1; P 5 1.48 3 1027). of EGF to APPL1-positive endosomes.
Cluster group 10 contained several endosomal regulators such as
Rabankyrin-5 (ANKFY1)26, Rabenosyn-5 (ZFYVE20)27 and Hrs New design principles of the endocytic system
(HGS), indicating that EGF endocytosis is particularly susceptible to Our analysis also showed that various endosomal parameters are
depletion of PI(3)P effectors. On the basis of these results, we predict significantly correlated and, therefore, functionally linked. For both
that many new components of the endocytic core machinery are in EGF (Fig. 5a–c) and TF, we observed a negative correlation between
these cluster groups. mean size and number of endosomes, a positive correlation between
Signalling pathways exert distinct effects on the endocytic system. the number of endosomes and their mean distance from nucleus and
Here we confirmed the activity of mitogen-activated protein kinase a negative correlation between endosome size and distance from
(MAPK), Ca21, integrin/cell adhesion and mTOR signalling path- nucleus. Total internal cargo and cargo concentration per endosome
ways19,28 and uncovered the activity of several new ones, TGF-b/ (Fig. 5d) as well as number of endosomes (Fig. 5e) were also strongly
activin, Wnt and Notch. Some (MAPK, Ca21, mTOR, Wnt) had a correlated positively. In contrast, there was no correlation between
wide range of effects on the endocytic system (enriched in several total internal cargo and endosome size (Fig. 5f) and their distance
cluster groups; not shown) but others produced very specific effects from nucleus (not shown).
(enriched in single or opposite groups). For example, in cluster The analysis of pair correlations indicate that the endocytic para-
groups 3 and 4 we found enrichment in the integrin/cell adhesion meters are functionally linked, but does not infer cause–effect rela-
pathway (P 5 0.012 in cluster group 3 and P 5 6.6 3 1024 in cluster tionships. To infer directionality in these relationships and learn
group 4; ITGA5, ITGB1, ITGA9, PAK1, MAPK9, MAPK1). general rules underlying the organization of the endocytic system,
Interestingly, the Notch and TGF-b pathways were enriched we performed a Bayesian network analysis. The structure of Bayesian
(P 5 0.026 and P 5 5.6 3 1023, respectively) in cluster group 8 networks is a directed acyclic graph where nodes denote the variables
(reduced total internal cargo for both EGF and TF). As we excluded of interest and edges correspond to logical dependencies between
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NATURE | Vol 464 | 11 March 2010 ARTICLES

a c d e f
RABEP1/EGF

Total internal cargo TF

Correlation coefficient = 0.51 40 Correlation coefficient = 0.15
30
30
Mean size TF

20 20
10 10
0 0
–10
–10
–20 20 μm Control/EGF 20 μm CCDC128/EGF 20 μm
–20 –15 –10 –5 0 5 10 15 20 25 –20 –10 0 10 20 30 40 RABEP1/EEA1
Mean size EGF Total internal cargo EGF

b g h
30 Correlation coefficient = 0.86 0.6 Control 0.6 CCDC128

colocalization
colocalization
0.5

Percentage
Percentage 0.5
Distance from

20 0.4 0.4
nucleus TF

10 0.3 0.3
0.2 0.2
0 20 μm 20 μm
0.1 0.1 Control/EEA1 CCDC128/EEA1 20 μm
–10 0.0 0.0
–20 EGF>EEA1 EGF>APPL EGF>EEA1 EGF>APPL RABEP1/APPL
–30
i
–20 –10 0 10 20 30 0.6 RABEP1
colocalization

0.5
Percentage

Distance from nucleus EGF 0.4

0.3
0.2
0.1
0.0
EGF>EEA1 EGF>APPL Control/APPL1 CCDC128/APPL1

Figure 4 | Regulators of transport of cargo to EEA1 vs APPL endosomes. reduced the endosomal staining of APPL1 determining an increase of the
a–c, Correlation between endosome size (a), endosome distance from nuclei cytoplasmic staining. Insets show higher magnifications to visualize better
(b) and total internal cargo (c) for both EGF and TF. The data are the endosomal (co)-localization. Merge insets show always EGF overlaid on
represented as scatter plots of z-score values. The density of the data point is the indicated endosomal marker. Scale bars, 10 mm. White arrows indicate
indicated in blue for low- and in red for high-density values. Pearson the presence of EGF in the APPL1 and EEA1-positive endosomes in the
correlation values (correlation coefficient) are indicated for each scatter plot. merge images. Green arrows indicate the presence of EEA1 on the indicated
d–f, Examples of knockdown of genes affecting the accumulation of EGF in endosomes in the EGF and EEA1 insets. Light blue arrows indicate the
APPL1-positive endosomes. HeLa cells silenced for the indicated genes were presence of APPL1 on the indicated endosomes in the EGF and APPL1
fixed and stained for the indicated markers. d, Mock-transfected control insets. g–i, Quantification of EGF colocalization with the markers indicated.
cells. e, Silencing of CCDC128 selectively affected EEA1 endosomes EGF-to-APPL colocalization is increased upon silencing of CCDC128 (h) and
(enlarged endosomes in the perinuclear region), determining the decreased upon silencing of RABEP1 (i) compared to control cells (g). Error
accumulation of EGF in APPL1-positive endosomes. f, Silencing of RABEP1 bars indicate standard error of the mean (s.e.m.).

them33. We constructed two Bayesian networks for EGF and TF the endocytic system. The dependencies between the triplet endo-
(Fig. 5g–h), where the nodes correspond to the ‘key’ phenotypic some distance from nucleus–number–size reflect a progression from
parameters and the edges to their pair correlation. The two networks many small peripheral to few large perinuclear endosomes. Genes
shared a similar structure. Surprisingly, for both EGF and TF the involved in protein ubiquitination were found enriched among the
parameter measuring endosome distance from nucleus was found regulators of this process (cluster group 4 P 5 0.032). We found
to influence other parameters, indicating a key regulatory role in RBX1 and DDB1, encoding components of a multiprotein complex

a d
30 Correlation coefficient = –0.61 30 Correlation coefficient = 0.78
Total internal

20 20
g h
Mean size

0.86
10
cargo

10
0 0
–10 EGF TF
–10
–20 Total Total
–20 Distance Distance
internal internal
–20 –10 0 10 20 –20 –10 0 10 20 30 from nucleus from nucleus
cargo cargo
Number of endosomes Cargo concentration
b e
30 Correlation coefficient = 0.42
20 Correlation coefficient = 0.61 0.16 0.02
Distance from

Total internal

20 0.61 –0.33 0.27

0.79 0.89 0.38
10
nuclei

10
cargo

0 0
–10 –10 Cargo 0.79 Cargo Endosome Cargo 0.88 Cargo Endosome
–20 –20 concentration content number concentration content number
–30
–20 –10 0 10 20 –20 –10 0 10 20 30
Number of endosomes Number of endosomes –0.05
c f 30
–0.43 –0.61 0.03 0.3 –0.68
30 Correlation coefficient = –0.36 Correlation coefficient = –0.27
Distance from

Total internal

20 20
10 10
nuclei

Endosome Endosome
cargo

0 0 size size
–10 –10
–20 –20
–30 0.51
–20 –10 0 10 20 30 –20 –10 0 10 20 30
Mean size Endosome size

Figure 5 | Design principles of the endocytic system. a–f, Analysis of endosome number (e) and weak correlation between total internal cargo and
correlations between different endocytic parameters. The data are endosome size (f). g–h, Bayesian networks of the endocytic parameters for
represented as in Fig. 4 and only correlations between EGF parameters are the genome-wide analysis of EGF and TF endocytosis. The nodes represent
shown: negative correlation between endosome size and number (a), positive individual endocytic parameters measuring EGF or TF endocytosis and the
correlation between number and distance from nuclei (b), negative numbers indicate the Pearson correlation values. In black the direct
correlation between endosome size and endosomes distance from nuclei correlations shared by the two networks, in red (present) and grey (absent)
(c), positive correlation between total internal cargo and mean endosome correlations that present differences between the two networks.
concentration (d), positive correlation between total internal cargo and
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ARTICLES NATURE | Vol 464 | 11 March 2010

including cullins catalysing ubiquitin ligation34 and other genes such act as intracellular signalling platforms32,35, the system might regulate
as FBXO22, FBXO30, FBXO46, FBXO6, USP31 and HECTD3, indi- the concentration of EGF and the number of EGF-positive endo-
cating a new role of protein ubiquitination in the endosomal spatio- somes to quantitatively control signalling responses, for example,
temporal progression. for different cell-fate decisions, as shown for the MAPK network44.
However, the two networks also presented some interesting differ- Regulating the density of receptor tyrosine kinases, and number and
ences. The negative dependency of endosome cargo content on dis- distribution of endosomes may be necessary to calibrate the concen-
tance from nucleus reflects a progressive concentration of EGF in tration of signalling molecules and their signalling output. In iden-
endosomes, in contrast to TF that is continuously removed and tifying genes that selectively affect cargo transport to APPL1-positive
recycled to the surface. A comparison of the triplet concentration– endosomes, we unveiled a sorting mechanism to distribute cargo to
size–content revealed some unexpected properties of the endocytic this compartment. This is the beginning of a more detailed molecular
system. The TF network has a structure typical of passively sorted characterization of APPL1 endosomes, their regulation of transport
membrane proteins, where cargo content depends on size and con- and their role in signal propagation32,35. However, a limitation of our
centration of cargo per endosome. In contrast, the mean concentra- approach remains the poor identification of highly homologous and
tion of EGF in the endosomes is adjusted so that the mean total functionally redundant genes (for example, RAB5A, B, C and so on).
content of EGF per endosome remains constant, independently of This could be overcome by combinations of several siRNAs, as for
endosome size (negative dependence of the size on the concentration CBL, CBLB and CBLC (Supplementary Table 4).
and no correlation between size and content). Such a mechanism This exploratory endeavour required uncommon resources for a
cannot be explained only by sorting of EGF into intra-luminal vesicles9, primary screen. Nevertheless, it was necessary to establish the condi-
because (1) this is inconsistent with the high correlation between size of tions for streamlining the technology and rendering such genomic
EGF and TF endosomes and (2) the knockdown of HGS (Hrs) and profiling easier and more affordable in the future. Several genes iden-
TSG101 (ref. 9) does not alter the size–concentration–content relation- tified in the screen are associated with human diseases, confirming the
ship. These data rather indicate the existence of a quasi-deterministic fundamental role of endocytosis in the pathogenesis of many human
mechanism tightly coupling fusion and fission events to regulate the diseases and providing novel potential drug targets. It will be important
concentration of EGF in each endosome. to determine which parameters of the QMPIA might act as physio-
Finally, the cell exerts a tighter control on the number of EGF- than pathological indicators reflecting disease-relevant alterations. When
TF-positive endosomes, as indicated by the stronger dependence on applied to primary cells and disease model systems, the screening plat-
total internal cargo (corrEGF 5 0.61 vs corrTF 5 0.16). In other words, form described here will reveal its true potential for drug discovery,
to increase cargo capacity the system in addition to concentrating through the identification of novel therapeutic targets, new mechan-
EGF in endosomes (as for TF) also generates new endosomes. isms of action, and for discerning therapeutic effects of small molecules
Altogether, these findings indicate that the cell regulates the number, from toxic ones.
size, loading and, therefore, the intracellular density of EGF-bearing
endosomes. These findings have profound implications for the role of METHODS SUMMARY
endosomes in signalling31,35,36. RNAi screen for endocytosis. HeLa cells were reverse transfected with three
genomic libraries (Ambion, Qiagen and a custom-made esiRNA library15) using
Discussion 20 nM siRNAs or 25 ng per well esiRNAs. Seventy-two hours after transfection,
cells were incubated with 100 ng ml21 EGF-Alexa 488 and 5 mg ml21 Transferrin–
As a first step towards a systems analysis of endocytosis, we profiled Alexa 647 (Molecular Probes) in serum-free medium for 10 min at 37 uC before
the human genome relative to a set of 58 parameters that quantita- fixation with formaldehyde. Thereafter, nuclei and cytoplasm were stained with
tively describe endocytic features. From this survey we could attri- 0.4 mg ml21 DAPI and 0.2 mM SYTO42 blue (Molecular Probes).
bute new functions to known and unknown (798) genes with respect Automated image acquisition. Triple-colour images were acquired in a fully
to EGF and TF endocytosis. Furthermore, we revealed unexpected automated and unbiased manner using a spinning disk confocal microscope
properties of the endocytic system. First, we uncovered the effect of (OPERA, Evotec Technologies-PerkinElmer) and a 340 water immersion
various signalling pathways, notably integrin/cell adhesion, TGF-b, objective (NA 5 0.9). Twelve images per well were collected to obtain a sufficient
Notch and Wnt, on both TF and EGF endocytosis. The activity of number of cells for reliable statistical analysis. Image correction and image
these pathways depends on endocytosis37–39, but our data further analysis were performed using custom designed image analysis software
(MotionTracking; see Supplementary Information).
indicate that they exert a feedback mechanism on the endocytic
EEA1-APPL1 endosome dissection of EGF trafficking. Genes of interest (78)
machinery. Interestingly, TGF-b, Notch and Wnt pathways have were screened to detect alterations in the EEA1- or APPL1-positive endosomes
been implicated in the regulation of epithelial–mesenchymal trans- with respect to EGF trafficking. Following siRNA transfection and EGF–Alexa
ition (EMT)40, cell-fate determination41 and tissue morphogenesis42 488 internalization, cells were stained with mouse monoclonal anti-EEA1 (BD
and given the established role of endocytosis in these events2,43, we Biosciences; Pharmingen) and rabbit polyclonal anti-APPL1 (ref. 31) antibodies
propose that part of their morphogenetic activity depends on their and quadruple-colour images were acquired as described (Methods).
effects on the endocytic pathway. Second, a surprising result was the Detection of surface receptors. To exclude changes in the level of surface recep-
low correlation between genes regulating TF and EGF endocytosis tors upon downregulation of Notch and TGF-b signalling pathways, cells were
(Figs 5g–h and 4a–c). Differences in the molecular machinery regu- transfected with si/esiRNAs targeting these genes and 1,034 randomly selected
lating cargo uptake and recycling have been reported30, but such a si/esiRNAs, incubated with 100 ng ml21 EGF–Alexa 488 and 5 mg ml21 TF–Alexa
647 (Molecular Probes) in serum-free medium for 30 min on ice, fixed and
degree of diversity is unprecedented. Third, our analysis provided
stained with DAPI-SYTO42 as described earlier. Signals were enhanced by stain-
insights into the design principles underlying the endocytic system. ing with a rabbit anti-Alexa 488 (Molecular Probes) and mouse monoclonal
The fact that the endosome distance from the nucleus is a key antibodies against the ectodomain of human TFRC (BD Biosciences;
regulatory parameter demonstrates directly that the spatio-temporal Pharmingen) without permeabilization. Triple-colour images (ten per well) were
progression of endosomes observed in living cells12 is not an epiphe- collected with a 320 water immersion objective (NA 5 0.7) as described earlier.
nomenon but has a fundamental role in the function of the endocytic
system. The many components of the actin and tubulin cytoskeleton Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
identified in the screen (cluster groups 3 and 4) provide insights into
the molecular regulation underlying this mechanism. Received 1 December 2008; accepted 17 December 2009.
An interesting aspect emerging from the comparison of the EGF Published online 28 February; corrected 11 March 2010 (see full-text HTML version
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23. Carlton, J. G. & Martin-Serrano, J. Parallels between cytokinesis and retroviral ‘Endotrack’, which received research funding from the European Community’s
budding: a role for the ESCRT machinery. Science 316, 1908–1912 (2007). Sixth Framework Programme.
24. Hill, E., Clarke, M. & Barr, F. A. The Rab6-binding kinesin, Rab6-KIFL, is required
Author Contributions C.C., Y.K. and M.Z. conceived the project and M.Z. directed
for cytokinesis. EMBO J. 19, 5711–5719 (2000).
it. C.C. and D.K. developed the endocytosis assay under the guidance of M.Z. and
25. Baluska, F., Menzel, D. & Barlow, P. W. Cytokinesis in plant and animal cells:
E.F.; M.S. performed the screen and image acquisition. Y.K. developed the QMPIA
endosomes ’shut the door’. Dev. Biol. 294, 1–10 (2006).
with the help of C.C. and J.C.R. and performed all computational analysis of the
26. Schnatwinkel, C. et al. The Rab5 effector Rabankyrin-5 regulates and coordinates
screening data. N.S. developed the clustering and pheno-score algorithms and
different endocytic mechanisms. PLoS Biol. 2, e261 (2004).
together with C.C. performed the analysis of the clusters. C.R.B., under the
27. Nielsen, E. et al. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 supervision of B.H., performed the si/esiRNAs remapping and bioinformatics
and recruited to endosomes through a FYVE finger domain. J. Cell Biol. 151, analysis. CC. and Y.K performed the Bayesian Network analysis. F.B. provided the
601–612 (2000).
esiRNA library. R.H., under the supervision of M.S.M. and W.E.N., provided IT
28. Galvez, T. et al. siRNA screen of the human signaling proteome identifies the support for the use of the computer cluster located at the TUD. C.C.,Y.K. and M.Z.
PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin wrote the manuscript.
uptake. Genome Biol. 8, R142 (2007).
29. Sigismund, S. et al. Clathrin-independent endocytosis of ubiquitinated cargos. Author Information Reprints and permissions information is available at
Proc. Natl Acad. Sci. USA 102, 2760–2765 (2005). www.nature.com/reprints. The authors declare no competing financial interests.
30. Motley, A., Bright, N. A., Seaman, M. N. & Robinson, M. S. Clathrin-mediated Correspondence and requests for materials should be addressed to M.Z.
endocytosis in AP-2-depleted cells. J. Cell Biol. 162, 909–918 (2003). (zerial@mpi-cbg.de).

249
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08779

METHODS incubated for 72 h at 37 uC and 5% CO2. Thereafter, the cells were incubated with
siRNA and esiRNA libraries and remapping to RefSeq 28. Three genomic 100 ng ml21 EGF–Alexa 488 and 5 mg ml21 transferrin–Alexa 647 (Molecular
RNAi libraries, two commercial siRNA libraries (Ambion Silencer Genome wide Probes) in serum-free medium for 10 min at 37 uC. Cells were subsequently
siRNA Library V.3 and Qiagen Human Whole Genome siRNA Library V.1) and washed with PBS and fixed with 3.7% formaldehyde. Nuclei and cytoplasm were
a custom-made esiRNA library15 were screened. The Ambion library included stained with 0.4 mg ml21 DAPI and 0.2 mM SYTO42 blue (Molecular Probes).
51,880 siRNAs targeting 22,527 genes in the human genome and was designed For the analysis of protein downregulation, cells were grown in 96-well plates
partly on the basis of the RefSeq database Release 15 and partly on the ENSEMBL and transfected with 20 nM siRNAs (final concentration) with 0.3 ml per well
database. The Qiagen library, which included 91,956 siRNAs targeting 22,732 Oligofectamine (Invitrogen) in a total volume of 100 ml. After 72 h total protein
genes, was entirely designed on the basis of the RefSeq database (kinase/ extracts were prepared and analysed by quantitative western blotting using
phosphatase and gpcr subsets: Refseq Release 18, Druggable Genome Set: antibodies against EEA1, ZFYVE20 and ANKFY1.
Refseq Release 17, the rest of the Whole Genome Set and the Predicted Genes Automated spinning disk confocal imaging. Twelve images per well were col-
Set RefSeq Release 12). The esiRNA library was prepared as described previ- lected with a 340 water immersion objective (NA 5 0.9) in a fully automated and
ously13 and included 17,188 esiRNAs targeting 16,722 human genes, designed on unbiased manner with a spinning disk confocal microscope (OPERA, Evotec
the basis of the ENSEMBL (Release 36) database. Ambion and Qiagen validated Technologies-Perkin Elmer). Triple-colour images were acquired in two sequen-
768 and 3,073 siRNAs by qRT–PCR, respectively. Out of these, 740 (Ambion) tial exposures by three different CCD cameras: the Alexa 488 signal was collected
and 3,072 (Qiagen) were shown to be efficient (.70% reduction) at silencing in a first exposure using 488 nm laser light and a 540/75 band-pass filter; Alexa 647
their corresponding target mRNAs, providing an overall estimated efficiency of and DAPI-SYTO 42 blue signals were collected in a second exposure using 635 nm
.95% for the Ambion and .99% for the Qiagen libraries, respectively. The and 405 nm laser light and with 700/90 and 450/50 band-pass filters, respectively.
sequences of all 3 libraries were remapped on the RefSeq Release 28 database by Images were subsequently corrected (MotionTracking; see Supplementary
BLAST. The sequence of an siRNA or esiRNA was mapped to a given gene on the Information) for uneven field illumination and chromatic aberrations according
basis of the following three conditions: (1) 19/19 bases match on a region on the to reference images. For reference, images of 2.5 mm multicolour beads, and
mRNA, (2) matching with all transcripts (including differentially spliced images of reference dyes and dark field images were collected using the Opera
mRNAs) of a given gene, (3) absence of matching with any other transcripts Adjustment Plate (Evotec Technologies-Perkin Elmer).
from other genes. All siRNAs/esiRNAs that did not meet these conditions were Three-dimensional imaging of the endosome distribution of the cell was per-
discarded from further analysis. After re-mapping to RefSeq Release 28, the 3 formed on a small set of 27 siRNAs (targeting 10 genes of the human genome).
libraries covered 33,223 genes of which 28,279 encode for proteins or contain an Using the same microscope and automated imaging protocols as above 10 focal
open reading frame, corresponding to 62% coverage of the transcriptome and planes with 0.5 mm steps were acquired.
73% of the proteome. EEA1-APPL1 endosome dissection of EGF trafficking. A smaller set of 78 genes
HT RNAi transfection and endocytic assay. HeLa cells were reverse transfected was further screened to detect alterations in the EEA1- or APPL1-positive endo-
with 20 nM siRNAs or 25 ng per well esiRNAs (final concentration) with 0.2 ml per somes with respect to EGF trafficking. For this, cells were transfected, EGF–Alexa
well Oligofectamine (Invitrogen) in a total volume of 50 ml. For HT-transfection a 488 was internalized and cells were fixed as above. Cells were then permeabilized
‘Freedom Evo’ pipetting robot (Tecan) equipped with a 384-fixed-needle-head, with 0.1% saponin and 0.2% fish gelatin (as blocking agent) and subsequently
an 8-needle-pipettor and a gripper was used under a customized biological safety stained with mouse monoclonal anti-EEA1 (BD Biosciences Pharmingen) and
cabinet. A fully automated workflow prepared transfection complexes in 384 wells rabbit polyclonal anti-APPL1 (ref. 31) antibodies. Fluorescently conjugated goat
at a throughput of 5 plates/50 min (manuscript submitted). For cell seeding and anti-rabbit–Alexa 568 and goat anti-mouse–Alexa 647 secondary antibodies
dispensing the assay solutions the MTP dispensers WellMate and Multidrop 384 (Molecular Probes) revealed the antigen signals and DAPI-SYTO 42 blue stained
(Thermo Scientific) were used; wash procedures were performed using the Power the nuclei and cytoplasm. Quadruple-colour confocal images were collected
Washer 384 (Tecan). (OPERA, Evotec Technologies-PerkinElmer) in two sequential exposures:
Briefly, 10 ml of 200 nM siRNAs or 5 ng ml21 esiRNAs were mixed with 10 ml of Alexa 488 and Alexa 647 signals were collected in a first exposure using 488 nm
pre-diluted Oligofectamine (40 ml ml21 OptiMem) for 15 min to allow complex and 635 nm laser light and with 540/75 and 690/50 band pass filters; Alexa 568 and
formation. Subsequently, 10 ml of this mixture were placed in 384-well DAPI-SYTO 42 blue signals were collected in a second exposure using 405 nm and
CellCarrier plates (Evotec Technologies). HeLa cells (1000 cells per well, counted 561 nm laser light and with 450/50 and 605/40 band-pass filters, respectively.
with CASY Model TT, Schärfe System) were seeded in 40 ml per well. Plates were Reference images were acquired and image correction performed as before.

©2010 Macmillan Publishers Limited. All rights reserved

 Vol 464 | 11 March 2010 | doi:10.1038/nature08756

ARTICLES
The primary transcriptome of the major
human pathogen Helicobacter pylori
Cynthia M. Sharma1, Steve Hoffmann2, Fabien Darfeuille3,4, Jérémy Reignier3,4, Sven Findeiß2, Alexandra Sittka1,
Sandrine Chabas3,4, Kristin Reiche5, Jörg Hackermüller5, Richard Reinhardt6, Peter F. Stadler2,5,7,8,9 & Jörg Vogel1,10
Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human
pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA
sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach
(dRNA-seq) selective for the 59 end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start
sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes,
indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and
by genome-wide antisense transcription. We also discovered an unexpected number of ,60 small RNAs including the
e-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and
trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary
transcriptomes of many living species.

About 50% of all humans are infected with Helicobacter pylori, a micro- Fig. 1). We primarily analysed H. pylori strain 26695 grown to mid-
aerophilic, Gram-negative e-proteobacterium that thrives in the acidic logarithmic phase (ML2/1 libraries), or under acid stress at pH 5.2
environment of the stomach, and is associated with severe inflam- (AS2/1) resembling the host environment. To increase data depths,
mation, peptic ulcer disease and gastric cancer1,2. The 1.67 megabase bacteria were also grown in contact with responsive gastric epithelial
pairs genome of H. pylori strain 26695 carries 1,576 open reading cells (AG2/1) or non-responsive liver cells (HU2/1), or in cell
frames (ORFs), but surprisingly few genes of transcriptional regula- culture medium alone (PL2/1) (Supplementary Fig. 2). Following
tors3,4. Moreover, only ,55 transcriptional start sites (TSS; compiled in 454 pyrosequencing, ,217 million bases of cDNA (Supplementary
Supplementary Table 1) were known in this important human patho- Table 2) were mapped to the H. pylori chromosome (Fig. 1a).
gen, and the principal organization of its transcriptome remained to be dRNA-seq confirmed the known acid induction10–12 of major
elucidated. Small noncoding RNAs (sRNAs), an otherwise abundant H. pylori virulence loci such as the urease (ure) operon or the cag
class of post-transcriptional regulators in bacteria5,6, also seemed to be pathogenicity island (Fig. 1b), as evident from a three- to fourfold
lacking in H. pylori, perhaps reflecting the absence of the common higher cDNA coverage of the ureA, ureI or cag16 transcripts in the
sRNA chaperone, Hfq, in all e-proteobacteria7. AS2 vs ML2 libraries (Supplementary Table 3). Crucially, the
Global analyses using tiling arrays and RNA-seq have provided exonuclease treatment revealed TSS by means of a characteristic redis-
invaluable insights into the gene expression patterns and sRNA output tribution of a gene’s cDNAs towards a sawtooth-like profile with an
of diverse bacteria, including several pathogens8,9. Yet, these studies elevated sharp 59 flank. For example, the AS1 cDNAs clustered
did not directly detect TSS owing to a lack of discrimination between towards the nuclease-protected primary 59 end of ureA mRNA, match-
primary and processed transcripts. Specifically, primary transcripts ing the known TSS13,14, whereas the AS2 cDNAs were uniformly dis-
including most mRNAs and sRNAs carry a 59 tri-phosphate tributed over ureA (Fig. 1b). Thus, the selective destruction of
(59PPP) group, whereas processed transcripts such as mature ribo- processed 59P transcripts enriches TSS-specific cDNAs (Supplemen-
somal and transfer RNAs (rRNA, tRNA) have a 59 mono-phosphate tary Fig. 2). Similar patterns mark the cagA TSS15 and reveal primary
(59P). Using a novel dRNA-seq approach to selectively identify native and processed 59 ends of tRNA-Phe (Fig. 1c). Altogether, 69 H. pylori
59PPP ends we present a single-nucleotide resolution map of the TSS determined by independent methods were matched by dRNA-seq
primary transcriptome of H. pylori and unravel the unexpectedly with high accuracy (Fig. 1d).
complex RNA output from this small and compact genome.
Genome-wide TSS and operon maps
Differential RNA-seq Annotation of 59 ends enriched in (1) vs (2) libraries and satisfying
Our dRNA-seq approach discriminates primary from processed 59 other plausible criteria identified a total of 1,907 TSS (Supplementary
ends by sequencing differential cDNA library pairs: one library Table 4). These were grouped into five categories (Fig. 2a): primary
denoted (2) from untreated total bacterial RNA, and the other TSS having the most cDNAs within #500 bp upstream of annotated
(1) enriched for primary transcripts by terminator exonuclease mRNA start codons or processed sRNAs; secondary TSS associated
treatment that degrades 59P but not 59PPP RNA (Supplementary with the same gene but with fewer cDNAs; internal TSS within an
1
Max Planck Institute for Infection Biology, RNA Biology Group, D-10117 Berlin, Germany. 2University of Leipzig, Department of Computer Science & Interdisciplinary Centre for
Bioinformatics, D-04107 Leipzig, Germany. 3INSERM U869 and 4Université de Bordeaux, F-33076 Bordeaux Cedex, France. 5Fraunhofer Institute for Cell Therapy and Immunology,
RNomics Group, D-04103 Leipzig, Germany. 6Max Planck Institute for Molecular Genetics, D-14195 Berlin, Germany. 7Max Planck Institute for the Mathematics in Sciences, D-04103
Leipzig, Germany. 8University of Vienna, Institute for Theoretical Chemistry, A-1090 Vienna, Austria. 9The Santa Fe Institute, Santa Fe, 87501 New Mexico, USA. 10University of
Würzburg, Institute for Molecular Infection Biology, D-97080 Würzburg, Germany.

250
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

a log2 c
mapped reads (–) cDNA, no treatment

Leading strand
18 (+) cDNA, 5′PPP enriched
(–)
0
(+)
tRNA
rRNA AUG
mRNA
cagA

Lagging strand
18
(–)
0
(+)

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.67

b tRNA-Phe
Urease operon cag pathogenicity island
RNase P cleavage
ML –

Leading strand
ML +
d
50
Relative score

AS – 87%

Number of TSS
0.4 0.1 40
0.2 AS + 0.05
0 0 30
cag11 cag12 cag16 cag17
cagA 20
cag10 cag23
ureH ureF ureE ureI ureB ureA 10
ca 33
ca g13

8
9

ca 20
ca 21
2

cag24
5
4

HP0068
05

g1
g1

g2

g2
g1

g1
Relative score

g
g
HP

tRNA-Val

Lagging strand
ca
ca

ca

ca
ca

0 0 0
<–100 <–2 –2 –1 0 1 2 >2 >100
–0.2 ML – –0.1
–0.4 –0.2 Difference (nt)
ML +

AS –

AS +
cag22–cag18 suboperon
cag25–cag18 operon
00

00

00

00

0
00

00

00

00

00

00

00

00

00

00

00
,0

,0

,0

,0

4,

6,

8,

0,

2,

4,

6,

8,

0,

2,

4,
72

74

76

78

56

56

56

57

57

57

57

57

58

58

58
Figure 1 | H. pylori TSS revealed by dRNA-seq. a, Combined cDNA reads and are consistent for all libraries of the same strand (maximum of 0.1 for
without (blue, (2) libraries) or with (orange, (1) libraries) terminator leading strand; minimum of 20.2 for lagging strand). c, Schematic drawing
exonuclease treatment mapped to and plotted as log2 values over the H. of cDNA enrichment patterns (here in AS2/1 libraries) at primary 59 ends
pylori chromosome. All libraries were adjusted to same scale. Vertical bars of cagA mRNA (top) or tRNA-Phe precursor (bottom). Exonuclease
indicate tRNA (green), rRNA (blue) or mRNA (grey) gene clusters. b, cDNAs treatment (AS1 library; red curve) shifts the cagA cDNAs towards the
of mid-log growth (ML2/1) and acid stress (AS2/1) libraries mapped onto nuclease-protected 59-end, yielding a sawtooth-like profile with an elevated
urease operon (left) or cag pathogenicity island (right) annotations in sharp 59 flank that matches the published TSS15. In contrast, the mature
forward and reverse direction (genome position at the bottom). Black and (RNase P-cleaved) 59 end of tRNA-Phe is predominant in the AS (2) library
grey arrows denote published (ureA13,14, ureI49,50, cagA/B15) and newly (black curve). d, Histogram indicating observed distances between 69 TSS
identified TSS, respectively. Annotation of cagB (open symbol) according to identified by dRNA-seq vs independent experimental approaches
H. pylori strain G27. Dotted lines exemplify the primary cag25–cag18 operon (Supplementary Table 1). Of these TSS 87% matched within 62 nt tolerance
and its associated cag22–cag18 suboperon transcribed by the internal TSS in (all 22 TSS mapped by 59 RACE in Supplementary Fig. 3 match with 61 nt).
cag23. Scales give a relative score (% mapped reads per genome position)

annotated gene on the same strand; antisense TSS situated inside or revealed an extended Pribnow box (tgnTAtaAT) as the 210 motif of
within #100 bp of an oppositely encoded gene; and orphan TSS the housekeeping s80 in H. pylori (Fig. 2b; for s28 and s54, see Sup-
without annotated genes in proximity. Consequently, multiple asso- plementary Tables 6–8 and Supplementary Fig. 5). Intriguingly, the
ciations of TSS are not uncommon (Fig. 2a). 235 motif is replaced by a periodic AT-rich signal upstream of
H. pylori was thought to lack the extensive operon structure3,16 typical position 214. Thus, consistent with earlier analysis of much fewer
of other bacterial genomes. We complemented our TSS map with promoters in H. pylori20 or related Campylobacter jejuni21, our global
DOOR17 and conventional RNA-seq analyses to assign 87.5% of all H. TSS map shows that transcription in e-proteobacteria initiates at
pylori genes to 337 primary operons (Supplementary Table 5), present- extended Pribnow boxes downstream of periodic AT-rich stretches.
ing the first operon map based on knowledge of transcriptional ini-
tiation. dRNA-seq readily detected internal TSS of downstream Massive antisense transcription
cistrons within longer primary operons, predicting 126 additional sub- The H. pylori transcriptome is highly complex (Fig. 2a), and 27%
operons and 66 monocistrons overlapping the 39 part of polycistrons in of the primary TSS are also antisense TSS, indicating that—similar to
H. pylori. Independent validation of such suboperonic signals (Sup- E. coli22—antisense transcription occurs across the entire H. pylori
plementary Fig. 4) confirmed an acid-induced internal TSS in cag23 genome (Fig. 3a, Supplementary Fig. 6 and Supplementary Table 9).
(Fig. 1b), supporting the known upregulation of cag22–cag18 from Because the antisense TSS are uncorrelated with local GC content
the primary cag25–18 operon under acid stress10. Likewise, internal (Fig. 3a), they are unlikely to reflect promiscuous transcription ini-
TSS reveal potential uncoupling of suboperons starting with ftsH (cell tiation at AT-rich hexamers. Furthermore, analysis of biological repli-
division) or copP (copper transport) from the physiologically unrelated cates (ML2/1 libraries), control experiments using actinomycin D to
HP1067 (cheY) chemotaxis gene in the HP1067–HP1074 operon18. suppress unspecific cDNA synthesis (Supplementary Information),
Bacterial promoters usually contain specific sequences for binding and independent northern blot probing (Supplementary Fig. 7),
of RNA polymerase (RNAP)-associated s factors, for example, the consistently show that the many detected antisense TSS are not
235 (TTGACA) and 210 (TATAAT) boxes of the housekeeping s70 artefacts of library construction. Rather, their high number might
in Escherichia coli19. Correspondingly, motif searches at the 1,907 TSS indicate that transcriptional initiation is an important cause of global
251
©2010 Macmillan Publishers Limited. All rights reserved
ARTICLES NATURE | Vol 464 | 11 March 2010

a b 1,000
P I P P,I O
S 800
Gene 1 Gene 2 Gene 3 Gene 4

Counts
600
<500 <100 <100
400
A A
200
Secondary Internal
0
119 439 –20 –15 –10 –5
Primary Antisense Extended –10 box position relative to TSS
812 31 244 969
2
0 36 12
451 686
0 2

Bits
1
0 Orphan
142 50
3 0
38 0

–40
–39
–38
–37
–36
–35
–34
–33
–32
–31
–30
–29
–28
–27
–26
–25
–24
–23
–22
–21
–20
–19
–18
–17
–16
–15
–14
–13
–12
–11
–10
–9
–8
–7
–6
–5
–4
–3
–2
–1
+1
216
5′ 3′

c d Reannotation
30 Old AUG TGNTATAAT SD AUG
26695
Leaderless mRNA HPAG1
SD sequence 25 J99
2 Hac
Bits

1 Consensus

Number of mRNAs
0 20

15

8
01
P1
H
Relative score
10 pgsA rocE htrA
0.2
5 0.1 AS –
0
0 AS +
–400 –200 –100 –80 –60 –40 –20
5′ UTR length (nt) 1,079,000 1,079,500 1,080,000 1,080,500 1,081,000 1,081,500

Figure 2 | TSS annotation and 59 mRNA structure. a, Top: representation of which 1,645/1,906 (,86%) lie 211, 210 and 29 bp relative to TSS (top,
categories for TSS annotation based on expression strength and genomic histogram of distribution; bottom, logo of upstream TSS sequences). The
context: primary (P), secondary (S), internal (I), antisense (A), or orphan dotted line indicates the upstream periodic AT-stretch signal. See
(O). Bottom: Venn diagram showing overlap among TSS categories. Many of Supplementary Table 7 for statistical analysis. c, Frequency of individual 59
the 1,907 H. pylori TSS associate with multiple categories (see TSS in gene 2 UTR length based on 825 TSS (primary and secondary) of mRNAs. 59 UTR
above), yielding 2,496 TSS associations in total (Supplementary Table 4). lengths ,10 nt (red bars) reveal 34 leaderless mRNAs. The inset shows the
Antisense TSS were detected for 721/1,576 (,46%) of annotated ORFs; SD sequence motif of H. pylori. d, Re-annotation of rocE gene prompted by a
primary TSS for 717 ORFs; 106 secondary TSS for 98 ORFs; 428 internal TSS newly discovered TSS downstream of its originally annotated start codon
for 363 ORFs. Primary TSS commonly reside in upstream ORFs: 142/812 (grey box). Reads of AS2/1 libraries mapping to the pgsA-rocE-HP1018-
(,17%) primary TSS are also internal; 142/439 (,32%) internal TSS are htrA region are shown below an alignment of rocE sequences (59 region) of
also primary. 216/812 primary TSS (,27%) are antisense TSS, revealing diverse Helicobacter strains. The highly conserved re-annotated AUG, its SD
profound global antisense transcription. b, Motif searches upstream of and the 210 box of the new TSS are boxed.
H. pylori TSS reveal extended Pribnow boxes (210 signal: tgnTAtaAT) of

antisense transcription in H. pylori, in addition to possible imperfect ,6 nt (median distance) upstream of start codons as the consensus
termination22. Shine–Dalgarno sequence in H. pylori (Fig. 2c). We found correlations
At least one antisense TSS is associated with ,46% of all ORFs, of 59UTR length with cellular function. For example, nucleotide- and
including many housekeeping genes such as valS (valyl-tRNA synthe- nucleoside-related genes almost invariably have ,30-nt-long 59UTRs,
tase) or rpl21 (ribosomal protein L21), although there is no general as if optimized for translation. In contrast, regulatory genes of cellular
bias towards core or variable H. pylori genes23 (data not shown). processes such as cell division, pathogenesis or transformation possess
Moreover, ,28% of the tRNAs, and the 59 leaders of 23S and 16S significantly longer 59UTRs (Supplementary Fig. 8). Structural align-
rRNA precursors have antisense TSS. Notably, as H. pylori lacks ment of the H. pylori 59UTRs with Rfam database families detected a
endoRNases E/G and other common processing factors of stable thiamine pyrophosphate riboswitch upstream of pnuC. In support of
RNAs3, antisense-guided cleavage involving the dsRNA-specific the previously predicted transcriptional attenuation mechanism30, both
RNase III (ref. 3) might compensate for this paucity. RNA-seq and northern blot probing observed a short (,100 nt) tran-
We identified antisense TSS for 22 of 34 putative phase-variable script from this candidate riboswitch (Supplementary Fig. 9). Although
genes24 featuring homopolymeric tracts and dinucleotide repeats, no other known riboswitches were detected, conforming to their general
and with functions in lipopolysaccharide biosynthesis, surface struc- paucity in Gram-negative species, there are 337 UTRs long enough
ture and DNA restriction/modification (Supplementary Table 10). (.60 nt) to harbour novel cis-regulatory RNA structures31.
Of these, the two fucT copies (HP0651/0379) encode the fucosyl- Although leaderless mRNAs are considered rare and primarily
transferases that modify the major Lewis antigen of H. pylori. Our phage-associated in Gram-negative species32, we found that ,2.2% of
discovery of antisense TSS adjacent to the 59 poly(C) tracts involved all H. pylori mRNAs have a 59UTR ,10 nt (Supplementary Table 11).
in switching on/off the fucT genes by slipped-strand mispairing25,26 At 26 genes, including the dnaA, recR and hemH housekeeping genes,
raises the possibility of antisense regulation of surface structure transcription initiates exactly at an AUG start codon essential for stable
variations and host interactions. Moreover, acid-stress-induced ribosome binding of leaderless mRNAs32.
antisense RNAs opposite to known acid-stress-repressed genes (for Some primary TSS lay downstream of previously annotated start
example, rpl21, HP1186, HP0637; refs 10, 11) would indicate similar codons, as exemplified by rocE (Fig. 2d). Yet, sequence conservation
control at low pH (Supplementary Fig. 7). among Helicobacter strains strongly supported the rocE TSS mapped
by dRNA-seq, as well as a new start codon ,30 bp downstream.
59 UTRs and leaderless mRNAs Similarly, we propose corrections for 18 more genes (Supplementary
The 59UTR (untranslated region) ranging from TSS to start codon Table 12), complementing re-annotations by genome comparison33.
determines the translational efficiency of messengers. In striking accor-
dance with the structurally observed ribosome contacts27, our TSS An unexpected wealth of Helicobacter sRNAs
annotation reveals that most (,50%) of the 59UTRs are 20–40 nucleo- Previous annotations in H. pylori predicted stable tRNAs, rRNAs,
tides (nt) in length (Fig. 2c), and support the AAGGag motif28,29 located transfer-messenger RNA (tmRNA), RNase P and signal recognition
252
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 ARTICLES

a 20
experiments (Supplementary Figs 10–14 and Supplementary
log-normalized

Antisense sRNA Small ORF Sense in ORF 6S RNA

Table 14), among these are all five members of a ,200 nt sRNA
expression

10

0
family whose multiplicity is reminiscent of the Qrr sRNAs that con-
10
trol quorum sensing and virulence in Vibrio bacteria34.
20
Plasticity zone
6S We have also identified 6S RNA (,180 nt), an abundant and
GC content

0.45 ubiquitous riboregulator of RNAP. 6S RNA notoriously failed to be

0.35 discovered in the e-subdivision31,35,36, perhaps because it is expressed
0.25 Plasticity zone cag antisense to HP1219 (Fig. 3b,c), a poorly conserved hypothetical ORF
0 0.5 1.0 1.5 1.67 (Supplementary Fig. 15). Structural probing experiments in vitro
b pRNA* pRNA c (Fig. 3d) and conservation analysis (Supplementary Fig. 16) indicate
0.3 0.6 0.8 1.2 1.7 2.0 A600
AAUAAGGCUAGGAGGAU AACGAACUUGCC
HP1219 (hypothetical) 6S that H. pylori 6S RNA adopts the characteristic structure of a long
ML– hairpin with a central asymmetric bulge by which E. coli 6S RNA
ML+
5S
sequesters RNAP35–37. To disentangle itself, RNAP uses 6S RNA as
0.1
AS– template for transcription of 14–20 nt RNA products (pRNAs)37.
We detected two classes of pRNAs in H. pylori (Fig. 3b), one starting
Relative score

0.05 AS+
0

1,295,300 1,295,400 1,205,500 1,295,600 1,295,700

with the corresponding bulge-internal adenosine of E. coli pRNAs37,
0
–6 ML– and the other (pRNA*) originating from the opposite strand as previ-
–12
ML+ ously observed with certain 6S RNA mutants in vitro (K. Wassarman,
AS– personal communication). Our in vivo detection of pRNAs in a
AS+ remote relative of E. coli shows that 6S RNA regulation of RNAP
purD yhcG
6S RNA
activity is a widely conserved mechanism.
Several of the new H. pylori sRNAs seem as abundant as 6S RNA
d (Fig. 3a), and most of them are conserved at the sequence level among
Le as T1
Lead T2
++
ad +
e
e
RNas

90
Helicobacter species but not outside the e-subdivision (data not
H
RN
T1
O
C

shown). In global structural clustering analysis assaying conservation

G151
G143 of functional secondary structure rather than primary sequence, we
G130
G121 110 observed that whereas the c-proteobacterial sRNAs of E. coli and
G108
G100
70 Salmonella form large groups of similar structures, the H. pylori
G85 Lead(II) sRNAs fall in small groups exhibiting specific structural motifs
G77
G71
RNase T2
RNase T1
(Supplementary Fig. 17). Thus, except for 6S RNA and housekeeping
G64 RNAs, e-proteobacteria including H. pylori might have evolved a
G59
G54
unique sRNA repertoire.
pRNA* Many sRNAs in other bacteria repress trans-encoded mRNAs by
G44
G41 pRNA short base-pairing in the 59UTR5,6. Here, the TargetRNA program38
G37 predicted an interaction of the abundant HPnc5490 sRNA with the
G32
59UTR of tlpB encoding a canonical chemotaxis receptor of H. pylori
G29
(Fig. 4a). The involvement of accessible loop residues of HPnc5490,
and the calculated RNA duplex strength (DG 5 234.3 kcal mol21)
G19 added confidence to this prediction. An HPnc5490 deletion mutant
G17
was generated, and observed to have increased levels of the ,60 kDa
G12 170 TlpB protein (Fig. 4b) and the tlpB-HP0102 operon mRNA, yet
G9
10 normal expression of the remaining canonical chemotaxis receptor
genes, tlpA and tlpC (Fig. 4c). Thus, HPnc5490 probably regulates
5′ 3′
tlpB as a trans-antisense RNA.
Figure 3 | Discovery of H. pylori sRNAs including 6S RNA. a, Relative We identified a family of six structurally related ,80 nt sRNAs
expression of candidate and validated sRNAs according to log2 values of read expressed antisense to small ORFs of homologous 22–30 amino acid
numbers (first 50 nt downstream of TSS) of all sequenced libraries (upper peptides (Fig. 4d, e and Supplementary Fig. 18), henceforth referred
part) compared to local GC content (lower part) plotted across the H. pylori
to as IsoA1-6 (RNA inhibitor of small ORF family A) and aapA1-6
genome. b, Reads in the ML and AS libraries show a highly expressed 180 nt
sRNA candidate, now H. pylori 6S RNA, in the yhcG–purD intergenic region (antisense RNA-associated peptide family A), respectively. Five of the
and opposite to annotated (yet questionable) ORF HP1219. Determined aapA ORFs produced stable mRNAs in vivo (Fig. 4e). In vitro trans-
sequences of detected 6S RNA-specific pRNA/pRNA* including their 6S lation assays yielded the expected small peptides, except for aapA2
RNA-borne TSS are shown at the top. c, Northern blot detection of 6S RNA mRNA whose Shine–Dalgarno sequence is mutated in strain 26695
(and 5S rRNA as loading control) from exponential growth to late stationary (not shown). Furthermore, translation of aapA1 or aapA3 was
phase in BHI medium. d, In vitro structure probing (left) of 59end-labelled strongly and specifically inhibited in the presence of the cognate
6S RNA of H. pylori. C, T1 and OH identify no reaction, digestion under IsoA1 or IsoA3 sRNAs (Fig. 4f), thus revealing candidates of cis-
denaturing conditions with nuclease T1 (cleaved G residues given to the antisense regulation in H. pylori.
left), or alkali, respectively. The right four lanes reveal cleavages induced by
The AapA peptides are conserved in other H. pylori strains
RNase T1, T2, or lead(II), whose positions are indicated in the derived
secondary structure of 6S RNA (right panel) by black, red, and grey circles, (Supplementary Fig. 19) and might interact with membranes, as
respectively. The template nucleotides of the detected pRNA and pRNA* suggested by their predicted high hydrophobicity and a-helical struc-
sequences are framed in black or blue, respectively. ture (Fig. 4d and Supplementary Fig. 20), as well as similarities to
Antimicrobial Peptide Database39 entries such as human defensin
particle RNA (SRP RNA), all of which were here confirmed to be LL-37. Therefore, the aapA–isoA loci might be toxin-antitoxin
expressed. Moreover, the dRNA-seq data predicted hundreds of addi- systems that slow down growth of H. pylori or other organisms in
tional sRNA candidates (denoted HPnc) from intergenic regions, the gastric mucosa, protect against phages, or facilitate the proposed
antisense to ORFs, and also sense within ORFs (Fig. 3a; Supplemen- (though controversial) altruistic autolysis of H. pylori40 which was
tary Fig. 10 and Supplementary Table 13). We have so far validated postulated to involve as yet unknown ,3.5 kDa hydrophobic pep-
the expression of ,60 new sRNAs by independent northern blot tides41. Whereas the AapA peptides remain to be detected in vivo, our
253
©2010 Macmillan Publishers Limited. All rights reserved
ARTICLES NATURE | Vol 464 | 11 March 2010

interaction map43. The data provide new insights into the organiza-

0
a b c

49
c5
tion of the H. pylori transcriptome, and provide a framework for

Pn
tlpB
WT ΔHPnc5490

T
ΔH
HP0101 HP0102 HP0104

W
10
9
better analysis of individual genes. Knowledge of the vast majority

Relative mRNA expression

5′–CG CA–3′
GGGGGGGGGGGUG
||:|||||:||||
175 8 of H. pylori TSS and operons will help evaluate unclear phenotypes of
7
3′–UU
CCUCCCCCUCCAC
UG–5′ 80 6
transposon insertions44,45, and eliminate unwanted antisense tran-
HP1043 HP1044 5 scripts in heterologous expression constructs of H. pylori vaccine
HPnc5490
58 TlpB
4
3
candidates. Altogether, ,100 sRNAs are known in E. coli5, the model
2 organism of bacterial RNA research. Corrected by genome size, H.
46
5′
3′ 1 pylori rivals E. coli despite the lack of a conserved Hfq protein.
0
Combined with the success of artificial antisense RNAs in H. pylori46,

C
10

tlp
tlp

tlp
P0
our results show that many RNA-mediated regulations are yet to be

H
discovered in e-proteobacterial pathogens.
d aapA1 e
Other RNA-seq studies detected termini of bacterial transcripts8
omp27 deoD

7
but could not unequivocally assign TSS due to lack of 59 group
H S

H S
uh

uh
L

L
AG

AG
AS

AS
PL

PL
M

M
isoA1 331 242
aapA2 242 aapA1
190
aapA5 discrimination. As 59PPP ends mark native transcripts in all eubac-
HP1175 omp27
190
147
110 teria, dRNA-seq should help improve the genome annotations of
isoA2
HP1432 aapA3
110 IsoA5 other organisms, alone or through metatranscriptomics47. It could
67
HP1433 67
IsoA1
also complement the popular CAGE48 technique for eukaryotic
isoA3
aapA4
mRNAs because their cap structure blocks terminator exonuclease.
34
HP1533 242 Moreover, an improved dRNA-seq protocol might permit detection
tnpB aapA6
isoA5 190
IsoA2 147
of processed 59 hydroxyl ends that unlike the more prominent 59P
aapA5 67
110 ends are not presently captured in the (2) library. Semi-quantitative
omp2 IsoA6
HP0022
isoA5
331 analysis of TSS coverage revealed considerable gene expression
242 aapA3 67
aapA6 190 changes in H. pylori grown along with eukaryotic cells (Supplemen-
nusA
HP1517 67
IsoA3 tary Table 16). Thus, dRNA-seq has the potential to unravel gene
isoA6 HP1516
331
expression in pathogens and perhaps also their hosts during
aapA4
4,000 bp
242
f infections.
Is 1
3
oA
oA

1 30 190 –
M

Is

AapA1 110
3.5 aapA1
AapA3
AapA5 IsoA4
METHODS SUMMARY
3.5 aapA3
AapA6
AapA4
67 Details of bacterial growth conditions, cDNA library construction, TSS annota-
tion, sRNA validation, and biocomputational as well as statistical analyses, as
Figure 4 | Trans and cis regulatory sRNAs. a, Genomic context of the operon well as other methods are provided as Supplementary Information. For dRNA-
(dotted line) encoding chemotaxis receptor, TlpB, and of HPnc5490 sRNA, seq, total RNA was extracted from H. pylori grown under various growth con-
together with the predicted 13 bp HPnc5490–tlpB RNA duplex (grey ditions, and treated with Terminator-59-phosphate-dependent exonuclease
background). A consensus structure of HPnc5490 homologues shown below (Epicentre Biotechnologies) to deplete processed RNAs. The cDNA libraries
reveals presentation of the pairing residues by an accessible loop region. (vertis Biotechnologie AG) were constructed for RNA 2/1 prior exonuclease
b, SDS–PAGE showing accumulation of TlpB protein (identified by mass treatment for each growth condition without size fractionation of RNA,
spectrometry) in wild-type vs HPnc5490-deficient H. pylori bacteria in mid- before cDNA synthesis. 225,000–540,000 cDNAs per library were sequenced
log growth. Marker bands (kDa) to the left. c, Quantitative real-time PCR on a Roche FLX machine, and mapped to the H. pylori 26695 genome. For each
analysis of HPnc5490-dependent expression of four H. pylori mRNAs. Wild- library, graphs representing the number of mapped reads per nucleotides were
type mRNA levels (grey bars) are set to 1, and fold-regulation upon HPnc5490 calculated and visualized using the Affymetrix Integrated Genome Browser. To
deletion is indicated by black bars. Error bars indicate standard deviations corroborate operon predictions, we also analysed a total of ,43,000,000 strand-
among three technical replicates. d, Genomic locations of IsoA1–6 RNA specific cDNAs obtained from randomly fragmented RNA by Solexa (Illumina)
(black boxes) and associated peptide-encoding aapA mRNAs (grey). IsoA2 sequencing.
overlaps with hypothetical ORF HP1176. In strain 26695, aapA2 lacks a
Received 6 August; accepted 14 December 2009.
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557–572 (2002). Acknowledgements We thank F. Seifert; H. Hamoutene and B. Timmermann for
28. Vanet, A., Marsan, L., Labigne, A. & Sagot, M. F. Inferring regulatory elements technical support; M. Schmid for mass spectrometry analysis; H. De Reuse, A. van
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signals. J. Mol. Biol. 297, 335–353 (2000). M. Droege for pyrosequencing support. J.V. and R.R. are supported by NGFN1 grants
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with improved accuracy in locating start codons. DNA Res. 8, 97–106 (2001). Regulatory RNAs in Prokaryotes (Grants VO8751/2, VO8751/4; STA850/7-1). S.H.
30. Rodionov, D. A., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S. Comparative was supported by a formel.1 grant of the University of Leipzig, the Freistaat Sachsen
genomics of thiamin biosynthesis in procaryotes. New genes and regulatory (LIFE project), the German Research Foundation IZBI (BIZ6/1-4) and Volkswagen
mechanisms. J. Biol. Chem. 277, 48949–48959 (2002). Stiftung (I/82 720). F.D. is supported by the French Agence Nationale de la
31. Weinberg, Z. et al. Identification of 22 candidate structured RNAs in bacteria Recherche (ANR-07-JCJC-0104-01), the French Association de la Recherche contre
using the CMfinder comparative genomics pipeline. Nucleic Acids Res. 35, le Cancer (ARC) and La Ligue Nationale contre le Cancer (LNCC). We thank D. Rose
4809–4819 (2007). for his supporting work and S. Washietl for a pre-release of the RNAcode software.
32. Brock, J. E., Pourshahian, S., Giliberti, J., Limbach, P. A. & Janssen, G. R. Ribosomes Author Contributions C.M.S., F.D., P.F.S. and J.V. designed the research; C.M.S.,
bind leaderless mRNA in Escherichia coli through recognition of their 59-terminal F.D., A.S., J.R., J.V. and S.C. performed all wet lab work. C.M.S., S.H., S.F., K.R., J.H.
AUG. RNA 14, 2159–2169 (2008). and P.F.S. conducted biocomputational analyses; R.R. carried out sequencing. J.V.
33. Boneca, I. G. et al. A revised annotation and comparative analysis of Helicobacter wrote the manuscript, which all authors commented on, and supervised the
pylori genomes. Nucleic Acids Res. 31, 1704–1714 (2003). project. Author information and raw data are available from C.M.S, P.F.S. and J.V.
34. Lenz, D. H. et al. The small RNA chaperone Hfq and multiple small RNAs control
quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69–82 (2004). Author Information Raw data are available from the NCBI Short Read Archive
35. Barrick, J. E., Sudarsan, N., Weinberg, Z., Ruzzo, W. L. & Breaker, R. R. 6S RNA is a (http://www.ncbi.nlm.nih.gov/Traces/sra) under accession number SRA010186.
widespread regulator of eubacterial RNA polymerase that resembles an open Reprints and permissions information is available at www.nature.com/reprints.
promoter. RNA 11, 774–784 (2005). The authors declare no competing financial interests. Correspondence and
36. Wassarman, K. M. & Storz, G. 6S RNA regulates E. coli RNA polymerase activity. requests for materials should be addressed to J.V.
Cell 101, 613–623 (2000). (joerg.vogel@uni-wuerzburg.de).

255
©2010 Macmillan Publishers Limited. All rights reserved
 Vol 464 | 11 March 2010 | doi:10.1038/nature08857

LETTERS
Confirmation of general relativity on large scales from
weak lensing and galaxy velocities
Reinabelle Reyes1, Rachel Mandelbaum1, Uros Seljak2,3,4, Tobias Baldauf2, James E. Gunn1, Lucas Lombriser2
& Robert E. Smith2

Although general relativity underlies modern cosmology, its quantity DSgm(R) is directly proportional to the observable quantity
applicability on cosmological length scales has yet to be stringently for galaxy–galaxy lensing9, the tangential shear, which is a statistical
tested. Such a test has recently been proposed1, using a quantity, measure of the weak distortion of shapes of background galaxies due to
EG, that combines measures of large-scale gravitational lensing, the gravitational deflection of light by matter along the line of sight.
galaxy clustering and structure growth rate. The combination is Here Sgm(R) is the projected surface mass density at a transverse radius
insensitive to ‘galaxy bias’ (the difference between the clustering R and S  gm(,R) is its mean value within the region bounded by R;
of visible galaxies and invisible dark matter) and is thus robust to DSgm(R) therefore measures an excess surface mass density. The scale
the uncertainty in this parameter. Modified theories of gravity R0 sets the minimum length scale that contributes to the ADSD.
generally predict values of EG different from the general rela- In analogy with the second line of equation (2), the galaxy–galaxy
tivistic prediction because, in these theories, the ‘gravitational slip’ ADSD is defined as
(the difference between the two potentials that describe perturba- " ð
2 #
tions in the gravitational metric) is non-zero, which leads to 2 R R0
U gg (R):rc 2 dR’ R’wgg (R’){wgg (R)z wgg (R0 )
changes in the growth of structure2 and the strength of the gravi- R R0 R
tational lensing effect3. Here we report that EG 5 0.39 6 0.06 on
where rc is the critical matter density of the Universe and wgg is the
length scales of tens of megaparsecs, in agreement with the general
projected two-point galaxy correlation function, which is a measure
relativistic prediction of EG < 0.4. The measured value excludes a
of how strongly galaxies are clustered together. Because the ADSDs
model1 within the tensor–vector–scalar gravity theory4,5, which
are insensitive to length scales less than R0, they have the advantage of
modifies both Newtonian and Einstein gravity. However, the rela-
being robust to nonlinear, small-scale effects such as the stochasticity
tively large uncertainty still permits models within f( ) theory6,
and scale dependence of galaxy bias. In practice, we choose the mini-
which is an extension of general relativity. A fivefold decrease in
mum scale R0 to be large enough to suppress these systematic effects
uncertainty is needed to rule out these models. but small enough to preserve a high signal-to-noise ratio in the mea-
Operationally, we define the probe of gravity EG as a combination surement of EG.
of three observable quantities: Another distinct advantage of using EG to test gravity is its insensi-
1 U gm (R) tivity to both the galaxy bias parameter, b, and the amplitude of
EG (R)~ ð1Þ matter perturbations1. This is true to first order in general relativity,
b U gg (R)
as well as in modified theories of gravity. In particular, in general
Here b is the redshift distortion parameter7, which is a measure of the relativity, equation (1) simplifies to EG 5 Vm,0/f, where Vm,0 is the
radial anisotropy in the clustering pattern reconstructed by galaxy present-day matter density parameter. Thus, unlike in previous
redshift surveys. Growth of large-scale structure over cosmic time attempts to constrain the theory of gravity on cosmological length
induces coherent galaxy motions that give rise to the observed aniso- scales10, we are not required to use additional observations and
tropy. In the regime where perturbations are linear, b is equal to the assumptions to estimate galaxy bias, and are able to obtain results
ratio of the dimensionless linear growth rate of structure, f, and the that are more robust.
galaxy bias parameter, b. The galaxy–matter and galaxy–galaxy annular We measure Ugm(R) and Ugg(R) from a sample of 70,205 luminous
differential surface densities8 (ADSDs), Ugm(R) and Ugg(R), are derived red galaxies11 (LRGs), from the Sloan Digital Sky Survey12, that satisfy
from observations of large-scale gravitational lensing and galaxy selection criteria13 used for large-scale structure studies. We use the
clustering, respectively. They have been constructed to suppress the value b 5 0.309 6 0.035 measured14 from a galaxy sample selected in
small-scale contributions to these observations. In particular, the the same way. Our sample covers 5,215 square degrees of sky and the
galaxy–matter ADSD is defined as redshift range z 5 0.16–0.47 (the redshift of the radiation emitted by a

2 distant galaxy is a measure of the time of emission). The mean redshift,
R0
U gm (R):DSgm (R){ DSgm (R0 ) z 5 0.32, corresponds to a look-back time of 3.5 3 109 yr, when the
R universe was about 77% of its current size, and is already well into the
ð
2 ð2Þ
2 R R0 accelerated phase of the cosmic expansion. The sample spans a large co-
~ 2 dR’ R’Sgm (R’){Sgm (R)z Sgm (R0 ) moving volume, 1.02h23 Gpc3, where h ; H0/(100 km s21 Mpc21) is a
R R0 R
dimensionless form of the current Hubble parameter, H0.
where DSgm(R) ; S  gm(,R) 2 Sgm(R) is a measure of the cross- Figure 1 shows the average galaxy–galaxy lensing profile, DSgm(R)
correlation between the distribution of galaxies and dark matter. The (Fig. 1a), and the projected two-point galaxy correlation function,
1
Princeton University Observatory, Peyton Hall, Princeton, New Jersey 08544, USA. 2Institute for Theoretical Physics, University of Zurich, Zurich 8057, Switzerland. 3Physics and
Astronomy Department and Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA. 4Institute for Early Universe, Ewha University, Seoul
120-750, South Korea.

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NATURE | Vol 464 | 11 March 2010 LETTERS

10 0.6

ΔΣ gm (h Mpc–2)

GR + ΛCDM

EG
0.4
1 f( )

TeVeS
0.2

0.1 2 4 6 8 10 20 40
1 2 4 6 8 10 20 40
R (h–1 Mpc)
b
Figure 2 | Comparison of observational constraints with predictions from
general relativity and viable modified theories of gravity. Estimates of
100 EG(R) with error bars (1s) including the statistical error in the measurement
of b (ref. 14). The grey shaded region is the 1s envelope of the mean EG on
scales R 5 10h21–50h21 Mpc, where the systematic effects are least
wgg (h–1 Mpc)

important (Supplementary Information). The horizontal line shows the

mean prediction of general relativity, EG 5 Vm,0/f(z), at the effective redshift
of the measurement, z 5 0.32. On the right-hand side of the panel, labelled
10 vertical bars show the predicted ranges from three different gravity theories:
general relativity (GR) plus L cold dark matter (LCDM) model
(EG 5 0.408 6 0.029 (1s)); a class of cosmologically interesting models in
f( ) theory with Compton-wavelength parameters21 B0 5 0.001–0.1
(EG 5 0.328–0.365); and a tensor–vector–scalar (TeVeS) model1 designed to
match existing cosmological data and to produce a significant enhancement
1 of the growth factor (EG 5 0.22, shown with a nominal error bar of 10% for
1 2 4 6 8 10 20 40
R (h–1 Mpc) clarity).

Figure 1 | Probes of large-scale structure measured from 70,000 LRGs. We take the average of EG(R) over scales R 5 10h21–50h21 Mpc,
a, b, Observed radial profiles for two complementary probes, galaxy–galaxy accounting for correlations in the data, and find it to be
lensing (a) and galaxy clustering (b), are shown for scales ÆEGæ 5 0.392 6 0.065 (1s) (grey shaded region in Fig. 2). The 16%
R 5 1.2h21–47h21 Mpc (open circles). The error bars (1s) are estimated error in EG is dominated by the 11% statistical error in b and the 12%
from jackknife resampling of 34 equal-area regions in the sky. Profiles
statistical error in the galaxy–galaxy lensing signal. In addition, there
measured from mock galaxy catalogues are also shown (solid curves). To
generate the mock galaxy catalogues, we use a standard five-parameter halo is a 5% lensing calibration uncertainty15. As detailed in the Sup-
occupation distribution (HOD) model with two parameters related to the plementary Information, systematic effects on EG are least important
assignment of central galaxies and three parameters related to the on length scales R . 10h21 Mpc, so the results are most robust there.
distribution of satellite galaxies (see Supplementary Information for more We note that the average for R 5 2h21–50h21 Mpc yields a result,
details). To fix the HOD model parameters, we require the galaxy number ÆEGæ 5 0.40 6 0.07, consistent with that above.
density to match the observed value and find the best joint fit to the observed The general relativistic prediction is EG 5 Vm,0/f(z) 5 0.408 6 0.029
galaxy–galaxy lensing and galaxy clustering profiles. Despite this tuning, it is at redshift z 5 0.32, where f(z) < Vm(z)0.55 < 0.629 and Vm(z) is the
remarkable that this simple model is able to reproduce both the overall shape matter density parameter at redshift z. The allowed range is determined
and particular features of the observed profiles.
by the size of current uncertainties on Vm,0 5 0.2565 6 0.018 (ref. 19).
The data are consistent with this prediction over the range of scales we
wgg(R) (Fig. 1b), measured from the LRG sample for scales consider (Fig. 2, solid line and GR 1 LCDM bar). Unfortunately, pro-
R 5 1.2h21–47h21 Mpc. To achieve a high signal-to-noise ratio in viding model-independent constraints on the gravitational slip is com-
the lensing profile, we stack together shape measurements15 of more plicated, because changes in the gravitational slip will in turn affect the
than 3 3 107 source galaxies (see Supplementary Information for rate of growth of structure. What is clear is that there is no evidence for
details). To calculate wgg(R), we use a standard method of counting a non-zero gravitational slip from our data. Thus, we find no deviation
galaxy pairs and comparing the result with that for a randomly dis- from general relativity on length scales 1011 times greater than those for
tributed sample16. which classical tests20 have been performed.
Figure 2 shows our estimate of EG(R), with 1s error bars that We also compare our constraint on EG with predictions from two
include the error in the measurement of b. We choose the minimum viable modified theories of gravity: tensor–vector–scalar theory5 and
scale, R0 5 1.5h21 Mpc, to be close to the typical virial radius of the f( ) theory6 (Fig. 2, TeVeS and f( ) bars). Models of f( ) theory21 that
haloes of the most massive LRGs, above which we expect the distri- are designed to reproduce the observed cosmic expansion
bution of galaxies to trace that of the dark matter, but our results are history with a specific model for the gravitational slip predict that
not very sensitive to this particular choice of R0. To estimate errors in EG 5 0.328–0.365 (Supplementary Information). The data favour
EG(R) while accounting for any correlations between radial bins, we slightly higher values, but are consistent with this predicted range.
use jackknife resampling of 34 galaxy subsamples from equal-area These models can be tested in the near future; limits on EG will
regions in the sky. To obtain numerical corrections accounting for improve as a result of the larger data sets and better control of sys-
the effect of scale-dependent galaxy bias and other systematic effects, tematic errors allowed by the next generation of galaxy surveys.
we use a suite of dark-matter simulations17 that have been populated Nevertheless, even with the current limits, we can tentatively rule
with galaxies using the HOD model18 that best reproduces the obser- out particular models. For example, a particular tensor–vector–scalar
vations (Fig. 1 and Supplementary Information). The correction model1 predicts that EG 5 0.22, which is lower than the observed
factors that we obtain are well below the statistical uncertainty in EG. value by more than 2.5s. Whether this result rules out the entire class
257
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

of tensor–vector–scalar models is an open issue22, but it in any case 14. Tegmark, M. et al. Cosmological constraints from the SDSS luminous red galaxies.
Phys. Rev. D 74, 123507 (2006).
serves as a concrete demonstration that our measurement of EG 15. Mandelbaum, R. et al. Systematic errors in weak lensing: application to SDSS
presents a new and non-trivial challenge to both existing and future galaxy-galaxy weak lensing. Mon. Not. R. Astron. Soc. 361, 1287–1322 (2005).
proposals of modifications to general relativity. 16. Landy, S. D. & Szalay, A. S. Bias and variance of angular correlation functions.
Astrophys. J. 412, 64–71 (1993).
Received 3 August 2009; accepted 19 January 2010. 17. Smith, R. E. Covariance of cross-correlations: towards efficient measures for
large-scale structure. Mon. Not. R. Astron. Soc. 400, 851–865 (2009).
1. Zhang, P., Liguori, M., Bean, R. & Dodelson, S. Probing gravity at cosmological 18. Seljak, U. Analytic model for galaxy and dark matter clustering. Mon. Not. R.
scales by measurements which test the relationship between gravitational lensing Astron. Soc. 318, 203–213 (2000).
and matter overdensity. Phys. Rev. Lett. 99, 141302 (2007). 19. Dunkley, J. et al. Five-year Wilkinson Microwave Anisotropy Probe observations:
2. Daniel, S. F., Caldwell, R. R., Cooray, A., Serra, P. & Melchiorri, A. Multiparameter likelihoods and parameters from the WMAP data. Astrophys. J. Suppl. Ser. 180,
investigation of gravitational slip. Phys. Rev. D 80, 023532 (2009). 306–329 (2009).
3. Uzan, J. P. & Bernardeau, F. Lensing at cosmological scales: a test of higher 20. Will, C. M. The confrontation between general relativity and experiment. Space
dimensional gravity. Phys. Rev. D 64, 083004 (2001). Sci. Rev. 148, 60–71 (2009).
4. Milgrom, M. A modification of the Newtonian dynamics—implications for 21. Song, Y.-S., Hu, W. & Sawicki, I. The large scale structure of f(R) gravity. Phys.
galaxies. Astrophys. J. 270, 371–389 (1983). Rev. D 75, 044004 (2007).
5. Bekenstein, J. D. Relativistic gravitation theory for the modified Newtonian 22. Ferreira, P. G. & Starkman, G. D. Einstein’s theory of gravity and the problem of
dynamics paradigm. Phys. Rev. D 70, 083509 (2004). missing mass. Science 326, 812–815 (2009).
6. Carroll, S. et al. Is cosmic speed-up due to new gravitational physics? Phys. Rev. D
Supplementary Information is linked to the online version of the paper at
70, 083509 (2004).
www.nature.com/nature.
7. Hamilton, A. J. S. in The Evolving Universe (ed. Hamilton, D.) 185–275 (Astrophys.
Space Sci. Library 231, Kluwer, 1998). Acknowledgements R.M. was supported for the duration of this work by NASA
8. Baldauf, T., Smith, R. E., Seljak, U. & Mandelbaum, R. An algorithm for the direct through a Hubble Fellowship grant awarded by the Space Telescope Science
reconstruction of the dark matter correlation function from weak lensing and Institute, which is operated by the Association of Universities for Research in
galaxy clustering. Phys. Rev. D (submitted). Astronomy, Inc., for NASA. U.S. acknowledges the Swiss National Foundation. T.B.
9. Bartelmann, M. & Schneider, P. Weak gravitational lensing. Phys. Rep. 340, acknowledges support by a grant from the German National Academic Foundation
291–472 (2001). during the initial phase of this project.
10. Guzzo, L. et al. A test of the nature of cosmic acceleration using galaxy redshift
Author Contributions R.R., R.M., U.S. and J.E.G. worked on the observational
distortions. Nature 451, 541–544 (2008).
analysis, with R.R. doing most of the computations. T.B. and R.E.S. worked on the
11. Eisenstein, D. et al. Spectroscopic target selection for the Sloan Digital Sky Survey:
numerical simulations, with T.B. calculating the correction factors. L.L. worked on
the luminous red galaxy sample. Astron. J. 122, 2267–2280 (2001).
the theoretical predictions for comparison with the observations.
12. York, D. G. et al. The Sloan Digital Sky Survey: technical summary. Astron. J. 120,
1579–1587 (2000). Author Information Reprints and permissions information is available at
13. Eisenstein, D. et al. Detection of the baryon acoustic peak in the large-scale www.nature.com/reprints. The authors declare no competing financial interests.
correlation function of SDSS luminous red galaxies. Astrophys. J. 633, 560–574 Correspondence and requests for materials should be addressed to R.R.
(2005). (rreyes@astro.princeton.edu).

258
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Vol 464 | 11 March 2010 | doi:10.1038/nature08864

LETTERS
Deviations from a uniform period spacing of gravity
modes in a massive star
Pieter Degroote1, Conny Aerts1,2, Annie Baglin3, Andrea Miglio4, Maryline Briquet1, Arlette Noels4, Ewa Niemczura5,
Josefina Montalban4, Steven Bloemen1, Raquel Oreiro1, Maja Vučković1, Kristof Smolders1, Michel Auvergne3,
Frederic Baudin6, Claude Catala3 & Eric Michel3

The life of a star is dominantly determined by the physical pro- with a span of 137 days has been obtained for the star by the
cesses in the stellar interior. Unfortunately, we still have a poor Convection Rotation and Planetary Transits (CoRoT4) satellite. A
understanding of how the stellar gas mixes near the stellar core, linear downward trend was removed from the data, after outliers
preventing precise predictions of stellar evolution1. The unknown were excluded from the light curve. The duty cycle of the final set
nature of the mixing processes as well as the extent of the central of measurements analysed here is 88.6% and the noise has an ampli-
mixed region is particularly problematic for massive stars2. tude of 1 mmag (Fig. 1). The light curve reveals the presence of
Oscillations in stars with masses a few times that of the Sun offer numerous oscillation modes.
a unique opportunity to disentangle the nature of various mixing Stellar oscillations have been found to occur at different stages of
processes, through the distinct signature they leave on period spa- stellar life, for a range of stellar masses5. The best known case is that of
cings in the gravity mode spectrum3. Here we report the detection the solar oscillations, which are acoustic modes with periods near
of numerous gravity modes in a young star with a mass of about 5 min (refs 6–9). The seismic interpretation of the detected solar
seven solar masses. The mean period spacing allows us to estimate oscillations led to a drastic improvement in the knowledge of the
the extent of the convective core, and the clear periodic deviation internal structure of the Sun10,11. Meanwhile, similar acoustic modes
from the mean constrains the location of the chemical transition have been detected in various types of distant stars12–14. Gravity
zone to be at about 10 per cent of the radius and rules out a clear- modes, on the other hand, probe much deeper layers inside stars
cut profile. and in principle allow the study of the core properties of stars far
The young massive star HD 50230 is poorly studied, but it is better than acoustic modes. Although such modes have been detected
known to have spectral type B3V and a visual magnitude of 8.95. in massive stars similar to HD 5023015,16, where they have typical
HD 50230 is in its central nuclear-burning phase, just like the Sun, periods of half a day to a few days, their seismic potential could
transforming hydrogen into helium in its core (the so-called main not be exploited because the number of detected modes in one star
sequence phase). This important evolutionary phase covers about was far too low.
90% of the life of the star, and the detailed internal structure of the Frequency analysis of HD 50230’s CoRoT light curve resulted in a
star during this phase determines its ultimate fate1,2. This star is at the rich frequency spectrum (see Supplementary Information for an
limiting mass of ending either as core-collapse supernova or as a outline of the analysis). We detected hundreds of gravity modes with
white dwarf, enriching the interstellar medium with helium and periods roughly in the range of half a day to five days and amplitudes
heavy metals in the former case and carbon in the latter. A high- of millimagnitude level, as well as tens of high-frequency acoustic
quality continuous photometric light curve with 32-s sampling and modes with periods in the range of one to ten hours and with

–10 1.0
Δ Brightness (mmag)

–5 0.5
Δ Flux (%)

0 0

5 –0.5

10 –1.0
2,860 2,880 2,900 2,920 2,940 2,960 2,980
Time (days)

Figure 1 | The light curve of HD 50230 measured by the CoRoT satellite. Main panel, the light curve. Inset, magnified view of the boxed part of the light
curve. The residuals resulting from the frequency analysis are plotted in grey.
1
Instituut voor Sterrenkunde, KU Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium. 2IMAPP, Department of Astrophysics, Radboud University Nijmegen, PO Box 9010, 6500 GL
Nijmegen, The Netherlands. 3LESIA, UMR8109, Université Pierre et Marie Curie, Université Denis Diderot, Observatoire de Paris, 92195 Meudon, France. 4Institut d’Astrophysique et
de Géophysique de l’Université de Liege, Allée du 6 Aout 17, 4000 Liege, Belgium. 5Astronomical Institute, Wroclaw University, Kopernika 11, 51-622 Wroclaw, Poland. 6Institut
d’Astrophysique Spatiale (IAS), Batiment 121, F-91405, Orsay Cedex, France.

259
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

0.20 than what is imposed by the convection theory is required23. Such an

additional mixing could be the result of convective overshooting,
that is, the extension of convective motions above the boundary given
0.15 by the local treatment of convection used in the stellar modelling (the
mixing length theory). Other mixing processes include turbulent
Amplitude (%)

diffusion or mixing induced by rotation. Theory predicts that the

0.10 relative importance of these various mixing processes can be separated,
as they leave a distinct marker on the spacing structure of gravity
modes3.
0.05
The deviations from the constant period spacing we determined
for HD 50230 allow us to evaluate the characteristics of the mixing.
To achieve this, the fundamental parameters of the star need to be
estimated. For this purpose, several ground-based high-resolution
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 spectra of HD 50230 were recorded with the CORALIE24 spectro-
Period (days) graph attached to the 1.2-m Euler telescope at the La Silla Obser-
Figure 2 | Extracted frequency spectrum in the g mode region. The vertical vatory in Chile, and with the High Efficiency and Resolution
dashed lines denote the members of the detected period spacing. Mercator Echelle Spectrograph (HERMES25) attached to the 1.2-m
Mercator telescope at La Palma. These follow-up spectra revealed a
considerably lower amplitudes of several micromagnitudes. This decimal logarithmic gravity of 3.8 6 0.2 (c.g.s. units) and a projected
high number of gravity mode frequencies is compatible with our equatorial surface rotational velocity below 5 km s21 from the syn-
non-adiabatic excitation computations for an appropriate stellar thesis of line profiles based on atmosphere models. The effective
model based on an increase of the metal opacity at temperatures temperature of the star was estimated to be 17,500 6 1,500 K from
around 200,000 K (ref. 17). The oscillation period content in the data its spectral energy distribution, based on archival photometry and
was extracted using classical and short-time Fourier analysis tech- space spectroscopy measured with the International Ultraviolet
Explorer (see Supplementary Information for a description of the
niques, combined with nonlinear fitting of the light curve5, and a
fitting procedure). These results from classical spectroscopy are in
search for a recurrent period spacing was conducted18. A group of
full agreement with the spectral type of B3V.
eight consecutive periods with an almost constant spacing of 9,418 s
Stellar structure models in accordance with the observed fun-
was detected in the data (Fig. 2). Monte Carlo simulations showed
damental parameters of HD 50230 were computed with the Code
that the probability of a coincidental occurrence of such spacing
Liégeois d’Évolution Stellaire (CLÉS26). We considered models with-
among eight consecutive oscillation periods within the list of
out extra mixing, as well as with instantaneous mixing due to core
detected frequencies is below 0.1%. Small deviations of up to 200 s
overshooting over a distance parametrized as 0.1–0.3 local pressure
from the constant period spacing occur (Fig. 3). We interpret these as scale heights, or with parametrized turbulent mixing3. Under the
being due to the presence of a region of varying chemical composi- assumption of solar metallicity and dipolar oscillation modes, the
tion outside the convective core3. This region is progressively built up mean spacing of 9,418 s led us to conclude that the overshoot para-
during the main sequence by the boundary of the convective core meter needs to be at least 0.2 in the radiative regions adjacent to the
receding towards the centre. core to be compatible with the observed effective temperature (Fig. 4).
The interior structure of massive stars basically consists of a con- Lowering the metallicity or increasing the degree of the modes
vective core surrounded by a radiative envelope. With this simplified increases the amount of extra mixing needed to fit the observed effec-
assumption, theory19 predicts the occurrence of a constant period tive temperature.
spacing among high-order gravity modes of the same degree. Such a The observed deviations from constant period spacing (Fig. 3) can
spacing has been found and interpreted previously in white dwarfs, be characterized by a periodic behaviour3. Its periodicity gives an
which are stellar remnants at the end of the life of stars like the indication of the evolutionary status in the main-sequence phase.
Sun20–22. They had hitherto not been detected in main-sequence stars. The current observations suggest that the period of the oscillatory
The deviations from the equidistant period spacing carry informa- part is equal to 2,450 s, indicating that about 60% of the initial
tion on the details of the physical conditions in the stellar interior,
which are otherwise inaccessible for observational testing. Obser- 12,000
vations of binaries and stellar clusters show that a mixed zone larger
11,000
Mean perios spacing (s)

9,800 10,000

9,000
9,700
8,000
9,600
7,000
ΔPeriod (s)

9,500
6,000
9,400
5,000
9,300 4,000
4.40 4.35 4.30 4.25 4.20 4.15 4.10 4.05 4.00
9,200 Log10 [effective temperature (K)]

9,100 Figure 4 | The influence of the size of the convective mixing region on the
1.0 1.1 1.2 1.3 1.4 1.5 1.6
Period (days)
mean value of the period spacing. Dashed line, the location of the observed
period spacing and the estimate of the effective temperature. Solid lines, the
Figure 3 | The components and values of the g mode period spacing. Filled mean period spacing value as a function of the effective temperature, for
squares, the values of the period components (error bars, s.e.m.). Grey line, a overshoot parameters of 0 (black), 0.2 (dark grey) and 0.3 (light grey), and
sinusoidal fit with decreasing amplitude, with a period of 2,450 s and different masses (8–4 solar masses from left to right). The metallicity was
maximum amplitude of 240 s. fixed at Z 5 0.02.
260
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

hydrogen in the centre of the star has already been consumed. The 15. Waelkens, C. Slowly pulsating B stars. Astron. Astrophys. 246, 453–468 (1991).
16. Krisciunas, K. A new class of pulsating stars. Bull. Am. Astron. Soc. 25, 1442–1443
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shape of the chemical composition gradient27. The observed small 17. Dupret, M. A. Nonradial nonadiabatic stellar pulsations. Astron. Astrophys. 366,
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21. Winget, D. E. et al. Whole earth telescope observations of the DBV white dwarf
As a next step, the pressure modes of HD 50230 could in principle GD358. Astrophys. J. 430, 839–849 (1994).
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Without the establishment of a pressure mode frequency spacing, pulsating DA white dwarfs. II — Mode trapping in compositionally stratified
such seismic exploitation of the pressure mode frequencies requires models. Astrophys. J. Suppl. Ser. 80, 369–401 (1992).
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14. Michel, E. et al. CoRoT measures solar-like oscillations and granulation in stars Correspondence and requests for materials should be addressed to P.D.
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 Vol 464 | 11 March 2010 | doi:10.1038/nature08876

LETTERS
Transmission of electrical signals by spin-wave
interconversion in a magnetic insulator
Y. Kajiwara1,2, K. Harii1, S. Takahashi1,3, J. Ohe1,3, K. Uchida1, M. Mizuguchi1, H. Umezawa5, H. Kawai5, K. Ando1,2,
K. Takanashi1, S. Maekawa1,3 & E. Saitoh1,2,4

The energy bandgap of an insulator is large enough to prevent A flow of spin angular momentum is called a spin current2. In
electron excitation and electrical conduction1. But in addition to solids, there are two types of carriers for non-equilibrium spin cur-
charge, an electron also has spin2, and the collective motion of spin rents. One is a conduction electron2,12,13 (Fig. 1a). The other is col-
can propagate—and so transfer a signal—in some insulators3. This lective motion of magnetic moments (Fig. 1b)—spin waves14,15,
motion is called a spin wave and is usually excited using magnetic comprising magnetostatic and exchange spin-wave modes14,15.
fields. Here we show that a spin wave in an insulator can be gen- Here we call a spin current carried by spin waves a ‘spin-wave spin
erated and detected using spin-Hall effects, which enable the direct current’ (see Supplementary Information section A for details).
conversion of an electric signal into a spin wave, and its sub- Extensive studies of conduction-electron spin currents in metals
sequent transmission through (and recovery from) an insulator and semiconductors have clarified that the currents have a critical
over macroscopic distances. First, we show evidence for the trans- problem; they disappear within a very short distance, typically hun-
fer of spin angular momentum between an insulator magnet dreds of nanometres16. In contrast, it has been shown that a spin-
Y3Fe5O12 and a platinum film. This transfer allows direct conver- wave spin current may persist for much greater distances because it is
sion of an electric current in the platinum film to a spin wave in the carried by the collective motion of spins coupled by exchange inter-
Y3Fe5O12 via spin-Hall effects4–11. Second, making use of the trans- action14,15. Significantly, a spin-wave spin current exists even in mag-
fer in a Pt/Y3Fe5O12/Pt system, we demonstrate that an electric netic insulators, in which its decay is typically suppressed. This is
current in one metal film induces voltage in the other, far distant, because the decay is caused mainly by conduction electrons, which
metal film. Specifically, the applied electric current is converted are absent in insulators. For instance, in the magnetic insulator
into spin angular momentum owing to the spin-Hall effect7,8,10,11 in Y3Fe5O12, the spin-wave decay length can be several centimetres3
the first platinum film; the angular momentum is then carried by a and thus the waves are propagated over a relatively long distance;
spin wave in the insulating Y3Fe5O12 layer; at the distant platinum Y3Fe5O12 is an ideal conductor for spin-wave spin currents even
film, the spin angular momentum of the spin wave is converted though it is an insulator for electric currents.
back to an electric voltage. This effect can be switched on and off To make use of the spin-wave spin currents in insulators, it is
using a magnetic field. Weak spin damping3 in Y3Fe5O12 is necessary to find methods for getting a d.c. spin current into and
responsible for its transparency for the transmission of spin angu- out of the insulators. We show that this can be done by using spin
lar momentum. This hybrid electrical transmission method poten- pumping and spin-transfer torque (STT). Here, spin pumping refers
tially offers a means of innovative signal delivery in electrical to the transfer of spin angular momentum from magnetization-
circuits and devices. precession motion to conduction-electron spin9,17–19, a phenomenon

Figure 1 | Two types of non-equilibrium spin currents in solids. a, A pumping detection mechanism in the present system. If the magnetization
schematic illustration of a conduction-electron spin current: spin angular (M) dynamics in the Y3Fe5O12 layer (on the top face of the block) pumps a
momentum JS carried by electron diffusion. b, A schematic illustration of a spin current JS into the Pt layer, the current generates electromotive force
spin-wave spin current: spin angular momentum carried by collective ESHE via ISHE.
magnetic-moment precession. c, A schematic illustration of the spin-
1
Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan. 2Department of Applied Physics and Physico-Informatics, Keio University, Yokohama 223-8522,
Japan. 3CREST, 4PRESTO, Japan Science and Technology Agency, Sanbancho, Tokyo 102-0075, Japan. 5FDK Corporation, Shizuoka 431-0495, Japan.

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©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

which allows generation of a spin current from magnetization Pt layer is used as a spin-current detector, in which the inverse spin-
motion. STT is, in contrast, the reverse process of this spin pumping, Hall effect9–11 (ISHE) converts a spin current into electromotive force
that is, the transfer of angular momentum from conduction-electron ESHE via the spin–orbit interaction. In ISHE, when a spin current
spin to magnetization20–22: the magnetization receives torque by carries the spin polarization s along the spatial direction JS, ESHE is
absorbing a spin current20–22. These two phenomena enable the given by9,11 (Fig. 1c):
mutual conversion of angular momentum between conduction-elec-
tron spin and magnetization. However, up to now, experiments on ESHE // JS 3 s (1)
these phenomena have been limited to electric conductors. In this
Letter we show that by using Pt/Y3Fe5O12 films, both phenomena23 ISHE is known to be enhanced in heavy noble metals such as Pt (refs
occur even at insulator/metal interfaces, and the phenomena allow 9, 11).
transmission of a d.c. electric signal through the insulator for a long During the measurements, a static in-plane magnetic field H is
distance in a controllable manner at room temperature. applied and the sample is placed at the centre of a 9.44 GHz micro-
First, we show evidence for spin pumping across a Pt/garnet-type wave cavity. When H fulfils the ferromagnetic spin-wave resonance
Y3Fe5O12 interface. Y3Fe5O12 is a ferrimagnetic insulator whose (SWR) conditions14,15, precession of magnetization is induced. If this
charge gap is 2.7 eV. Owing to this huge gap, Y3Fe5O12 exhibits very precession pumps spin currents into the Pt layer, then voltage is
high resistivity (,1012 V cm at room temperature, greater than that generated in the Pt layer via ISHE; spin pumping in this system is
of air). Also, the magnetization damping is very small: sensitively detected by measuring the voltage difference V between
a < 6.7 3 1025, where a is the Gilbert damping coefficient19,24 for the ends of the Pt layer9,11.
the sample used in the present study (Fig. 2b). Figure 2a is a schematic Figures 2c and 2d are the SWR spectrum and the H dependence of
illustration of the experimental set-up. The sample is a bilayer film dV/dH, respectively, both measured with microwaves applied and
composed of a single-crystal Y3Fe5O12 layer and a Pt layer. Here, the with the external magnetic field perpendicular (h 5 90u) to the dir-
ection across the electrodes. In the SWR spectrum, many resonance
a c signals appear, each dispersion corresponding to a SWR mode14,15 in
Microwaves SWR V
the Y3Fe5O12 layer. These resonance fields are much greater than the
θ = 90º in-plane magnetization saturation field (HC 5 20 Oe) of this
dP/dH (a.u.)

V H Y3Fe5O12 film. In the dV/dH spectrum, as shown in Fig. 2d, multiple

H 0 peaks appear at these SWR fields, indicating that electromotive force
θ
is induced in the Pt layer concomitant with SWR in the Y3Fe5O12
Y3Fe5O12 layer. Figure 2g shows the microwave-power dependence of the max-
Pt imum peak values Vmax in the V(H) curves. The experimental data
b d (filled circles) are well reproduced by a curve (solid line) calculated
4
0.5 ISHE from a longitudinal spin-pumping model in which the spin accu-
V (µV)

N=1
θ = 90º
dV/dH (μV Oe–1)

SWR 3
7
5
0
mulation in the Pt layer is taken into consideration (see
dP/dH (a.u.)

2.5 2.7
H (kOe) Supplementary Information section B for details). This voltage signal
0 was found to disappear in a Cu/Y3Fe5O12 system, where the Pt layer is
0
V H replaced by a Cu layer in which the spin–orbit interaction is very
weak22, indicating the important role of spin–orbit interaction, or
–0.5 ISHE, in the voltage generation. The voltage signal also disappears in
4.73 4.75 2.5 2.7
H (kOe) H (kOe) a Pt/SiO2/Y3Fe5O12 system, where the Y3Fe5O12 and the Pt layers are
e f g separated by a thin (10 nm) film of insulating SiO2, and also in a Pt/
4 Gd3Ga5O12 system, where the Y3Fe5O12 layer is replaced by a para-
Vmax(θ)/Vmax(θ = 90°)

ISHE 1 Exp. Exp.

90º Sinθ Cal. magnetic Gd3Ga5O12 layer. These last two results indicate that direct
contact between the magnet Y3Fe5O12 and Pt is necessary for the
V (μV)

V (μV)

0 0 2
0 observed voltage generation; electromagnetic artefacts are irrelevant.
2
θ = –90º By changing the field direction h, the V signal for Pt/Y3Fe5O12 dis-
–1 appears at h 5 0 (Fig. 2e, f) and then changes its sign at 290u , h , 0.
2.5 2.6 2.7 –90º 0 90º 0 0.2 0.4 0.6 This behaviour is consistent with equation (1), and is direct evidence
H (kOe) θ P (mW)
for ISHE induced by spin pumping from the insulator Y3Fe5O12.
Figure 2 | Spin pumping in Pt/Y3Fe5O12. a, Schematic illustration of the The spin pumping in Pt/Y3Fe5O12 can be attributed to the small
experimental set-up. The sample is a Pt/Y3Fe5O12 bilayer film, 3 mm but finite spin-exchange interaction17,19 between a conduction elec-
3 1 mm, composed of a 1.3-mm-thick garnet-type Y3Fe5O12 layer and a 10- tron in Pt and a localized moment in Y3Fe5O12, or to the mixing
nm-thick Pt layer. The voltage (V) electrodes attached to the Pt film are conductance of the conduction electrons23 at the interface. By taking
3 mm apart. H denotes the in-plane external magnetic field. b, SWR
spectrum measured when a magnetic field is perpendicular to the film.
this interaction into account in coupled equations for the magnet-
Arrows, fitting results for the exchange spin-wave resonance fields29. N, spin- ization and spin accumulation (the Landau–Lifshitz–Gilbert equa-
wave mode number along the direction perpendicular to the film. Spectral tion24 and the Bloch equation with spin diffusion17,19), we obtain the
width and a for the N 5 1 mode was estimated via a fitting procedure using pumped spin current, which when combined with ISHE9–11 allows
Lorentz-type dispersion functions (Supplementary Information section E). calculation of V as a function of the spin-exchange interaction at the
P and H, the microwave absorption intensity and the strength of interface. From the experimental values of V together with values of
H, respectively. c, Ferromagnetic SWR spectrum for the Pt/Y3Fe5O12 film at microwave field strength and the spin-Hall angle for Pt (ref. 11), the
h 5 90u (h is defined in a). d, H dependence of dV/dH for the Pt/Y3Fe5O12 magnitude of the spin-exchange energy density at the interface is
film measured by applying 1 mW microwaves at h 5 90u. Inset, H estimated to be ,16 erg cm22; alternatively, the magnitude of the
dependence of V for the Pt/Y3Fe5O12 film at h 5 90u. Galvanomagnetic
mixing conductance23 is estimated as 3 3 1012 cm22 (see
effects24 in magnets are irrelevant to V, as Y3Fe5O12 is an insulator. e, H
dependence of V measured by applying the microwaves at various values of Supplementary Information section B for details).
h. f, h dependence of the maximum peak values Vmax in the V(H) curves The above observation of the spin pumping in Pt/Y3Fe5O12 sug-
measured with application of microwaves. g, Microwave-power dependence gests the possibility of the reverse process: STT20–22 acting on the
of Vmax. The experimental data (filled circles) are well reproduced by a curve insulator Y3Fe5O12. Second, we demonstrate STT across the Pt/
(solid line) calculated from a longitudinal spin-pumping model. Y3Fe5O12 interface using the same system as follows. We applied an
263
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

electric current J to the Pt layer (Fig. 3a). In the Pt layer, J is converted magnetization spontaneously oscillates20–22 with the eigenfrequen-
into a spin current via the spin-Hall effect7,8,11,22. This spin current cies14 and emits electromagnetic waves. As the magnetization damp-
exerts STT on the magnetization in the Y3Fe5O12 layer. This is the ing in Y3Fe5O12 is very small3, this cancellation is achieved by a small
reverse process of the aforementioned spin pumping effect. When amount of STT. In contrast, when J , 0 (J . 0) at h 5 90u
J . 0 (J , 0) at h 5 90u (h 5 290u), STT is antiparallel to the mag- (h 5 290u), STT and the damping torque are parallel and cannot
netization damping torque in the Y3Fe5O12 layer22 (Fig. 3b). Here, h be cancelled out22 (Fig. 3c).
represents the angle between J and the magnetization direction of the We measured the power spectra S(j), where j is electric current
Y3Fe5O12 layer. In this case, if the magnitude of STT is greater than density, of microwaves emitted from the Pt/Y3Fe5O12 film using an
that of the damping torque, the damping is cancelled out by STT. antenna while applying an electric current to the Pt layer. In Fig. 3d,
This means that when a sufficient amount of current is applied, the we show DS(j) ; S(j) 2 S(2j) spectra at various values of j when
H 5 1.2 kOe (.HC). When the applied magnetic field is in the dir-
ection along the electric current, h 5 0, there are no emission signals
in DS(j). In contrast, when the field is perpendicular to the current,
a b Microwaves c h 5 90u, multiple peak signals appear in the spectra when j is greater
M M than 4.4 3 108 A m22 (;jc), as shown in Fig. 3d. The peak frequen-
H H cies in the spectra vary with the magnetic field (Fig. 3f and i). The
H DT solid curve in Fig. 3i represents the ferromagnetic resonance frequen-
θ STT
DT STT cies calculated from the Kittel equation (see Supplementary
Y3Fe5O12
J σ Information section C for details); these peaks evidently originate
M = Pt σ from the spontaneous magnetization oscillations induced when
M/Y3Fe5O12 SC
SC j . jc. The multiple peaks (see Fig. 3d) may be attributed to magneto-
static spin-wave modes which are excited simultaneously in the
d e f
M = Cu M = Pt θ = 90º
Y3Fe5O12 layer (see Supplementary Information section D for
M = Pt
S(j) – S(–j)

20 θ = 0 details), but the strong change in the whole spectral shape with cur-
j = 13.3 108 A m–2
20
20
4.5
20 rent and field strengths suggests the appearance of chaos25 or the
f 5.5
H = 1.15 kOe spin-diffusion-induced instability26 in magnetization dynamics. In
H = 1.2 kOe H = 1.2 kOe
θ = 90º θ = 90º the j dependence of the frequency-integrated intensities for the
S(j) – S(–j) (fW MHz–1)

S(j) – S(–j) (fW MHz–1)

j = 11.1 108 A m–2 1.10

DS(j) spectra, a clear threshold is observed at jc (Fig. 3g). This implies
10.0
1.05
that STT compensates the magnetization damping torque at j 5 jc.
8.9
The centroid frequency of the emission spectra decreases slightly with
1.00 the current, a tendency which follows that in current-injection-type
6.7
magnetization oscillations27,28. We checked that these peak signals
0.95
4.4 disappear both in Pt/Gd3Ga5O12 and Cu/Y3Fe5O12 (Fig. 3e and h)
3.3 0.90
films.
2.2
Finally, we show electric-signal transmission in the insulator
0.85 Y3Fe5O12 film by making use of these phenomena together.
0
0 0 0 Figure 4a is a schematic illustration of the experimental set-up.
4.5 5 5.5 4.5 5 5.5 4 4.5 5 Two Pt films (i and o) are sputtered on a single-crystal Y3Fe5O12 film
f (GHz) f (GHz) f (GHz) and an electric current is applied to the Pt film i. The distance
g h i between the films i and o is 1 mm, shorter than the spin-wave decay
5.2
length in Y3Fe5O12 crystals3. In this set-up, the electric current
W(j) – W(–j) (pW)

f0 (GHz)

Exp.
8
applied to the Pt film i induces magnetization oscillation in the
(GHz)

Cal.
5
0 10 Y3Fe5O12 layer due to the STT across the Pt/Y3Fe5O12 interface, as
1 j (108 A m–2) 1
f0

4 demonstrated in Fig. 3. This magnetization oscillation then propa-

M = Pt M = Cu M = Pt gates in the Y3Fe5O12 layer via a spin-wave spin current. Figure 4b
shows a numerical calculation of this propagation. When the oscil-
0 5 10 0 5 10 0 1 2
H (kOe) lation reaches the second interface, it generates electric voltage in the
j (108 A m–2) j (108 A m–2)
Pt film o via spin pumping and ISHE, as demonstrated in Fig. 2. As
Figure 3 | Magnetization oscillation induced by spin-transfer torque in Pt/ shown in Figs 1c and 3b, these effects are activated when j . 0 (j , 0)
Y3Fe5O12. a, Schematic illustration of the experimental set-up; the sample at h 5 90u (h 5 290u)9,22. We measured the voltage generated in the
film is that shown in Fig. 2a. h denotes the angle between the external in- Pt film o while applying electric currents to the Pt film i.
plane magnetic field H and the direction along the electric current J in the Pt Figures 4c and d show the voltage difference V between the ends of
layer. b, c, Schematic illustrations of the directions of spin-transfer torque
the Pt film o as a function of the electric current density j in the Pt film
(STT) acting on the magnetization (M) and the magnetization-damping
torque (DT) of the Y3Fe5O12 layer at h 5 90u when J . 0 (b) and J , 0 (c). J, i. When the magnetization of the Y3Fe5O12 layer is along the electric
amplitude of J; s, spin polarization of the spin current (SC) induced from current, h 5 0 and 180u, no signal appears in V (Fig. 4c). At h 5 0 and
J by the spin-Hall effect in the Pt layer. d, e, DS(j) ; S(j) 2 S(2j) spectra for 180u, the above spin-transfer and voltage-generation mechanisms are
various values of the current density j in the Pt layer at h 5 90u for Pt/ inactive, and this result also confirms that the two Pt films are
Y3Fe5O12 (d) and Cu/Y3Fe5O12 (e) films when H 5 1.2 kOe. S(j), the well isolated electrically. When h 5 90u (h 5 290u), in contrast, the
frequency f power spectrum of the microwaves (see Methods). In DS(j), voltage V signal appears with application of a current
background noise is eliminated as the antisymmetric component with j . 6.0 3 108 A m22 (j , 26.0 3 108 A m22), as shown in Fig. 4d.
respect to j is extracted. Inset to d, DS(j) spectrum at h 5 0 for the Pt/ We found that there is a clear threshold at 6.0 3 108 A m22 in the j
Y3Fe5O12 film measured with application of j 5 11.1 3 108 A m22 when
dependence of V (Fig. 4d). This threshold current density is compar-
H 5 1.2 kOe. f, DS(j) spectra for various magnetic field strengths H for the
Pt/Y3Fe5O12 film measured when j 5 13.3 3 108 A m22 at h 5 90u. able to that for the current-induced magnetization oscillation shown
g, h, Values of the frequency integral from 4.5 GHz up to 5.5 GHz of the DS(j) in Fig. 3g. All these results are consistent with the prediction that
spectra for the Pt/Y3Fe5O12 film (shown in d), and for the Cu/Y3Fe5O12 film electric-signal transmission in the Y3Fe5O12 is activated when j . jjcj
(shown in e), respectively, as functions of j. i, H dependence of the centroid (j , 2jjcj) at h 5 90u (h 5 290u), where jjcj is a threshold current
frequencies f0 of the DS(j) spectra shown in f. density. If the distance between the Pt films were less than
264
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a b c d 1.2
Pt/Y3Fe5O12

V (nV)
Pt film i
V 1 H = 2.3 kOe 1 θ = 90º
Y3Fe5O12 Pt (o) ESHE
1
0 H (kOe) 3
Pt (i) H
θ θ = 0º

V (nV)

V (nV)
J 0 0
1 mm
θ = 180º
y
z
y x x –1 –1 θ = –90º Pt/Y3Fe5O12
JS H = 2.3 kOe
0 0.5 1.0
|my,z| (a.u.) –10 –5 0 5 10 –10 –5 0 5 10
j (108 A m−2) j (108 A m−2)

Figure 4 | Electric-signal transmission via spin-wave spin currents. a, A Landau–Lifshitz–Gilbert equation30 at room temperature. In the calculation,
schematic illustration of the experimental set-up. The sample is a 1.3-mm- STT22 that compensates the magnetization-damping torque at the Pt film
thick single-crystal Y3Fe5O12 (111) film on which two separate 15-nm-thick i/Y3Fe5O12 interface (dashed rectangle) is taken into consideration. Time
Pt films (i and o) are sputtered. The distance between the Pt films is 1 mm. average is taken for 1 ms (Supplementary Information section F). c, d, V as a
The surfaces of the Y3Fe5O12 layer, Pt film i and Pt film o are rectangular function of j in the Pt film i at h 5 0u (red curve in c), h 5 180u (blue curve in
shapes of area (mm2) 35, 27.5 and 0.5, respectively. The distance between the c), h 5 90u (red curve in d) and h 5 290u(blue curve in d). h is defined in
voltage electrodes (V) attached to the Pt film o is 5 mm. b, In-plane spatial a. An in-plane magnetic field of 2.3 kOe is applied. Inset to d, V at
distribution of the time average of the magnetization-precession amplitude j 5 16.6 3 108 A m22 as a function of H (0.2 kOe , H , 3 kOe) when
| my,z | in the Y3Fe5O12 layer numerically calculated using a stochastic h 5 90u.

nanometres, electron tunnelling could convey spin angular Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
momentum; in the present macroscopic-sized system, in contrast,
the tunnelling is clearly irrelevant. We confirmed that the electric- Received 28 September 2009; accepted 1 February 2010.
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25. Stiles, M. D., Xiao, J. & Zangwill, A. Phenomenological theory of current-induced Grant-in-Aid for Scientific Research in Priority Area ‘Creation and control of spin
magnetization precession. Phys. Rev. B 69, 054408 (2004). current’ (19048028) from MEXT, Japan, a Grant-in-Aid for Scientific Research (A)
26. Wigen, P. E., Doetsch, H., Ming, Y., Baselgia, L. & Waldner, F. Chaos in magnetic from MEXT, Japan, the global COE for the ‘Materials integration international
garnet thin films. J. Appl. Phys. 63, 4157–4159 (1988). centre of education and research’ and ‘High-level global cooperation for
27. Rippard, W. H., Pufall, M. R., Kaka, S., Russek, S. E. & Silva, T. J. Direct-current leading-edge platform on access spaces (C12)’ from MEXT, Japan, a Grant for
induced dynamics in Co90Fe10/Ni80Fe20 point contacts. Phys. Rev. Lett. 92, Industrial Technology Research from NEDO, Japan, and Fundamental Research
027201 (2004). Grant from TRF, Japan.
28. Krivorotov, I. N. et al. Large-amplitude coherent spin waves excited by spin-
polarized current in nanoscale spin valves. Phys. Rev. B 76, 024418 (2007). Author Contributions Y.K., K.H., K.U. and K.A. performed the measurements and
29. Laulicht, I., Suss, J. T. & Barak, J. The temperature dependence of the analysed the data; J.O. carried out the numerical analysis; S.T., S.M. and E.S.
ferromagnetic and paramagnetic resonance spectra in thin yttrium-iron-garnet provided the theoretical analysis; H.U. and H.K. contributed to the sample
films. J. Appl. Phys. 70, 2251–2258 (1991). fabrication; Y.K., K.H, K.U., M.M. and K.T. contributed to the experimental set-up;
30. Brown, W. F. Jr. Thermal fluctuations of a single-domain particle. Phys. Rev. 130, Y.K., S.T., J.O., K.U., M.M., H.U., K.T., S.M. and E.S. wrote the manuscript; all authors
1677–1686 (1963). discussed the results and commented on the manuscript; and E.S. planned and
supervised the project.
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature. Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Acknowledgements We thank K. Sato, Y. Suzuki, Y. Tserkovnyak, G. Tatara, Correspondence and requests for materials should be addressed to E.S.
T. Ishibashi and K. M. Itoh for discussions. This work was supported by a (saitoheiji@imr.tohoku.ac.jp).

266
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08876

METHODS electric connections were made with the microprobes. An electric current was
Film preparation. A single-crystal Y3Fe5O12 (111) film was grown on a 0.5-mm- applied to the Pt film i with a voltage-function generator (NF WF1946B) and a
thick Gd3Ga5O12 (111) single-crystal substrate by liquid phase epitaxy. For the hand-made current amplifier. The voltage signal in the Pt film o was led to a
film growth, we used PbO-B2O3 flux around 1,200 K. Then, a 10-nm-thick Pt storage scope (Tektronix TDS1001) and to a lock-in amplifier (NF LI5640) via a
layer was sputtered on the Y3Fe5O12 layer through a metal mask using a sputter- preamplifier (DL-instruments 1201). Precise I–V measurements were performed
ing and etching system (K-Science KE604TT1-S2B). Immediately before the with the lock-in amplifier by modulating sinusoidally the input d.c. current and
sputtering, the surface was cleaned through the metal mask by Ar-ion bombard- then by integrating the lock-in signal with respect to the current. Before the
ment in a vacuum. The chemical composition and crystalline structure of the precise measurement, we performed rough measurements to see the overall
film were confirmed by X-ray fluorescence spectroscopy and X-ray diffractome- behaviour of V; we measured d.c. voltage V using the storage scope with chan-
try, respectively. The magnetization-saturation field for the Y3Fe5O12 layer was ging input current without sinusoidal modulations.
estimated by measuring the magnetic-field dependence of a magneto-optical Micromagnetic simulation. We numerically solved the Landau–Lifshitz–
Kerr effect signal induced by He-Ne laser light (632.8 nm) in a cross-Nicol Gilbert equation:
configuration. dM a dM
Spin pumping measurements shown in Fig. 2. The Pt/Y3Fe5O12 sample system ~{c(Heff |M)z M| ztstt
dt Ms dt
was placed near the centre of a TE011 microwave cavity at which the magnetic-
field component of the microwave mode is maximized while the electric-field by using the object-oriented micromagnetic framework31 extended to the fourth
component is minimized. During the measurements, the microwave mode with order Runge-Kutta method. Here, c 5 1.76 3 107 Oe21 s21 is the gyromagnetic
a frequency of 9.44 GHz was exited in the cavity, and an external static magnetic ratio, 4pMs 5 1,956 Oe is the saturation magnetization and a 5 6.7 3 1025 is
field H was applied along the film. The microwave power was less than 10 mW, a the Gilbert damping coefficient. The field Heff is an effective magnetic field
value lower than the saturation of the ferromagnetic resonance absorption for which includes an applied magnetic field Hdc 5 2.3 kOe, an exchange field
the present sample. The microwave absorption was detected using a spectro- Hex 5 (2A/Ms2)=2M due to the ferromagnetic coupling where the exchange stiff-
meter (JEOL JES-FA200) connected to the cavity. For voltage measurements, ness A 5 4.73 3 10212 J m21, the demagnetization field Hdemag and the random
two 100-nm-thick 0.5-mm-wide Pt electrodes were sputtered on the ends of the magnetic field due to the thermal effect30. tstt 5 cM 3 (s 3 M) represents the spin
Pt film. A twisted pair of thin coated Cu wires (0.08 mm in diameter) is con- torque term due to the transmission of angular momentum from Pt film i to
nected to the electrodes with silver paste. The voltage difference between these Y3Fe5O12 via an exchange interaction (see Supplementary Information section B
two wires was measured using a lock-in amplifier (NF LI5640) and a volt meter for details). When the static field Hdc is applied, the spin torque term tstt points
(Keithley 2000) via an amplifier (DL-instruments 1201). to the direction opposite to the damping vector M 3 dM/dt. For realizing the
Microwave emission measurements shown in Fig. 3. A gold coplanar-wave- experimental condition where the input is the threshold current, the parameter c
guide antenna, whose centre-conductor width is 100 mm, was attached to the Pt was chosen so that the magnitude is that of the damping term. We calculated the
surface of the Pt/Y3Fe5O12 sample film. The microwave signal received by the time average of the out-of-plane component of the magnetization. The gradation
antenna was led to an amplifier (Miteq AFS44-00102000-30-10P-44) via a micro- of the colour indicates that the spin-wave spin current propagates in the x
wave probe (Picoprobe 40A-GSG-100-VP-NM). The amplified microwave signal direction.
was analysed and recorded by a spectrum analyser (Anritsu MS2687B).
Non-local I–V measurements shown in Fig. 4. The sample film was fixed on a 31. Donahue, M. J. & Porter, D. G. OOMMF v1.2a3 Object Oriented MicroMagnetic
sample stage of a micro-prober system (Nagase GRAIL-103054LVMG) and Framework Software (NIST, 2004).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08863

LETTERS
Tunable polymer multi-shape memory effect
Tao Xie1

Shape memory polymers are materials that can memorize temporary polymer shrank by about 26% and reached an equilibrium length
shapes and revert to their permanent shape upon exposure to an (Supplementary Fig. 1). The shrinkage was not related to water loss
external stimulus such as heat1, light2,3, moisture4 or magnetic field5. (see Supplementary Information), but was due to the removal of
Such properties have enabled a variety of applications including residual stress/strain from the polymer processing step. X-ray scatter-
deployable space structures6, biomedical devices7,8, adaptive optical ing analysis further suggests that the stress relaxation occurred in the
devices9, smart dry adhesives10 and fasteners11. The ultimate poten- ionic phase of the polymer (Supplementary Fig. 2). The annealing also
tial for a shape memory polymer, however, is limited by the number led to polymer darkening, yet the infrared spectra of PFSA before and
of temporary shapes it can memorize in each shape memory cycle after annealing (Supplementary Fig. 3) appear nearly identical. This
and the ability to tune the shape memory transition temperature(s) suggests that the primary polymer structure remained intact during
for the targeted applications. Currently known shape memory annealing, as supported by a previous study showing20 that the ther-
polymers are capable of memorizing one or two temporary shapes, mal degradation of PFSA starts at 300 uC. In addition, more recent
corresponding to dual- and triple-shape memory effects (also count- work on the chemical durability of PFSA suggested that its end-groups
ing the permanent shape), respectively11–13. At the molecular level, are the most unstable moieties on the polymer21, the thermal decom-
the maximum number of temporary shapes a shape memory poly- position of which is the probable source for the darkening observed
mer can memorize correlates directly to the number of discrete here. Given the high molecular weight of PFSA17 and the correspond-
reversible phase transitions (shape memory transitions) in the poly- ing low end-group concentration, minor degradation in polymer end-
mer11–13. Intuitively, one might deduce that multi-shape memory groups would not be detectable by infrared21 or thermo-gravimetric
effects are achievable simply by introducing additional reversible analysis20, nor is it expected to affect the polymer’s thermomechanical
phase transitions. The task of synthesizing a polymer with more than properties.
two distinctive and strongly bonded13 reversible phases, however, is The equilibrium length after annealing defines the permanent
extremely challenging. Tuning shape memory effects, on the other shape for PFSA and its shape memory performance was evaluated
hand, is often achieved through tailoring the shape memory transi- using thermal mechanical analysis. In a typical thermally activated
tion temperatures, which requires alteration in the material com- dual-shape memory cycle, a shape memory polymer is deformed at
position14–16. Here I show that the perfluorosulphonic acid ionomer an elevated temperature (deformation temperature, Td) and the
(PFSA), which has only one broad reversible phase transition, deformed temporary shape is fixed upon cooling. When heated to
exhibits dual-, triple-, and at least quadruple-shape memory effects, a recovery temperature (Tr), the permanent shape is recovered. The
all highly tunable without any change to the material composition. dual-shape memory effect can thus be quantified on the basis of the
PFSA is a commercial thermoplastic polymer with a polytetrafluor- percentage of shape fixation (shape fixity, Rf) and shape recovery
oethylene backbone and perfluoroether sulphonic acid side chains (shape recovery, Rr), calculated using equations (1) and (2) shown
(Fig. 1a). Owing to its proton conducting capability, PFSA has been in the Methods Summary.
extensively studied as proton exchange membranes for fuel cells17. Accordingly, PFSA was found to exhibit excellent dual-shape
Besides fuel cells, PFSA has also been used in a number of other appli- memory performance when deformed and recovered at 140 uC (that
cations including chlor-alkali cells, sensors and actuators. Generally, is, Td and Tr both above the upper end of the glass transition), with
PFSA possesses an ionic cluster phase and a crystalline phase17. Above both Rf and Rr approaching 100% (Fig. 1c). Shape fixing and recovery
room temperature (25 uC), PFSA typically shows two thermally rever- can also be carried out near the peak (Supplementary Fig. 4,
sible transitions17. These two transitions (below 150 uC and above Td 5 Tr 5 100 uC) or the onset (Fig. 1d, Td 5 Tr 5 60 uC) of the glass
240 uC) correspond respectively to the short-range segmental motions transition, with Rf and Rr above 97% in both cases. Consecutive shape
(glass transition) within a static electrostatic network and the onset of memory cycling experiments (Td 5 Tr 5 80 uC) show that shape
the long-range molecular mobility due to destabilization of the elec- memory performance is highly reproducible (Supplementary Fig. 5).
trostatic network18. Although a glass transition is usually used for a Additionally, upon consecutive cycling at the annealing temperature of
shape memory transition1, the temperature persistence of an electro- 140 uC, PFSA experienced almost no deterioration in either Rf or Rr
static network has also proved beneficial in the design of shape memory (Supplementary Fig. 6), confirming that indeed the darkening due to
polymers19. For PFSA, its combination of a glass transition and a mech- annealing did not affect the thermomechanical response of PFSA.
anism for setting a permanent shape (the temperature-persistent elec- A prototype dual-shape memory cycle occurs with both shape fixa-
trostatic network) motivated us to explore its shape memory tion and recovery above the shape memory transition to maintain
properties. optimal shape fixing and recovery properties. Although recent studies
The PFSA used in this study is Nafion, which has an equivalent have demonstrated the feasibility and benefits (for example, thermal
weight (average molecular mass per mole of ionic groups in g mol21) healing) of shape fixation near22,23 or even below24 the onset of the
of 1,000. It possesses a broad glass transition from ,55 uC to ,130 uC shape memory transition, Rf under such conditions is not typically
(Fig. 1b). Before evaluating its shape memory performance, the as- reported. Presumably, undesirable instantaneous recovery (spring
received PFSA film was first annealed at 140 uC, upon which the back) may have occurred, leading to a compromised Rf, as confirmed
1
Mail Code: 480-106-710, Chemical Sciences and Materials Systems Laboratory, General Motors Research and Development Center, 30500 Mound Road, Warren, Michigan 48090-
9055, USA.

267
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

by a recent example for which an Rf of 83% was reported24. So, that PFSA is tunable simply by deforming the same polymer at different
PFSA maintained near-perfect shape fixing and recovery performance temperatures, again, without compromising Rf.
at any temperature above the onset of its glass transition is rather The multi-stage recovery (Fig. 1e) further indicates that the polymer
unexpected. can memorize multiple temporary shapes in a single shape memory
Although the strain imparted at a temperature as low as 60 uC can cycle, that is, the multi-shape memory effect. Here, it is important to
be fully recovered at the same temperature (Fig. 1d), the deformation note that currently known triple-shape memory polymer systems
strain introduced at 140 uC was unable to recover fully at lower tem- possess two well separated phase transitions and that the two tem-
peratures. As shown in Fig. 1e, for PFSA deformed at 140 uC, increas- porary shapes are introduced above and between both transition tem-
ing the recovery temperature in a staged manner led to staged peratures, respectively11–13. Tuning the triple-shape memory effect for
recovery behaviour. Previous work has shown that shape recovery such systems would require varying of the ratio between the two
at a certain temperature may depend on the deformation temper- reversible phases or changing the reversible phase transition tempera-
ature at which the shape fixing occurred22,23, but a well-defined multi- tures11–13, which cannot be realized without changing the material
stage (beyond two-stage) recovery such as that shown in Fig. 1e has composition. PFSA, in contrast, has only one broad phase transition
not previously been realized. In contrast to the staged recovery, a and its triple-shape memory effect, therefore, should not be limited to
continuous shape recovery behaviour was observed when the two discrete temperature regions.
recovery temperature was increased in a continuous fashion to The triple-shape memory effect for PFSA is demonstrated in
140 uC for three samples deformed at 60, 80 and 100 uC, respectively Fig. 2a. The permanent shape S0 was deformed at 140 uC and fixed
(Supplementary Fig. 7). In those cases, the shape recovery at 68 uC to yield the first temporary shape S1, which was further
curve shifted to the right (higher temperatures) for PFSA with a deformed at 68 uC and fixed at 20 uC to yield the second temporary
higher Td. shape S2. Upon reheating to 68 uC, the recovered first temporary
The above experimental results clearly suggest that the polymer shape S1rec was obtained. Further heating to 140 uC yielded the
memorizes not just the strain, but also the thermomechanical history. recovered permanent shape S0rec. Quantitatively, two triple-shape
For instance, the polymer memorizes the deformation temperature, memory cycles are illustrated in Fig. 2b (with two deformation tem-
showing the so-called temperature memory effect25. As illustrated in peratures of 140 uC and 53 uC) and Supplementary Fig. 9 (with two
Supplementary Fig. 8a–c, when the deformation occurs at a certain Td deformation temperatures of 90 uC and 53 uC).
within the glass transition, the subsequent iso-strain stress recovery Equations (3) and (4) (see Methods Summary) were used to cal-
experiment shows that the peak of recovery stress appears at a tem- culate the Rf and Rr values for the triple-shape memory cycles. As
perature roughly the same as the corresponding Td. Compared to the such, a notable difference between Fig. 2b and Supplementary Fig. 9
earlier example of the polymer temperature memory effect25, this lies in the first-shape fixity (Rf(S0RS1) being 83.5% and 74.5%,
effect for PFSA occurs in a temperature range with both Rf and Rr respectively), suggesting that the first-shape fixity is closely related
approaching 100%. In addition, whereas adjusting the recovery stress to the difference between the two deformation temperatures in the
for known shape memory polymer systems is typically accomplished corresponding triple-shape memory cycle. Shape recoveries, on the
by changing the rubbery modulus through the adjustment of other hand, were above 95% in all cases. Together, Fig. 2a and b and
the crosslinking density8,14–16 or inclusion of reinforcing fillers3,26, Supplementary Fig. 9 confirm that the triple-shape memory effect for
Supplementary Figs 8a–c suggest that the peak recovery stress of PFSA can be realized at any two arbitrary temperatures above the

a b 1.0 c 140
0.9
3 40
(CF2CF2)m CF2CF 0.8 110

Temperature (ºC)
n
30

Stress (MPa)
O
log[(E′) (MPa)]

0.6
Strain (%)

2 0.6 80
tanδ

CF2
CF CF3 20
0.4
O 50
0.3
1 0.2 10
CF2
CF2 20
0.0 0
SO3H 0.0
0 –10
20 40 60 80 100 120 140 0 20 40 60 80
Temperature (ºC) Time (min)

d e 120
140
80 9
60 100 1.0
110
Temperature (ºC)

Temperature (ºC)

80
Strain (%)

Stress (MPa)
Stress (MPa)

6
40 60
Strain (%)

60 80
3 40 0.5
20 40 50
20
0
0 20
0 20 0.0
0 10 20 30 40 50 0 50 100 150 200 250 300
Time (min) Time (min)

Figure 1 | Structure, dynamic mechanical, and dual-shape memory modulus E9. c, Dual-shape memory cycle at Td 5 Tr 5 140 uC. Rf: 100%, Rr:
properties of PFSA. a, Structure of PFSA (m 5 5.56 for an equivalent weight 96.6%. d, Dual-shape memory cycle at Td 5 Tr 5 60 uC. Rf: 98.6%, Rr: 97.7%.
of 1,000; n is the number of repeating units). b, Dynamic mechanical analysis e, Multi-staged shape recovery (Td 5 140 uC).
curve of PFSA. tand is the ratio between the loss modulus E99 and the storage
268
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a a

S0 S1
S0 S1 S2 S3 S2rec S1rec S0rec

b Fixing Recovery
S2 15
140
εS3, load 140
εS3
120 12
120

Temperature (°C)
100 εS2, load 9

Stress (MPa)
S1rec S0rec 100

Strain (%)
80 εS2 εS2, rec
80 6
b Fixing Recovery 60
εS1, load
60
120 40 εS1, rec 3
εS2, load εS1
εS2 40
140 20
100 10 εS0 εS0, rec 0
0 20
80 Temperature (ºC)

Stress (MPa)
30 60 90 120 150 180
Strain (%)

100 Time (min)

8
60 εS1, load
εS1, rec Figure 3 | Quadruple-shape memory properties of PFSA. a, Visual
εS1
40 demonstration. S0: permanent shape; S1: first temporary shape (Td1:
60 4 140 uC); S2: second temporary shape (Td2: 107 uC); S3: third temporary
20 shape (Td3: 68 uC); S2rec: recovered second temporary shape (Tr1: 68 uC);
εS0, rec S1rec: recovered first temporary shape (Tr2: 107 uC); S0rec: recovered
εS0
0 20 0 permanent shape (Tr3: 140 uC). b, Quantitative thermal mechanical cycle
(Td1 5 Tr3 5 140 uC, Td2 5 Tr2 5 90 uC, Td3 5 Tr1 5 53 uC).
40 60 80 100 120 140 160
Rf(S0RS1): 58.7%, Rf(S1RS2): 57.1%, Rf(S2RS3): 96.1%,
Time (min)
Rr(S3RS2): 100.0%, Rr(S2RS1): 99.6%, Rr(S1RS0): 93.0%.
Figure 2 | Triple-shape memory properties of PFSA. a, Visual
demonstration. b, Quantitative thermal mechanical cycle
(Td1 5 Tr2 5 140 uC, Td2 5 Tr1 5 53 uC). Rf(S0RS1): 83.5%,
strong and temperature-dependent molecular interactions (for
Rf(S1RS2): 96.7%, Rr(S2RS1): 97.4%, Rr(S1RS0): 94.6%. example, self-complementary hydrogen bonding27,28) or composi-
tional heterogeneity (for example, compositional gradient) in the
polymer structure. Some aspects of tunable multi-shape memory
onset of the glass transition, provided that the two temperatures are effects may even be applicable to semi-crystalline shape memory
not too close. polymers. Crystallization of such polymers can be induced by either
The versatile shape memory properties for PFSA are also reflected in cooling or strain3,24,29 and the structural perfection of crystallites (and
a quadruple-shape memory effect. As demonstrated in Fig. 3a, starting hence their corresponding melting transitions) depends on the ther-
as a permanent shape S0, PFSA can memorize three temporary shapes momechanical history29. Using a combination of temperature and
(S1, S2 and S3) in the same shape memory cycle. Subsequent heating strain control during the shape fixing step, it is potentially possible to
to the relevant temperatures led to the recovered shapes (S2rec, S1rec manipulate the melting transition, which would lead to tunable
and S0rec). Quantitatively (Fig. 3b), whereas the Rr values at all three shape recovery behaviour. The above concepts, althought they are
recovery stages were above 93%, the first and second Rf values were yet to be verified experimentally, suggest strategies for expanding
around 60%. Theoretically, multi-shape memory effects beyond tunable multi-shape memory effects to a broad range of polymers,
quadruple- are feasible. In fact, the multi-stage recovery (Fig. 1e) itself the availability of which could significantly expand the technical
can be interpreted as memorizing five temporary shapes through a potential for shape memory polymers.
one-step programming process, with each recovery step representing
the recovery of one temporary shape12. If more and more temporary
METHODS SUMMARY
shapes were to be fixed individually into the PFSA instead of the one-
All quantitative shape memory properties were evaluated in a tensile and force
step programming process, the Rf for each individual fixing step would
controlled mode in a typical thermal mechanical analysis set-up under ambient
be further compromised because the fixation of more temporary humidity conditions (relative humidity of 22–34%). The heating and cooling
shapes demands shape fixation at a temperature closer to the corres- rates were both 5 uC per minute. Rf and Rr for the dual-shape memory effect were
ponding deformation temperature. calculated using equations (1) and (2) below:
The versatile shape memory properties demonstrated above for
Rf ~100%|e=eload ð1Þ
PFSA stem from its broad glass transition. This broad transition
can be viewed as the collective contribution of an infinite number Rr ~100%|(e{erec )=e ð2Þ
of transitions, each corresponding to infinitely sharp transition tem-
peratures continuously distributed across the broad transition. In the where eload represents the maximum strain under load, e is the fixed strain after
context of shape memory effects, these sharp transitions can be cooling and load removal, and erec is the strain after recovery.
Equations (1) and (2) are expanded to equations (3) and (4) to calculate Rf and
understood as individual memory elements. Depending on the
Rr for the triple-shape and quadruple-shape memory effects.
deformation temperature(s) during the shape memory cycles, a vari-
able number of memory elements are activated for the memory func- Rf (X?Y )~100%|(ey {ex )=(ey, load {ex ) ð3Þ
tion, leading to the tunable shape memory effects. Therefore, other
ionomers with broad glass transitions are likely to show similar shape Rr (Y ?X)~100%|(ey {ex, rec )=(ey {ex ) ð4Þ
memory effects. Amorphous shape memory polymers possessing a where X and Y denote two different shapes, respectively, ey, load represents the
broad thermally reversible transition are another class of candidates. maximum strain under load, ey and ex are fixed strains after cooling and load
In such cases, the broad thermal reversible transition may arise from removal, and ex,rec is the strain after recovery.
269
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

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14. Xie, T. & Rousseau, I. A. Facile tailoring of thermal transition temperatures of Acknowledgements I thank I. Rousseau, H. Kia, P. Krajewski, X. Huang and
epoxy shape memory polymers. Polymer 50, 1852–1856 (2009). M. Verbrugge for comments, D. Eckel for Infrared analysis, and R. L. Speer Jr for
15. Liu, C. & Mather, P. T. Thermomechanical characterization of a tailored series of X-ray analysis.
shape memory polymers. J. Appl. Med. Polym. 6, 47–52 (2002).
16. Liu, C. et al. Chemically cross-linked polycyclooctene: synthesis, characterization, Author Information Reprints and permissions information is available at
and shape memory behavior. Macromolecules 35, 9868–9874 (2002). www.nature.com/reprints. The author declares no competing financial interests.
17. Mauritz, K. A. & Moore, R. B. State of understanding of Nafion. Chem. Rev. 104, Correspondence and requests for materials should be addressed to the author
4535–4585 (2004). (tao.xie@gm.com).

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Vol 464 | 11 March 2010 | doi:10.1038/nature08905

LETTERS
A large atomic chlorine source inferred from
mid-continental reactive nitrogen chemistry
Joel A. Thornton1, James P. Kercher1{, Theran P. Riedel1,2, Nicholas L. Wagner3, Julie Cozic3,4, John S. Holloway3,4,
William P. Dubé3,4, Glenn M. Wolfe1,2, Patricia K. Quinn5, Ann M. Middlebrook3, Becky Alexander1
& Steven S. Brown3

Halogen atoms and oxides are highly reactive and can profoundly reaction (1b). Owing to efficient deposition of HNO3, reaction (1a)
affect atmospheric composition. Chlorine atoms can decrease the represents a terminal NOx sink in the lower troposphere, whereas
lifetimes of gaseous elemental mercury1 and hydrocarbons such as reaction (1b) recycles NOx through ClNO2 photolysis (reaction (2))
the greenhouse gas methane2. Chlorine atoms also influence cycles within a few hours after sunrise14.
that catalytically destroy or produce tropospheric ozone3, a green-
house gas potentially toxic to plant and animal life. Conversion of ClNO2 1 hn R Cl. 1 NO2 (2)
inorganic chloride into gaseous chlorine atom precursors within
the troposphere is generally considered a coastal or marine air phe- Models incorporating reactions (1b) and (2) differ in their predicted
nomenon4. Here we report mid-continental observations of the impacts, but generally show summer ozone concentrations in coastal
chlorine atom precursor nitryl chloride at a distance of 1,400 km urban areas to be enhanced by faster cycling of oxidants produced
from the nearest coastline. We observe persistent and significant from Cl . attacking hydrocarbons3,5.
nitryl chloride production relative to the consumption of its nitro- These previous studies have been restricted to coastal or marine
gen oxide precursors. Comparison of these findings to model pre- regions. However, our observations made near Boulder, Colorado,
dictions based on aerosol and precipitation composition data from an urban location in the middle of North America, demonstrate that
long-term monitoring networks suggests nitryl chloride production this chemistry extends well inland, and are consistent with calcula-
in the contiguous USA alone is at a level similar to previous global tions based on network observations of aerosol and precipitation
estimates for coastal and marine regions5. We also suggest that a composition.
significant fraction of tropospheric chlorine atoms6 may arise We made in situ measurements on 11–25 February 2009 at the
directly from anthropogenic pollutants. National Oceanic and Atmospheric Administration’s Kohler Mesa
Night-time reactions of nitrogen oxides are known to convert facility, just west and 150 m above Boulder, Colorado. The site, subject
inorganic chloride into chlorine atom (Cl .) precursors7 (for example, to large variability in pollutant levels, receives either the urban plume
Fig. 1). N2O5, a nocturnal NOx reservoir (NOx 5 NO 1 NO2), can from nearby cities, or much cleaner air from the Rocky Mountain
react on airborne particles to produce only HNO3 (reaction (1a)), or
both nitryl chloride (ClNO2) and nitrate (NO32) (reaction (1b)).

N2O5(g) 1 H2O(aq) R 2HNO3(aq) (1a)

HCl Acid HNO3 HCl
HNO3 Cl
displacement H2SO4
2 2 N2O5
N2O5(g) 1 Cl (aq) R ClNO2(g) 1 NO3 (aq) (1b) ClNO2
Acid precursor and
Inland NOx and NOx
chlorine emissions
The ClNO2 yield depends on water and chloride concentrations NOx chlorine emissions
within particles8. The latter is often sufficient for efficient ClNO2 Aeolian sources
production5. However, the moles of particulate chloride per volume Sea
spray
of air (pCl2) is typically small, and would limit reaction (1b) except
that gaseous HCl can almost always provide a larger reservoir
through equilibrium repartitioning to particles9. Depending on the Figure 1 | Schematic of chlorine activation by night-time NOx chemistry.
environment, soluble chloride or NOx availability and reactivity may The emphasis here is on inland ClNO2 production, although production also
limit ClNO2 production. The few prior observations of ClNO2 within occurs in coastal and marine regions. Globally, sea spray is the dominant
polluted marine air have shown it is produced in high yields during source of tropospheric inorganic chloride9, which can be transported as fine-
mode sea spray particles or gaseous HCl following acid displacement by
spring and summer, and exceeds previously predicted values by
HNO3 or H2SO4. Locally, other sources of chloride from industrial
factors of 2–30 (refs 5, 10, 11). activities9, biomass burning9, or transport of wind-blown soil dust15 may be
ClNO2 production has an impact on NOx and Cl budgets, both of important. Reactions of N2O5 on chloride-containing particles produce
which affect the troposphere’s oxidizing capacity12. Current atmo- ClNO2, which photochemically converts to chlorine atoms and NO2 in the
spheric chemistry models predict reaction (1a) accounts for 30–50% morning. The chlorine atoms react with hydrocarbons, returning to HCl or
of total NOx removal in polluted regions13, but they typically neglect forming an organo-chlorine compound.
1
Department of Atmospheric Sciences, 2Department of Chemistry, University of Washington, Seattle, Washington 98195, USA. 3Earth Systems Research Laboratory, National
Oceanic and Atmospheric Administration, Boulder, Colorado 80305, USA. 4Cooperative Institute for Research in Environmental Studies, Boulder, Colorado 80309, USA. 5Pacific
Marine Environmental Laboratory, National Oceanic and Atmospheric Administration, Seattle, Washington 98115, USA. {Present address: Department of Chemistry, Hiram College,
Hiram, Ohio 44234, USA.

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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

region. Typically residing above the urban nocturnal boundary layer, equations initialized with observations of NO2, O3, Sa, pCl2. The N2O5
the site is often minimally affected by direct, local night-time emissions. reaction probability and wClNO2 are adjusted between 0.005–0.03 and
It therefore allows overnight observation of the chemical evolution of 0.07–0.36, respectively, so the model matches the observed NO2, O3,
air masses characteristic of this region. More information on the site N2O5 and ClNO2 at a specific time. Model details and additional out-
and measurement methods is provided in Supplementary Information. put are given in Supplementary Information.
There are three salient features of the February 2009 time series The night of 15–16 February illustrates the time evolution of
(Fig. 2). First, ClNO2 production was routinely observed in a region ClNO2 under low wind, providing a model-to-measurement com-
of North America far removed from sea spray, but possibly affected by parison least affected by air mass variations due to transport. A con-
chloride transport from coastal areas9 or inland salt beds15, and by stant model wClNO2 of 0.14 adequately reproduces the observed
anthropogenic sources including combustion and transportation9. ClNO2 and pCl2 evolution (Fig. 3b), as well as NO2, O3 and N2O5
Second, ClNO2 mixing ratios consistently reached 100–450 parts per at specific times after sunset. These species are more sensitive to fresh
trillion by volume (p.p.t.v.) whenever the urban plume was sampled. emissions or land-surface interactions caused by slight changes in
These levels are unexpectedly large, reaching a third to a half of the transport, and therefore exhibit larger deviations from the model
maximum values observed in polluted coastal areas5,10. Third, consis- curve (Supplementary Information). This night did not show
tent with the above mechanism, ClNO2 was observed only at night or evidence of a chloride limitation, and had the highest 24-h average
in the early morning, and often correlated with N2O5 (Fig. 2, top). pCl2 and ClNO2 of the campaign. Most other nights did not allow
However, their relationship was variable, probably due to changes in: comparison to the box model with a single set of initial conditions
(1) the N2O5 heterogeneous loss rate, which depends on humidity, mainly because of transport effects (Supplementary Information).
particle composition and phase16, and surface area density (Sa); (2) air The box-model-derived wClNO2 are generally lower than calculated
mass age and non-aerosol losses of N2O5 (Supplementary Informa- from laboratory parameterizations8,18 using measured pCl2 and
tion); and (3) chloride availability. We observed similar levels of N2O5 estimated particle water17 (Supplementary Information). Several
and ClNO2 in February 2008 at a nearby location, suggesting year-to- possible reasons for the apparent discrepancy exist. First, although
year consistency (Supplementary Information). sufficient total chloride mass may exist, it probably partitions to
As expected, the 2009 observations also indicate total available a fraction of the particle surface area19, reducing the population
chloride for ClNO2 production is greater than pCl2 alone5 (Fig. 3a). average efficiency of reaction (1b) which our model-derived wClNO2
The inferred minimum abundance of total available chloride values represent. A more accurate calculation would require size-
(100–500 p.p.t.v.) is consistent with admittedly uncertain HCl obser- resolved particle composition and pH measurements, which are chal-
vations in other urban areas9, and with the pCl2 « HClg equilibrium lenging20. Additionally, the wClNO2 required in the model is different
predicted using thermodynamic models constrained by our measure- for different nights, and even decreases within some nights, suggest-
ments17 (Supplementary Information). ing significant variability in the factors controlling ClNO2 produc-
We performed time-dependent chemical box modelling to develop tion as well as a possible limitation by available chloride.
a quantitative relationship between measured pCl2 and the ClNO2 The existence of a mid-continental ClNO2 source was unknown
yield from reaction (1) (wClNO2 ) needed to explain the observations. before the observations we report here. Yet, the ingredients for
The box model integrates explicit N2O5, ClNO2 and pCl2 mass balance this source, NOx and particulate chloride, are known from various
500 500 500 Figure 2 | Time series of key quantities
observed in Boulder, Colorado, from 11 to 25
ClNO2 (p.p.t.v.)

400
ClNO2 (p.p.t.v.)

400 400
ClNO2 (p.p.t.v.)

February 2009. Main panels: from top to

300 300 300
bottom, ClNO2, N2O5, pCl2 shown in mixing
200 200 200 ratio units (p.p.t.v.) for comparison with gases,
100 100 100 and relative humidity (RH). Insets (top) are
0 0 0 point-to-point comparisons of ClNO2 to N2O5
0 400 800 1,200 0 400 800 1,200 0 400 800 1,200 on 13–14 February (left), 15–16 February
N2O5 (p.p.t.v.) N2O5 (p.p.t.v.) N2O5 (p.p.t.v.)
(centre) and 21–22 February (right). These nights
are characterized by high (85%), moderate (50%)
ClNO2 (p.p.t.v.)

400
and low (35%) RH; and high, moderate and low
ratios of ClNO2:N2O5, respectively. The
200
ClNO2:N2O5 ratio is not solely a function of RH.
For example, on 14 February we sampled air from
0 the urban surface layer containing fresh NOx
1,500 emissions which can titrate N2O5. See
N2O5 (p.p.t.v.)

Supplementary Information for additional

1,000 details.
500

0
pCl– (p.p.t.v.)

100

50

80
RH (%)

40

0
12 14 16 18 20 22 24
Days in February 2009 (local time)

272
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a b 600

200 ClNO2 ClNO2

500
4× pCl– 4 × pCl–

Mixing ratio (p.p.t.v.)

Mixing ratio (p.p.t.v.)

150 400

300
100

200

50
100

0 0
0 3 6 9 12 15 18 21 24 0 4 8 12 16
Hour of day (local time) Hours since sunset

Figure 3 | Observed and modelled relationships of ClNO2 and particulate pCl2 below the detection threshold, and solid lines represent model
chloride. a, 3-h averages of coincident ClNO2 and 4 3 pCl2 versus time of predictions. The model reasonably reproduces the night-time evolution of
day. Shading indicates the standard error of means; each bin contains more ClNO2 with a constant ClNO2 yield of 0.14, and matches the ozone, NO2 and
than 15 points. b, Observed and modelled evolution of ClNO2 and pCl2 over N2O5 observations 12 h after sunset. See Supplementary Information for a
the 12-h period from sunset on 15 February. Symbols as in a, stars denote more complete description of the model.

long-term databases to be ubiquitous across the North American wsClNO2 ðr Þ with a combination of precipitation and aerosol composi-
continent (and elsewhere). With these data sets, we now estimate tion measurements from the National Atmospheric Deposition
the magnitude of this halogen source across wider scales and com- Program (NADP)22 and the Interagency Monitoring of Protected
pare these estimates to multi-location observations of ClNO2. Visual Environments (IMPROVE) network23, respectively. This
Assuming lower tropospheric NOx is in steady-state, we calculate a approach provides wClNO2 consistent with all existing ClNO2 obser-
total annual ClNO2 production rate as a sum over seasonal averages vations in Boulder and coastal regions5,10, while reproducing the
(s) via the following equation: likely spatial distribution in total available chloride.
X XX Figure 4 shows the annual average fields of ENOx (Fig. 4a), fN2 O5
PClNO2 ~ s
PClNO 2
~ s
ENO x
(r)fNs 2 O5 (r)wsClNO2 (r) ð3Þ (Fig. 4b), wClNO2 (Fig. 4c), and the resulting PClNO2 (Fig. 4d). Based on
s s r
the seasonal values of these components, the variability in the Boulder
where r denotes a 1 3 1u grid cell in the contiguous US or its coastal and network data, and alternative approaches to equation (3), all of
s
regions, ENOx
(r) is the anthropogenic NOx emission rate from the which are presented in the Supplementary Information, we estimate
EDGAR database21, fNs 2 O5 ðr Þ is the fraction of NOx removed by N2O5 that PClNO2 for the contiguous US lies in the range 3.2–8.2 Tg yr21,
heterogeneous chemistry predicted by the GEOS-Chem global providing a photolytic Cl. source of 1.4–3.6 Tg Cl yr21. This US
model13 and wsClNO2 ðr Þ is the ClNO2 yield. We constrained ClNO2 source is far larger than the first global estimate of

a NOx emission rate b Fraction of NOx lost via N2O5

8

45 0.6
7.5

40 7
0.4

35 6.5

6 0.2
30
Latitude (°N)

5.5
c Yield of ClNO2 d Production rate of ClNO2
1
10
45

9
40
0.5
8
35

30 7

0
120 100 80 120 100 80
Longitude (°W) Longitude (°W)

Figure 4 | Annual average components of PClNO2 over the US. a, US NOx IMPROVE and NADP network observations; and d, the ClNO2 production
emissions (kg yr21) given by the EDGAR database with a logarithmic colour rate, PClNO2 , in g Cl yr21, obtained from the product of quantities shown in
scale starting at 105.5 kg NO2 yr21; b, annual average fraction of total nitrate panels a, b and c with a logarithmic colour scale starting at 106.5 g Cl yr21.
(0–2 km) formed by N2O5 reactions on particles predicted by the GEOS- Boulder, Colorado, is at approximately 40u N, 105u W.
Chem global model; c, annual average yield of ClNO2 calculated from
273
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

0.06 Tg Cl yr21 (ref. 24), and is similar to the recent 3.2 Tg Cl yr21 8. Behnke, W., George, C., Scheer, V. & Zetzsch, C. Production and decay of ClNO2
from the reaction of gaseous N2O5 with NaCl solution: bulk and aerosol
estimated for global coastal and marine regions5. More than half of experiments. J. Geophys. Res. D 102, 3795–3804 (1997).
the predicted ClNO2 production occurs over land, and 40% occurs 9. Graedel, T. E. & Keene, W. C. Tropospheric budget of reactive chlorine. Glob.
during winter (December–February). It also suggests that, in the US, Biogeochem. Cycles 9, 47–77 (1995).
an amount of NOx equivalent to 8–22% of that emitted cycles through 10. Kercher, J., Riedel, T. & Thornton, J. Chlorine activation by N2O5: simultaneous, in
ClNO2, potentially forming a non-negligible fraction of reactive nitro- situ detection of ClNO2 and N2O5 by chemical ionization mass spectrometry.
Atmos. Meas. Techn. 2, 193–204 (2009).
gen at night’s end, primarily during winter above the nocturnal sur- 11. Pechtl, S. & von Glasow, R. Reactive chlorine in the marine boundary layer in the
face layer (see Supplementary Information). outflow of polluted continental air: a model study. Geophys. Res. Lett. 34, L11813,
If the global distribution of pCl2 and NOx sources are similar to doi:10.1029/2007GL02976 (2007).
those in the US, as independent measurements suggest25, we estimate 12. Logan, J. A., Prather, M. J., Wofsy, S. C. & McElroy, M. B. Tropospheric chemistry:
a global perspective. J. Geophys. Res. 86, 7210–7254 (1981).
the global Cl. source from ClNO2 to be 8–22 Tg Cl. yr21, which is of 13. Alexander, B. et al. Quantifying atmospheric nitrate formation pathways based on
the same order as that inferred from methane isotopes in remote a global model of the oxygen isotopic composition (D17O) of atmospheric nitrate.
regions (25–35 Tg Cl . yr21)2,6. ClNO2 production will mostly occur Atmos. Chem. Phys. 9, 5043–5056 (2009).
in polluted regions; as such, Cl . from ClNO2 will react with other 14. Nelson, H. H. & Johnston, H. S. Kinetics of the reaction of Cl with ClNO and ClNO2
and the photochemistry of ClNO2. J. Phys. Chem. 85, 3891–3896 (1981).
hydrocarbons as well as methane. Thus, the Cl . source from ClNO2 15. Abuduwailli, J., Gabchenko, M. V. & Xu, J. R. Eolian transport of salts — a case
may be in addition to that inferred from methane isotopes2,6. study in the area of Lake Ebinur (Xinjiang, Northwest China). J. Arid Environ. 72,
Some fraction of the Cl. from ClNO2 may also convert to temporary 1843–1852 (2008).
reservoirs, such as HOCl and ClONO2, possibly enhancing Cl. pro- 16. Thornton, J. A., Braban, C. F. & Abbatt, J. P. D. N2O5 hydrolysis on sub-micron
organic aerosols: the effect of relative humidity, particle phase, and particle size.
duction not captured by our estimates11. Phys. Chem. Chem. Phys. 5, 4593–4603 (2003).
Although likely to be the most rigorous observationally con- 17. Wexler, A. S. & Clegg, S. L. Atmospheric aerosol models for systems including the
strained estimate of continental-scale ClNO2 production to date, ions H1, NH41, Na1, SO422, NO32,Cl2, Br2, and H2O. J. Geophys. Res. D 107,
the accuracy of the above approach (and probably any other) is 4207, doi:10.1029/2001JD000451 (2002).
18. Roberts, J. M., Osthoff, H. D., Brown, S. S. & Ravishankara, A. R. Laboratory
limited by significant uncertainty in fN2 O5 and wClNO2 that arise from measurements of ClNO2 yields from N2O5 reactions as a function of chloride
an incomplete understanding of N2O5 reactivity26,27 and of chloride molarity in various inorganic salt solutions. Geophys. Res. Lett. 36, L20808,
partitioning across the particle distribution19. More studies of the doi:10.1029/2009GL040448 (2009).
NOx–ClNO2–pCl2–HCl system around the globe are critical to 19. Moffet, R. C., de Foy, B., Molina, L. T., Molina, M. J. & Prather, K. A. Measurement
of ambient aerosols in northern Mexico City by single particle mass spectrometry.
refine these predictions. Atmos. Chem. Phys. 8, 4499–4516 (2008).
Our results imply that a significant fraction of the tropospheric Cl . 20. Keene, W. C. & Savoie, D. L. The pH of deliquesced sea-salt aerosol in polluted
source is anthropogenic, distributed over a relatively small area of the marine air. Geophys. Res. Lett. 25, 2181–2184 (1998).
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irradiated snow packs29, may also affect halogen activation far from Program (NRSP-3) (Illinois State Water Survey, Champaign, Illinois, 2010).
anthropogenic emissions, albeit on a smaller concentration scale. 23. Malm, W. C., Sisler, J. F., Huffman, D., Eldred, R. A. & Cahill, T. A. Spatial and
Therefore, past and future trends in continental NOx emissions seasonal trends in particle concentration and optical extinction in the United
States. J. Geophys. Res. D 99, 1347–1370 (1994).
may represent an important global influence on tropospheric 24. Erickson, D. J., Seuzaret, C., Keene, W. C. & Gong, S. L. A general circulation model
halogen sources that has largely gone unrecognized. based calculation of HCl and ClNO2 production from sea salt dechlorination:
NOx influences climate by directly and indirectly regulating oxid- reactive chlorine emissions inventory. J. Geophys. Res. D 104, 8347–8372 (1999).
ant budgets that determine the methane lifetime and affect aerosol 25. Zhang, Q. et al. Ubiquity and dominance of oxygenated species in organic aerosols
in anthropogenically-influenced Northern Hemisphere midlatitudes. Geophys.
formation30. Nocturnal processing of NOx is typically considered a Res. Lett. 34, L13801, doi:10.1029/2007GL029979 (2007).
reduction in the troposphere’s oxidizing capacity due to the conver- 26. Bertram, T. H. et al. Direct measurements of N2O5 reactivity on ambient aerosol
sion of NOx and ozone into soluble, largely non-reactive species (for particles. Geophys. Res. Lett. 36, L19803, doi:10.1029/2009GL040248 (2009).
example, HNO3). Widespread ClNO2 production instead renders 27. Brown, S. S. et al. Variability in nocturnal nitrogen oxide processing and its role in
regional air quality. Science 311, 67–70 (2006).
nocturnal NOx chemistry a potential source of oxidants and ozone 28. Singh, H. B. & Hanst, P. L. Peroxyacetyl nitrate (PAN) in the unpolluted atmosphere
in polluted regions, and may have as yet unrecognized influences in — an important reservoir for nitrogen oxides. Geophys. Res. Lett. 8, 941–944 (1981).
remote regions. Thus, anthropogenic NOx may have an even larger 29. Grannas, A. M. et al. An overview of snow photochemistry: evidence, mechanisms
effect on the oxidizing power of the lower troposphere than current and impacts. Atmos. Chem. Phys. 7, 4329–4373 (2007).
30. Shindell, D. T. et al. Improved attribution of climate forcing to emissions. Science
models estimate. 326, 716–718 (2009).
Received 2 October 2009; accepted 2 February 2010. Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
1. Donohoue, D. L., Bauer, D. & Hynes, A. J. Temperature and pressure dependent
rate coefficients for the reaction of Hg with Cl and the reaction of Cl with Cl: a Acknowledgements Funding for the ClNO2 observations and analysis was
pulsed laser photolysis-pulsed laser induced fluorescence study. J. Phys. Chem. A provided by NSF grants ATM-0633897 and ATM-0846183 to J.A.T. Boulder field
109, 7732–7741 (2005). measurements were supported in part by the NOAA Atmospheric Chemistry and
2. Platt, U., Allan, W. & Lowe, D. Hemispheric average Cl atom concentration from Climate Program. J.P.K. thanks the Camille and Henry Dreyfus Foundation for a
13
C/12C ratios in atmospheric methane. Atmos. Chem. Phys. 4, 2393–2399 (2004). postdoctoral fellowship in environmental chemistry. N.L.W. thanks the National
3. Knipping, E. M. & Dabdub, D. Impact of chlorine emissions from sea-salt aerosol Research Council for a postdoctoral fellowship.
on coastal urban ozone. Environ. Sci. Technol. 37, 275–284 (2003).
Author Contributions This was largely a collaborative effort. All authors
4. Von Glasow, R. & Crutzen, P. J. in The Atmosphere (ed. Keeling, R. F.) 21–64
contributed to the collection of observations during the 2009 intensive campaign
(Oxford Univ. Press, 2007).
described here. J.P.K., W.P.D., J.A.T. and S.S.B conceived of the need and initial
5. Osthoff, H. D. et al. High levels of nitryl chloride in the polluted subtropical marine
designs for the measurement campaign; J.A.T. performed much of the final
boundary layer. Nature Geosci. 1, 324–328 (2008).
analysis and modelling; T.P.R. performed the predictions of continental-scale
6. Allan, W., Struthers, H. & Lowe, D. C. Methane carbon isotope effects caused by ClNO2 production; and J.A.T. wrote the paper with significant input from all
atomic chlorine in the marine boundary layer: global model results compared with co-authors, especially S.S.B.
Southern Hemisphere measurements. J. Geophys. Res. D 112, D04306,
doi:10.1029/2006JD007369 (2007). Author Information Reprints and permissions information is available at
7. Finlayson-Pitts, B. J., Ezell, M. J. & Pitts, J. N. Formation of chemically active www.nature.com/reprints. The authors declare no competing financial interests.
chlorine compounds by reactions of atmospheric NaCl particles with gaseous Correspondence and requests for materials should be addressed to J.A.T.
N2O5 and ClONO2. Nature 337, 241–244 (1989). (thornton@atmos.washington.edu).

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Vol 464 | 11 March 2010 | doi:10.1038/nature08798

LETTERS
Antagonistic coevolution accelerates molecular
evolution
Steve Paterson1*, Tom Vogwill1*, Angus Buckling2, Rebecca Benmayor2, Andrew J. Spiers3, Nicholas R. Thomson4,
Mike Quail4, Frances Smith4, Danielle Walker4, Ben Libberton1, Andrew Fenton1, Neil Hall1
& Michael A. Brockhurst1*

The Red Queen hypothesis proposes that coevolution of interact- between this rapid phenotypic evolution and the underlying pattern
ing species (such as hosts and parasites) should drive molecular of molecular evolution has not been resolved. Crucially, it is possible
evolution through continual natural selection for adaptation and to separate bacteria and phage when transferring populations to fresh
counter-adaptation1–3. Although the divergence observed at some media14, which allows one partner to be held evolutionarily constant
host-resistance4–6 and parasite-infectivity7–9 genes is consistent while the other partner is allowed to evolve15–17. Initially isogenic,
with this, the long time periods typically required to study coevolu- replicate populations of P. fluorescens and W2 were propagated by
tion have so far prevented any direct empirical test. Here we show, serial transfer under two conditions: (1) evolution, in which the
using experimental populations of the bacterium Pseudomonas bacterial genotype was held constant and only the phage was allowed
fluorescens SBW25 and its viral parasite, phage W2 (refs 10, 11), to adapt, and (2) coevolution, in which both the bacterium and the
that the rate of molecular evolution in the phage was far higher phage were allowed to evolve adaptations and counter-adaptations.
when both bacterium and phage coevolved with each other than At the end of the selection experiment we obtained whole-genome
when phage evolved against a constant host genotype. Coevolution sequences of phage populations by high coverage second-generation
also resulted in far greater genetic divergence between replicate sequencing to determine the identity and frequency of mutations in
populations, which was correlated with the range of hosts that each population. Mutations were partitioned into synonymous and
coevolved phage were able to infect. Consistent with this, the most non-synonymous changes; very few synonymous mutations were
rapidly evolving phage genes under coevolution were those observed and only non-synonymous mutations were used in analyses
involved in host infection. These results demonstrate, at both the (see Supplementary Information; note that each indel (that is, inser-
genomic and phenotypic level, that antagonistic coevolution is a tions or deletions) was counted as one mutation regardless of its
cause of rapid and divergent evolution, and is likely to be a major length). From these data we calculated the number of sites that had
driver of evolutionary change within species. acquired mutations in each population relative to the ancestral ref-
According to the Red Queen hypothesis, biotic interactions are a erence W2 sequence (obtained as part of this study; see Supplementary
fundamental driver of molecular evolution2. The Red Queen hypo- Information), and from allele frequencies, the genetic distance of each
thesis posits that for a given species, its effective environment is likely population from the ancestral W2 sequence, the genetic divergence
to be comprised of the other species in the ecosystem, such that an among populations and the genetic diversity within each population.
adaptation increasing the fitness of one species necessarily causes a Coevolved phage populations showed twice the genetic distance
decline in fitness of those species with which it interacts1,3. Such from the ancestor as that of the evolved populations (average pairwise
coevolutionary interactions give rise to continual natural selection genetic distances: coevolved, 22.7 6 1.9 standard error (s.e.); evolved,
for adaptation and counter-adaptation by ecologically interacting 11.1 6 0.4 s.e.; t 5 6.64, d.f. 5 4.46, P , 0.01), shown also by the
species1,3, thereby driving molecular evolution2. Nowhere are such increased branch lengths for coevolved populations in the phylogen-
evolutionary dynamics thought to be so prevalent as in interactions etic tree in Fig. 1a. Similarly, coevolved populations had a greater
between hosts and virulent parasites, in which selection is strongly number of sites exhibiting mutations than evolved populations (co-
antagonistic yet closely coupled12. Comparative studies have found evolved, mean 52.8, range 46–60; evolved, mean 37.5, range 29–42;
particularly high rates of molecular evolution in genes associated likelihood-ratio test (LRT) 5 14.3, P , 0.001). Furthermore, far
with infection7–9 or resistance to infection4–6. However, there have greater genetic divergence was observed among replicate coevolved
been no direct empirical tests of whether antagonistic host–parasite populations than was observed among replicate evolved populations
coevolution accelerates molecular evolution in parasite genomes, (WST 5 0.45 for coevolved populations versus WST 5 0.06 for evolved
and whether such evolution is particularly rapid at genes determining populations (Supplementary Table 1), in which WST is a measure of
infectivity. the proportion of the total molecular variation attributable to differ-
Here we use experimental evolution of populations of the bac- ences among populations18). The tree in Fig. 1a also shows that rep-
terium Pseudomonas fluorescens SBW25 and its viral parasite, phage licate populations from the same treatment grouped together
W2. We have previously demonstrated that P. fluorescens and W2 genotypically. This topology is not due to co-ancestry as all popula-
undergo a persistent coevolutionary ‘arms race’ with reciprocal selec- tions were split at the start of the experiment. Instead, the topology
tion for the evolution of new resistance and infectivity phenotypes reflects parallel evolution: selection acting independently at the same
through time in bacteria and phage, respectively10,13, but the link sites among replicate populations. Thus, the tree reflects three key
1
School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK. 2Zoology Department, University of Oxford, South Parks Road,
Oxford OX1 3PS, UK. 3SIMBIOS Centre, Level 5 Kydd Building, University of Abertay Dundee, Bell Street, Dundee DD1 1HG, UK. 4Pathogen Genomics, The Wellcome Trust Sanger
Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
*These authors contributed equally to this work.
275
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

a b Phage infectivity traits. To address this prediction, we phenotypically char-

acterized the infectivity profile of each phage population. Specifically,
C4 we used cross-infection experiments to test whether phage from each
Scale population were able to infect hosts from all coevolved populations.
C1
We found that coevolved phage populations varied in terms of both

C4
C1
C3
C5

C6
C3 the range and identity of host genotypes that they were able to infect
E3 E2
C1 hosts (Fig. 1b), but that phage from evolved populations failed to infect any
E1 coevolved hosts (data not shown). Indeed, phenotypic divergence of
E6 the infectivity profile of coevolved phage populations closely
C6 C3 hosts
E5 matched the genetic divergence of the phage genomes as demon-
E4 strated by the similar topologies of trees constructed using genetic
C5 ref C4 hosts or phenotypic traits (Fig. 1b).
The increased rate of molecular evolution observed in the co-
C5 hosts
evolved populations was not distributed uniformly across the phage
genome (Fig. 2a). Four genes showed significantly increased molecular
evolution in coevolved versus evolved phage genomes. SBWP25_0036
C6 hosts (EMBL accession FN594518), which encodes a tail-fibre protein (gp49),
had a greater number of sites with mutations in the coevolved versus
Figure 1 | Genetic and phenotypic responses to selection. a, Phylogenetic evolved treatment, and, based on the allele frequencies of mutations at
tree for evolved (E1–6) and coevolved (C1, C3–6) phage populations and these sites, a substantially higher divergence from the ancestral genotype
ancestral reference genotype (ref) based on Euclidean distances calculated (number of mutational sites: coevolved, mean 17.6, range 15–20;
from the frequency and identity of mutations in each population. Scale bar evolved, mean 12.7, range 11–14; LRT 5 4.43, P , 0.05; pairwise
indicates a Euclidean distance of one. b, The phage-infectivity range based genetic distance: coevolved, 10.86 6 0.90 s.e.; evolved, 4.51 6 0.19 s.e.;
on the ability of each coevolved population to infect 20 bacterial clones from t 5 7.69, d.f. 5 4.38, P , 0.01). SBWP25_0027, which encodes a struc-
each host population. Infection by phage is shown in red, and resistance by tural protein (gp40), also had a greater number of sites with mutations
hosts is shown in grey. The dendrogram indicates phenotypic similarity
and a higher divergence from the ancestor in the coevolved than the
between phage populations.
evolved populations (number of mutational sites: coevolved, mean
evolutionary patterns. Specifically, the extent to which replicates: (1) 5.8, range 4–7; evolved mean 1.0, range 0–2; LRT 5 20.9, P , 0.001;
followed a similar trajectory away from the ancestral sequence, pairwise genetic distance: coevolved, 1.79 6 0.23 s.e.; evolved,
presumably as they adapted to laboratory conditions; (2) evolved 0.17 6 0.05 s.e.; t 5 7.69, d.f. 5 4.38, P , 0.01). SBWP25_0034 and
similarly among replicates within a treatment but differently in res- SBWP25_0035, which encode internal virion structural proteins
ponse to consistent differences between treatments; and (3) showed gp47 and gp48, respectively, also showed higher rates of molecular
independent evolution within each replicate, and at a far higher rate in evolution in coevolved populations, although to a lesser extent
the coevolved than the evolved treatment. than SBWP25_0027 and SBWP25_0036 (pairwise genetic distance:
Increased genetic divergence between parasite populations due to SBWP25_0034 coevolved, 1.21 6 0.13 s.e.; evolved, 0.34 6 0.06 s.e.;
coevolution is likely to be driven primarily by divergent selection on t 5 6.79, P , 0.01; SBWP25_0035 coevolved, 1.23 6 0.22 s.e.; evolved,
a b
Pairwise diversity

12 8
Pairwise distance per gene

per gene

10 6
SBWP25_0027

SBWP25_0032

SBWP25_0034

SBWP25_0035
SBWP25_0036

8 4
6 2
4
2
0
0 10 20 30 40
Position on phage genome (kb)

0 10 20 30 40
0.0 0.2 0.4 0.6 0.8 1.0
Position on phage genome (kb)
Frequency
c
SBWP25_0027 SBWP25_0036
C1
C3
C4
C5
C6
E1
E2
E3
E4
E5
E6
0 500 0 500 1,000 1,500
Length (bp)

Figure 2 | Patterns of molecular evolution in the W2 genome. a, b, Pairwise within each population are shown as bars underneath each coding sequence,
genetic distance between each phage population and the ancestral genotype with the colour of each bar indicating the frequency of each mutation within
(a), and genetic diversity within each phage population (b). Symbols denote each population (white, rare; red, common). c, Magnified view of identity
means 6 s.e.m. of replicate populations within the coevolved (magenta; and frequency of mutations in each population for SBWP25_0027 (gp40)
n 5 5) and the evolved (blue; n 5 6) treatments. The locations of mutations and SBWP25_0036 (gp49). bp, base pairs.
276
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

0.48 6 0.06 s.e.; t 5 3.61, P , 0.05). SBWP25_0027 and SBWP25_0036 and within populations. By contrast, populations adapting to a fixed
had a similar density of mutations (Fig. 2c) as each other, but, because host genotype showed a remarkable degree of parallel evolution, indi-
of its smaller size, SBWP25_0027 contributed less to the overall diver- cating genetic constraints on the evolutionary trajectories taken by
gence of the coevolved genomes from the ancestor than SBWP25_0036. replicate populations22,23. Coevolutionary interactions between species
Consistent with the observed evolution of the phage-infectivity range are likely therefore to be responsible for rapid evolutionary change
(Fig. 1b), all four of these proteins are predicted to be involved in host within species, potentially causing sufficient between-population
attachment19. In tailed bacteriophages, attachment is a two-step process genetic divergence to drive speciation itself24.
consisting of an initial reversible adsorption by tail fibres, followed by
irreversible adhesion by structural proteins20. The average size of dele- METHODS SUMMARY
tions in the tail fibre gene (SBWP25_0036) was positively correlated to Twelve replicate microcosms (30 ml glass universals containing 6 ml of King’s B
the number of bacterial genotypes the phage populations could infect (KB) broth) were inoculated with 107 isogenic cells of P. fluorescens SBW25 and
(infectivity range) (Supplementary Fig. 1), suggesting that tail fibres are 104 isogenic particles of phage W2. Cultures were propagated by 12 serial trans-
under strong directional selection for reduced protein length during fers in a static incubator at 28 uC. Transfers for the six coevolving populations
coevolution but that the precise genetic changes varied between popu- involved transferring 60 ml (1%) of culture to a fresh KB microcosm every 48 h.
lations. Shortened tail fibres also evolved in the evolution treatment, Transfers for the six evolving populations involved isolating phage populations
using 0.1 vol. chloroform and centrifuging at 14,000g for 2 min, and then inocu-
although to a lesser degree than under coevolution and without a
lating fresh microcosms with 60 ml (1%) of the phage population plus 107 ances-
concomitant increase in the infectivity range (Supplementary Fig. 1), tral SBW25 cells every 48 h. Every two transfers we estimated phage population
suggesting that to some extent shorter tail fibres may also be a general density by plating dilutions of each phage population onto KB agar plates with a
adaptation to laboratory conditions, perhaps through increasing semi-solid overlay bacterial lawn. At the end of the experiment phage DNA was
adsorption efficiency. SBWP25_0032, encoding a tail tubular protein isolated from each population25 and sequenced on a Roche 454 Titanium pyro-
(gp45), showed divergence from the ancestor in both evolved and sequencer. Reads were mapped to the W2 reference sequence and mutations were
coevolved treatments, but at different sites in each treatment. identified and their frequencies calculated using the Roche Newbler mapping
SBWP25_0027 (gp40) and SBWP25_0036 (gp49) accounted for most tool. We used all non-synonymous changes to construct a phylogenetic tree
of the divergence between replicate, coevolved phage populations using Euclidean genetic distance (the square root of pairwise differences), which
(Supplementary Fig. 2). is suggested as an appropriate metric for molecular variation data18. To deter-
mine phage population infectivity profiles, 20 independent bacterial colonies
Coevolved populations also showed higher genetic diversity within were isolated from each of the five coevolved populations by plating on KB agar,
populations than did evolved populations (Supplementary Table 1). and these were streaked against a perpendicular line of each phage population
This was predominantly due to variation in SBWP25_0027 (gp40), as that had been previously applied to a KB agar plate10. A bacterial colony was
SBWP25_0036 (gp49) displayed within-population diversity in both deemed susceptible if it showed any inhibition of growth after encountering the
treatments (Fig. 2b). This high genetic diversity at SBWP25_0036 is line of phage.
surprising given the apparent directional selection for reduced tail-fibre
Full Methods and any associated references are available in the online version of
protein length during coevolution. This suggests that SBWP25_0036
the paper at www.nature.com/nature.
polymorphisms may be transient and the result of recurrent con-
tinuing selective sweeps, and/or clonal interference. Alternatively, Received 8 September; accepted 23 December 2009.
both SBWP25_0036 and SBWP25_0027 may be subject to diversifying Published online 24 February 2010.
or fluctuating selection within populations. Together these genes 1. Van Valen, L. A new evolutionary law. Evol. Theory 1, 1–30 (1973).
(SBWP25_0036 and SBWP25_0027) are believed to control host 2. Van Valen, L. Molecular evolution as predicted by natural selection. J. Mol. Evol. 3,
adhesion19; thus, it is possible that diversity at these genes may deter- 89–101 (1974).
mine fine-scale host-specificity differences between phage genotypes. 3. Stenseth, N. C. & Smith, J. M. Coevolution in ecosystems: Red Queen evolution or
Such phenotypic differences between individual phage clones from stasis? Evolution 38, 870–880 (1984).
4. Clark, A. G. et al. Evolution of genes and genomes on the Drosophila phylogeny.
the same population have been observed in a previous study in this Nature 450, 203–218 (2007).
system16. 5. Hedrick, P. W. Evolutionary genetics of the major histocompatibility complex. Am.
Overall, our results are consistent with accelerated evolution in the Nat. 143, 945–964 (1994).
coevolution treatment that is driven by selective effects, rather than 6. Obbard, D. J., Jiggins, F. M., Halligan, D. L. & Little, T. J. Natural selection
purely demographic differences between treatments. Demographic drives extremely rapid evolution in antiviral RNAi genes. Curr. Biol. 16, 580–585
(2006).
effects, such as reduced generation time or population size, or 7. Blanc, G. et al. Molecular evolution of Rickettsia surface antigens: evidence of
reduced fidelity of DNA replication, would have led to a genome- positive selection. Mol. Biol. Evol. 22, 2073–2083 (2005).
wide increase in divergence and diversity, which was not observed. By 8. Mu, J. et al. Genome-wide variation and identification of vaccine targets in the
contrast, genetic divergence and diversity for most phage genes were Plasmodium falciparum genome. Nature Genet. 39, 126–130 (2006).
9. Barrett, L. G. et al. Diversity and evolution of effector loci in natural populations of
roughly similar in the two treatments (Fig. 2), indicating selection
the plant pathogen Melampsora lini. Mol. Biol. Evol. 26, 2499–2513 (2009).
under coevolution on specific infectivity genes/traits, such as that for 10. Buckling, A. & Rainey, P. B. Antagonistic coevolution between a bacterium and a
tail-fibre protein length or infectivity range (Supplementary Fig. 1). bacteriophage. Proc. R. Soc. Lond. B 269, 931–936 (2002).
Furthermore, whereas greater genetic divergence among coevolved 11. Brockhurst, M. A., Morgan, A. D., Fenton, A. & Buckling, A. Experimental
populations (Fig. 1a) could be explicable simply if coevolved popula- coevolution with bacteria and phage: the Pseudomonas fluorescens–W2 model
system. Infect. Genet. Evol. 7, 547–552 (2007).
tions are also smaller, and hence more susceptible to genetic drift, this
12. Woolhouse, M. E., Webster, J. P., Domingo, E., Charlesworth, B. & Levin, B. R.
explanation is incompatible with the higher genetic diversity Biological and biomedical implications of the co-evolution of pathogens and their
observed in SBWP25_0027 in coevolved populations (Fig. 2b). In hosts. Nature Genet. 32, 569–577 (2002).
line with this, there was no significant difference in phage population 13. Brockhurst, M. A., Morgan, A. D., Rainey, P. B. & Buckling, A. Population mixing
size between treatments over the course of the experiment (log10 accelerates coevolution. Ecol. Lett. 6, 975–979 (2003).
14. Morgan, A., Gandon, S. & Buckling, A. The effect of migration on local adaptation
(plaque-forming units (p.f.u.) ml21) averaged through time: coe- in a coevolving host–parasite system. Nature 437, 253–256 (2005).
volved, 7.39 6 0.14 s.e.; evolved, 7.51 6 0.08 s.e.; t 5 0.71, d.f. 5 9, 15. Morgan, A. & Buckling, A. Relative number of generations of hosts and parasites
P 5 0.5). does not influence parasite local adaptation in coevolving populations of bacteria
Our results highlight coevolution as a fundamental driver of mole- and phages. J. Evol. Biol. 19, 1956–1963 (2006).
cular evolution, and emphasize the utility of genome re-sequencing 16. Poullain, V., Gandon, S., Brockhurst, M. A., Buckling, A. & Hochberg, M. E. The
evolution of specificity in evolving and coevolving antagonistic interactions
for quantifying evolutionary dynamics in experimental evolution21. between a bacteria and its phage. Evolution 62, 1–11 (2008).
We directly demonstrate that antagonistic coevolution accelerates 17. Benmayor, R., Hodgson, D. J., Perron, G. G. & Buckling, A. Host mixing and disease
molecular evolution and can generate genetic divergence both between emergence. Curr. Biol. 19, 764–767 (2009).
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LETTERS NATURE | Vol 464 | 11 March 2010

18. Excoffier, L., Smouse, P. E. & Quattro, J. M. Analysis of molecular variance inferred Acknowledgements We are grateful to M. Begon and G. Hurst for comments on
from metric distances among DNA haplotypes: application to human previous drafts of this manuscript. We acknowledge funding from the Natural
mitocondrial DNA restriction data. Genetics 131, 479–491 (1992). Environment Research Council, the Wellcome Trust, the Leverhulme Trust and the
19. Ceyssens, P. J. et al. Genomic analysis of Pseudomonas aeruginosa phages LKD16 European Research Council. We are grateful for technical assistance from staff at
and LKA1: establishment of the WKMV subgroup within the T7 supergroup. the Centre for Genomic Research, University of Liverpool.
J. Bacteriol. 188, 6924–6931 (2006).
20. Calendar, R. L. The Bacteriophages (Oxford Univ. Press, 2005). Author Contributions M.A.B. and S.P. conceived and designed the study; T.V.
21. Barrick, J. E. et al. Genome evolution and adaptation in a long-term experiment performed selection experiments, infectivity assays and prepared samples for
with Escherichia coli. Nature 461, 1243–1247 (2009). population genomic sequencing; B.L. and M.A.B. performed further assays; R.B.
22. Bull, J. J. et al. Exceptional convergent evolution in a virus. Genetics 147, 1497–1507 prepared samples for ancestral genome sequencing; N.R.T., M.Q., F.S. and D.W.
(1997). performed ancestral genome sequencing and assembly; A.J.S. and N.R.T.
23. Wichman, H. A., Badgett, M. R., Scott, L. A., Boulianne, C. M. & Bull, J. J. Different performed genome annotation; S.P., T.V. and M.A.B. analysed the data; M.A.B.,
trajectories of parallel evolution during viral adaptation. Science 285, 422–424 S.P., A.B., A.J.S. and N.R.T. wrote the manuscript; T.V. and N.H. commented on the
(1999). manuscript; M.A.B., A.F. and S.P. supervised the research; S.P., N.H., A.B. and
24. Thompson, J. N. The Geographic Mosaic of Coevolution (Univ. of Chicago Press, M.A.B. obtained funding.
2005).
25. Santos, M. A. An improved method for the small scale preparation of Author Information The ancestral genome sequence of phage W2 has been
bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids submitted to the EMBL Nucleotide Sequence Database under accession number
Res. 19, 5442 (1991). FN594518. Reprints and permissions information is available at www.nature.com/
reprints. The authors declare no competing financial interests. Correspondence
Supplementary Information is linked to the online version of the paper at and requests for materials should be addressed to M.A.B.
www.nature.com/nature. (michael.brockhurst@liverpool.ac.uk).

278
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08798

METHODS Infectivity profiles. Twenty independent bacterial colonies were isolated from
Selection experiment. A single bacterial colony of P. fluorescens SBW25 was each of the five coevolved populations by plating on KB agar. Samples from each
isolated from laboratory stocks by plating on KB agar, and a single plaque of coevolved and evolved phage population were isolated using 0.1 vol. chloroform
W2 was isolated from an agar overlay of P. fluorescens infected with a sample of and centrifugation at 14,000g for 2 min to produce phage supernatants. An
W2 from laboratory stocks. Each was grown up overnight and used to found 12 infectivity profile for a phage population was determined by drying lines of
populations with 107 SBW25 cells and 104 phage particles. Cultures were pro- phage on KB agar plates, and perpendicularly streaking all isolated bacterial
pagated in 30-ml glass universals containing 6 ml KB media by serial transfer for colonies across it. A bacterial colony was deemed susceptible if it showed any
24 days in a static incubator at 28 uC. Transfers for the coevolved treatment inhibition of growth after encountering the line of phage10.
involved transferring 60 ml (1%) of culture to a fresh KB microcosm every Population genetic methods. The average number of pairwise genetic differences
48 h. The evolved treatment involved isolating phage populations using between each population and the ancestral genotype was calculated from the
0.1 vol. chloroform and centrifuging at 14,000g for 2 min, and then inoculating frequency of each mutation identified by the Newbler mapping tool. To minimise
fresh microcosms with 60 ml (1%) of the phage population plus 107 ancestral potential bias due to variation in sequence coverage between libraries, all non-
SBW25 cells every 48 h. Every two transfers, phage densities were estimated by synonyomous mutations identified were used, including potential sequencing
plating dilutions of each population onto KB agar plates with a semi-solid errors present at low frequency; although the results were essentially identical if
overlay bacterial lawn. analyses were limited to only high-confidence mutations or to non-synonymous
Molecular methods. The complete sequence of the ancestral W2 clone was and synonymous mutations combined. (Because the frequency of sequencing
obtained through a whole-genome shotgun library approach. W2 DNA was errors and synonymous mutations were low they contributed little to genetic
isolated from an infected SBW25 culture after lysis with 0.1 vol. chloroform, distance measures.) Indels, regardless of size, were counted as a single mutation.
centrifugation at 14,000g for 2 min, and extraction from the supernatant as The number of sites with mutations was calculated based on sites with a mutation
previously described25. The purified phage DNA was used to produce 2–4 kb frequency of .5%. This threshold was used to avoid including sequencing errors,
and 4–6 kb size-fractionated subclone libraries in pCR4-TOPO (Invitrogen). which were likely to have occurred, on average, at comparable magnitudes in both
Sequencing was performed using ABI BigDye Terminator cycle sequencing kits treatments (for further discussion of handling sequencing errors in heterogeneous
with sample analysis performed on ABI 3730xl instruments. The finished population samples see ref. 27). The significance of different genetic distances
sequence was generated from a total of 1,104 good quality reads (93% pass rate; between treatments was determined using Welch two sample t-tests, and for
867,704 bp; theoretical coverage of 320) including 363 finishing reads used to numbers of sites containing mutations using generalized linear models with a
close gaps and span low coverage regions. The genome was assembled using Poisson error distribution. A phylogenetic tree was constructed using Euclidean
Phrap and was tested by BLASTN against the P. fluorescens SBW25 genome genetic distance (the square root of pairwise differences), which is suggested as an
sequence to ensure that no chromosomal sequences had been incorporated into appropriate metric for molecular variation data18. We note that it is possible that a
the phage alignment. The sequence was annotated in Artemis26 (Wellcome Trust small number of the mutations that we observed could have arisen in the original
Sanger Institute) where putative coding sequences (CDS) were investigated by outgrowth from which all the populations were initiated; thus, some allelic vari-
BLASTP against the non-redundant (nr) NCBI protein database. Highest homo- ation could have been shared by all the populations at the beginning of the
logies were found to LKA1 bacteriophage, and CDS named accordingly. The selection experiment. However, any such effect would not be sufficient to explain
genome has been submitted to EMBL under accession number FN594518. our results, as replicate populations from the same treatment would only group
Phage DNA was extracted from selection experiment populations as earlier together within the tree (as observed in Fig. 1a) where selection increases the
(sufficient quality and quantity of DNA for subsequent pyrosequencing was frequency of the same allele independently in replicate populations in one treat-
obtained for five of six coevolved populations and for all evolved populations). ment but not in the other treatment. Analysis of molecular variation (AMOVA)
Shotgun single-stranded template DNA (sstDNA) libraries were prepared for was conducted to apportion molecular variation into components due to vari-
five coevolved populations (C1, C3–6) and six evolved populations (E1–6) by ation within populations, among populations and between treatments18. To
nebulising DNA and ligating multiplex identifier tags according to manufac- achieve this, AMOVA was conducted on populations in each treatment separately
turer’s instructions. Libraries were then sequenced on a Roche 454 Titanium and on all populations combined. All analyses were conducted in R v2.9 and
pyrosequencer. The average sequence coverage for each library ranged from Perl v5.8.
374–354. Reads were mapped to the W2 reference sequence and mutations were 26. Rutherford, K. et al. Artemis: sequence visualization and annotation. Bioinformatics
identified and their frequencies calculated using the Roche Newbler mapping 16, 944–945 (2000).
tool. Although some mutations were fixed in individual populations, no single 27. Barrick, J. E. & Lenski, R. E. Genome-wide mutational diversity in an evolving
mutation was fixed across all populations, indicating that the reference sequence population of Escherichia coli. Cold Spring Harb. Symp. Quant. Biol., doi:10.1101/
used was that of the ancestral genotype. sqb.2009.74.018 (23 September 2009).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08691

LETTERS
Compensatory evolution in mitochondrial tRNAs
navigates valleys of low fitness
Margarita V. Meer1, Alexey S. Kondrashov2, Yael Artzy-Randrup2,3 & Fyodor A. Kondrashov1

A long-standing controversy in evolutionary biology is whether or molecular level, because they maintain the high stability of the RNA
not evolving lineages can cross valleys on the fitness landscape that stem structures. Here we study whether or not such switches are also
correspond to low-fitness genotypes, which can eventually enable compensatory at the level of fitness.
them to reach isolated fitness peaks1–9. Here we study the fitness We obtained sequences, secondary structure predictions and
landscapes traversed by switches between different AU and GC alignments for 1,825 mitochondrial tRNAs consisting of 22 non-
Watson–Crick nucleotide pairs at complementary sites of mito- redundant sets from 83 mammalian species17, and subdivided them
chondrial transfer RNA stem regions in 83 mammalian species. into seven clades, each with an unambiguous phylogeny (see Methods).
We find that such Watson–Crick switches occur 30–40 times more For each clade we reconstructed the ancestral states for substitutions in
slowly than pairs of neutral substitutions, and that alleles corres- the stem secondary structures of mitochondrial tRNAs (mt-tRNAs)
ponding to GU and AC non-Watson–Crick intermediate states and calculated the number of Watson–Crick switches for each of the
segregate within human populations at low frequencies, similar to 22 mt-tRNAs (see Methods and Supplementary Table 1). We recorded
those of non-synonymous alleles. Substitutions leading to a Watson– a Watson–Crick switch when at a pair of interacting sites two taxa
Crick switch are strongly correlated, especially in mitochondrial harboured different Watson–Crick pairs of nucleotides and the switch
tRNAs encoded on the GT-nucleotide-rich strand of the mitochon- between them was caused by exactly two substitutions, as judged from
drial genome. Using these data we estimate that a typical Watson– the reconstructed ancestral states (Fig. 2). Out of a total of 2,867 sub-
Crick switch involves crossing a fitness valley of a depth of about stitutions in mt-tRNA stems of the seven clades, we observed 1,532
1023 or even about 1022, with AC intermediates being slightly more Watson–Crick switches of all the four types, with a vast majority of
deleterious than GU intermediates. This compensatory evolution them belonging to the AU–GC type. Thus, at least 55% of all substitu-
must proceed through rare intermediate variants that never reach tions observed in stem structures of mt-tRNAs are involved in Watson–
fixation2. The ubiquitous nature of compensatory evolution in mam- Crick switches. The prevalence of errors in our results caused by mul-
malian mitochondrial tRNAs and other molecules10,11 implies that tiple substitutions at each site must be low, as demonstrated by a small
simultaneous fixation of two alleles that are individually deleterious fraction (about 6% for the AU–GC type) of Watson–Crick switches
may be a common phenomenon at the molecular level. involving more than two substitutions (Table 1).
When fitness is affected by complex interactions between alleles at
different loci, an evolving lineage must navigate an epistatic fitness
landscape1,12,13. The most basic mode of epistasis is the compensatory Continuous Isolated
interaction of two diallelic loci (sites) at which each allele can confer a fitness ridges fitness peaks
a c
high fitness when combined with the suitable allele at the other
locus10. Nevertheless, even this basic mode of epistasis can lead to AC GC AC GC
fitness landscapes of two types (Fig. 1). The first is a continuous ridge
of high fitness that circumvents a maladapted genotype, commonly
AU GU AU GU
referred to as a Dobzhansky–Muller incompatibility10,14,15. Evolution
along continuous fitness ridges has the key role in speciation16 and in
compensation of pathogenic mutations in proteins and tRNAs10,11.
b d
The second is a pair of isolated fitness peaks separated by a fitness
AC GC AC GC
valley, first considered by Sewall Wright in the context of his shifting-
balance model1,2.
The importance of crossing fitness valleys in evolution remains AU GU AU GU
unknown1–9, and we study this issue with regard to the evolution of
complementary mitochondrial tRNA stem sites. The evolution of
High fitness Low fitness
RNA molecules can proceed through switches between different
Watson–Crick pairs of nucleotides at a pair of interacting sites.
Four kinds of Watson–Crick switch are possible. The most common
Figure 1 | Fitness landscapes resulting from an interaction between two
is the switch between an AU and GC Watson–Crick pair, or vice versa, complementary sites in an RNA stem structure. A switch between AU and
with the intermediate state of AC or GU (AU–GC type). The three GC Watson–Crick pairs can proceed on flat (a) or ascending (b) continuous
other types of switch are AU–UA, GC–CG and AU–CG, with the fitness ridges without a decrease in fitness, or through a fitness valley in
intermediate states being AA or UU, GG or CC, and AG or UC, which both possible intermediate variants have reduced fitness by the same
respectively. Watson–Crick switches involve compensation at the (c) or a different (d) amount.
1
Bioinformatics and Genomics Programme, Centre for Genomic Regulation, C/Dr Aiguader 88, Barcelona Biomedical Research Park Building, 08003 Barcelona, Spain. 2Life Sciences
Institute, Evolutionary Biology and Ecology Department, University of Michigan, Ann Arbor, Michigan 48109, USA. 3Howard Hughes Medical Institute and Department of Ecology and
Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 49109, USA.

279
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

a GC Eubalaena australis
b GC Artibeus jamaicensis
GC GC
GC GC Eubalaena japonica
GC Mystacina tuberculata
GC Balaena mysticetus GC
GC GC Caperea marginata AU Chalinolobus tuberculatus
AU
AU Balaenoptera bonaerensis AU Pipistrellus abramus
GC
GC GC Balaenoptera acutorostrata GC
GC Rhinolophus monoceros
GC Megaptera novaeangliae GC
GC GC
GC Balaenoptera physalus GC Rhinolophus pumilus
GC
GC
GC Eschrichtius robustus GC Rousettus aegyptiacus
GC
GC Balaenoptera brydei GC
GC GC Pteropus dasymallus
GC GC Balaenoptera borealis
GC
GC Balaenoptera musculus GC Pteropus scapulatus

c AU Acinonyx jubatus d AU Hemiechinus auritus

AU AU
AU AU Felis catus
AU Erinaceus europaeus
AU
AU Herpestes javanicus
AU Canis familiaris AU Echinosorex gymnura
AU
AU Ursus americanus GC Mogera wogura
AU AU AU Ursus arctos ?? GC
AU
GC GC Talpa europaea
AU Ursus maritimus
AU AU Arctocephalus forsteri GC Urotrichus talpoides
AU
AU Eumetopias jubatus GC
AU GC Sorex unguiculatus
AU Odobenus rosmarus GC
AU
GC Episoriculus fumidus
GC Halichoerus grypus GC
GC
GC Phoca vitulina GC Crocidura russula

Figure 2 | Watson–Crick switches in mammalian mt-tRNAs. Examples Watson–Crick switches were detected on terminal (a) and internal
from mt-tRNATyr in Mysticeti (a), mt-tRNAPhe in Chiroptera (b), mt- (b–d) branches; however, those switches that spanned the root of the tree
tRNAHis in Carnivora (c) and mt-tRNAPro in Eulipotyphla (d). (d) were not considered in our analysis.

To address the key question of whether Watson–Crick switches the two substitutions involved in a Watson–Crick switch and selec-
involve crossing fitness valleys, we studied substitutions and poly- tion favouring at least the second of them18,19 (Fig. 1b–d).
morphism of mt-tRNAs. We first investigated the evolutionary inter- For all AU«GC switches, we observed 568 cases in which the LCA
dependence of the two substitutions involved in an AU«GC was identical to the terminal state and 157 cases in which the LCA was
Watson–Crick switch. If the fitness landscape is a flat continuous identical to an intermediate state, resulting in a TIR of 3.6:1. In
ridge (Fig. 1a), both substitutions are neutral and they are expected addition to selection, the TIR can also be affected by unequal lengths
to occur independently of each other, without clustering on the of the lineages of the two species, changes in the mutation rate, and
phylogenetic tree18,19. The extent of clustering can be characterized incorrect identification of the LCA state18,19. To ascertain the impact
by the ratio of the numbers of terminal to intermediate states (the of these factors, we assembled pseudo-Watson–Crick pairs by choos-
terminal-to-intermediate ratio, TIR) in the last common ancestor ing all random pairs of sites from stems of different mt-tRNAs and
(LCA) of the species separated by a Watson–Crick switch. The applied a randomized sampling technique (see Methods). For these
LCA of the species separated by an AU–GC switch can either be randomly linked pairs of tRNA stem sites we obtained a TIR of 1:1.9
identical to one of them—that is, to the terminal state (AU or (1,248:2,316). A similar ratio of 1:2.2 (267,923:585,532) was obtained
GC)—or it can coincide with the state intermediate between these for random pairs of fourfold synonymous sites of different mito-
Watson–Crick pairs (AC or GU). When the LCA is identical to a chondrial protein-coding genes. Thus, a 3.6:1 TIR at interacting sites
terminal state, two substitutions must have occurred in the same exceeds the roughly 1:2 TIR observed in our randomization tests by
lineage, whereas when the LCA corresponds to an intermediate state, about sevenfold, and the difference is statistically significant
one substitution must have occurred on each of the two lineages (P , 0.001, Fisher’s t-test).
leading to the terminal states. If all substitutions were selectively Two kinds of fitness landscape can lead to such clustering of sub-
neutral, the TIR is expected to be 1:1 (LCA terminal:LCA intermedi- stitutions. First, an AU«GC switch can proceed, perhaps after a
ate) and would support the flat continuous ridge shown in Fig. 1a; a change in the fitness landscape caused by evolution elsewhere in
TIR with more frequent LCA terminal states indicates clustering of the genome, on an ascending continuous fitness ridge such that the

Table 1 | Number of Watson–Crick switches in mammalian mt-tRNAs

Type of compensatory switch Terminal ancestral state Intermediate ancestral state Ancestral state not reconstructed
and tRNA
Ancestral state AU Ancestral state GC Ancestral state GU Ancestral state AC Ancestral state in More than two
root of the tree substitutions
GU-rich tRNAs AU«GC 103 68 68 1 173 13
AC-rich tRNAs AU«GC 239 158 31 57 356 38
AU«CG Ancestral state AU Ancestral state CG Ancestral state AU Ancestral state CG
4 2 0 2 6 39
CG«GC Ancestral state CG or GC Ancestral state CC Ancestral state GG
0 0 0 2 3
AU«UA Ancestral state AU or UA Ancestral state UU Ancestral state AA
27 5 1 24 22

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©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

fitness of the intermediate state GU or AC is no less than the fitnesses be expected if compensatory evolution in GU-rich tRNAs proceeds
of the initial Watson–Crick state (Fig. 1b). Second, an AU«GC mostly through less deleterious GU intermediate states.
switch can proceed through a fitness valley (Fig. 1c, d), with the We corroborated the estimated coefficients of selection against GU
two compensatory substitutions fixed together in the same haplo- and AC intermediate states obtained from Kimura’s model2 with data
type2,3. These situations predict a different frequency of intermediate on the frequency of GU and AC alleles from 5,004 complete human
states in Watson–Crick switches. Because GU pairs are more stable mitochondrial genotypes. The AC and the GU alleles were substan-
than AC pairs in RNA stem structures20 and stability is a key factor tially rarer than synonymous alleles, with the AC alleles being slightly
connecting mt-tRNAs to fitness21–23, GU pairs should on average rarer and the GU alleles slightly more common than non-synonymous
confer a higher fitness than AC pairs. Thus, if AU«GC switches alleles (Fig. 4 and Supplementary Table 2). Because non-synonymous
traverse a continuous ascending ridge (Fig. 1b), evolution should substitutions in the mitochondria are under stringent selection25, both
occur preferentially through the GU intermediates and only rarely GU and AC intermediates must also be substantially deleterious, with
through the AC intermediates, whereas AU«GC switches between AC being slightly more so.
isolated peaks can occur through both GU or AC intermediates. Thus, the data suggest that Nes ? 1, where s is the coefficient of
Because we use a phylogenetic approach to reconstruct the ances- selection against an intermediate allele, so that we can ignore random
tral states, we cannot directly infer the intermediate state in a com- drift and reverse mutations from rare deleterious alleles and use the
pensatory switch (see Fig. 2 for examples). We therefore apply an approximation of p 5 m/s to estimate selection from polymorphism
indirect approach by using the strong strand-specific mutation bias data, where m is the rate of mutation and p is the average frequency of
present in mammalian mitochondria. In the GT-rich strand, the rates the segregating deleterious allele26. In mitochondria m has been esti-
of CRT and ARG transitions are increased about sixfold, and in the mated as 2 3 1025 (ref. 27) with cumulative strand-specific rates of
AC-rich strand there is a reciprocal increase in the rates of GRA and TRC and GRA mutations as 2.8 3 1026 and 1.5 3 1025 in AC-rich
TRC transitions24. Accordingly, GU-rich mt-tRNAs (eight mt- and GU-rich tRNAs, respectively24,28 (see Methods). We determined
tRNAs encoded on the AC-rich strand) have a roughly sixfold higher p as 1.7 3 1023 and 2.0 3 1024 for segregating GU and AC alleles,
CRU and ARG mutation rate than the URC and GRA rate, and respectively, in GU-rich tRNAs, and 3.9 3 1024 and 1.4 3 1023 for
this pattern is reversed in AC-rich mt-tRNAs (14 mt-tRNAs encoded GU and AC alleles in AC-rich tRNAs. Combining the estimates of m
on the GT-rich strand). Thus, the mutational bias in AC-rich tRNAs and p we obtain coefficients of selection against GU and AC inter-
favours an AC intermediate in AU«GC switches, whereas a GU mediates in stem structures of mt-tRNAs as about 8 3 1023 and
intermediate is favoured by mutation in GU-rich tRNAs (Fig. 3). about 1022, respectively (Supplementary Table 3), in good agree-
Thus, the continuous ascending ridge model (Fig. 1b) predicts that ment with the estimates based on the rate of evolution. Moreover,
AU«GC switches should be more common in GU-rich tRNAs and there is practically no overlap between the frequency distributions of
that the TIR should be similar in GU-rich and AC-rich tRNAs. In GU and AC alleles on the one hand, and of synonymous alleles on the
contrast, in the isolated peaks model, which can involve both GU and other, which rules out a substantial fraction of effectively neutral
AC intermediates, only a marginally higher rate of AU«GC switches intermediate states (Fig. 4). Data on eight other mammalian species,
in GU-rich tRNAs and a much higher TIR for AC-rich tRNAs2 are
expected. a 1.0
The prevalence of AU«GC switches was roughly the same in the
two strands, with 1.6 switches in each stem site in GU-rich mt-tRNAs AC alleles
and 1.4 in AC-rich mt-tRNAs (Table 1) rejecting the ascending con- GU alleles
tinuous fitness ridge model. In contrast, the TIR was substantially Non-synonymous alleles
higher in 14 AC-rich tRNAs than in 8 GU-rich tRNAs (397:88 (4.5:1) Fourfold synonymous alleles
Frequency

versus 171:69 (2.5:1), respectively), supporting the isolated peaks 0.5

model. This model was further supported by a 33:8 (4.1:1) TIR test
on non-AU«GC switches, in which the intermediate states are
expected to be less stable than the UG and AC pairs20.
As a further test of the applicability of the isolated peaks fitness
landscapes to mt-tRNAs, we applied Kimura’s model of compen-
satory evolution2 to data on the rate of Watson–Crick switches in 0
tRNAs and the rate of synonymous substitutions in mitochondrial
b 1.0
protein-coding genes. We found that the rates of AU«GC switches
in GU-rich and AC-rich tRNAs are 30-fold and 40-fold lower,
respectively, than the rate of synonymous evolution. Because Ne,
the effective population size, is about 10,000 in mammals, this
implies that the coefficient of selection against the intermediate states
Frequency

is about 5 3 1023 and about 8 3 1023 in GU-rich and AC-rich

0.5
tRNAs, respectively (Figure 1 in ref. 2), as long as the strength of
selection is uniform at all sites. This difference between strands is to

GU

0
0 1 2 3 4 5–10 >10
AU GC
Number of individuals carrying the minor allele

Figure 4 | Polymorphism frequency distribution of minor alleles in the

AC
human population. a, Derived alleles produced by high-rate mutation,
ARG and CRT on the heavy strand and GRA and TRC on the light strand.
Figure 3 | Mutation strand bias in mammalian mitochondria. Mutations b, Alleles produced by lower-rate mutations, ARG and CRT on the light
with an increased rate in GU-rich (red) and AC-rich (blue) tRNAs are shown. strand and GRA and TRC on the heavy strand.
281
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

in which the AC and GU alleles were also rarer than synonymous 7. Weinreich, D. M. & Chao, L. Rapid evolutionary escape by large populations from
local fitness peaks is likely in nature. Evolution 59, 1175–1182 (2005).
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8. Chen, Y. & Stephan, W. Compensatory evolution of a precursor messenger RNA
(Supplementary Table 4). secondary structure in the Drosophila melanogaster Adh gene. Proc. Natl Acad. Sci.
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evolution on a continuous fitness ridge if we assume that at a majority vertebrate introns using Kimura’s model of compensatory fitness interactions.
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switches occur mostly at a small fraction of sites at which such inter- incompatibilities in protein evolution. Proc. Natl Acad. Sci. USA 99, 14878–14883
mediates are effectively neutral. However, the similarity of estimates (2002).
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from the application of the Kimura model2 to evolutionary data on evolution in mammalian mitochondrial tRNAs. Nature Genet. 36, 1207–1212
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the one hand, and from frequencies of segregating alleles on the 12. Maynard Smith, J. Natural selection and the concept of protein space. Nature 225,
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or not such stepping stones can be used to cross wider fitness valleys
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METHODS SUMMARY 24. Tanaka, M. & Ozawa, T. Strand asymmetry in human mitochondrial DNA
Substitutions in stem structures of mt-tRNAs obtained from a mt-tRNA data- mutations. Genomics 22, 327–335 (1994).
base30 were reconstructed with the use of parsimony, maximum-likelihood and 25. Stewart, J. B. et al. Strong purifying selection in transmission of mammalian
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Bayesian methods. A Watson–Crick switch was recorded when two different taxa
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displaying one of the two Watson–Crick pairs were separated by exactly two
591–597 (2001).
substitutions. The average frequency of segregating minor alleles in Watson– 27. Howell, N. et al. The pedigree rate of sequence divergence in the human
Crick complementary stem sites was measured in 5,004 complete human mito- mitochondrial genome: there is a difference between phylogenetic and pedigree
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total mutation rate of 2 3 1025 from ref. 27, an estimate of about 90% of all 28. Malyarchuk, B. A., Rogozin, I. B., Berikov, V. B. & Derenko, M. V. Analysis of
mutations as transitions from ref. 28, and a combined bias of a sixfold increase in phylogenetically reconstructed mutational spectra in human mitochondrial DNA
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the AC-rich strand24. 29. McDonald, J. H. & Kreitman, M. Adaptive protein evolution at the Adh locus in
Drosophila. Nature 351, 652–654 (1991).
Full Methods and any associated references are available in the online version of 30. Popadin, K. Y., Mamirova, L. A. & Kondrashov, F. A. A manually curated database
the paper at www.nature.com/nature. of tetrapod mitochondrially encoded tRNA sequences and secondary structures.
BMC Bioinformatics 8, 441 (2007).
Received 3 June; accepted 16 November 2009.
Published online 24 February 2010. Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
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5. Wade, M. J. & Goodnight, C. J. Wright’s shifting balance theory: an experimental Author Information Reprints and permissions information is available at
study. Science 253, 1015–1018 (1991). www.nature.com/reprints. The authors declare no competing financial interests.
6. Coyne, J. A., Barton, N. H. & Turelli, M. Is Wright’s shifting balance process Correspondence and requests for materials should be addressed to F.A.K.
important in evolution? Evolution 54, 306–317 (2000). (fyodor.kondrashov@crg.es).

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doi:10.1038/nature08691

METHODS eight other mammalian species for which more than ten complete genomes were
Measuring evolution and polymorphism. For all stem structures of 22 mt-tRNAs available in GenBank.
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GC«CG and AU«CG, were recorded when two different taxa displayed one of 33. Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Wheeler, D. L.
the two Watson–Crick pairs and were separated by exactly two substitutions. GenBank. Nucleic Acids Res. 34, D16–D20 (2006).
For randomization of Watson–Crick pairs in stem sites, we assembled pseudo- 34. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high
Watson–Crick pairs by selecting all possible pairs of individual sites from stems throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
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sites from stems in mt-tRNAs such that each site in a pair was picked from a
36. Flynn, J. J., Finarelli, J. A., Zehr, S., Hsu, J. & Nedbal, M. A. Molecular phylogeny of
different tRNA. We then obtained the TIR measurement in such pseudo-
the carnivora (mammalia): assessing the impact of increased sampling on
Watson–Crick pairs on each of the seven phylogenetic trees in the same way resolving enigmatic relationships. Syst. Biol. 54, 317–337 (2005).
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substitutions occurring in two random sites on a given tree. The same procedure effect of missing data on supertree and supermatrix analyses of multiple gene
was followed for obtaining pseudo-Watson–Crick pairs from synonymous sites data sets. Mol. Phylogenet. Evol. 41, 165–181 (2006).
except that each site in a pseudo-Watson–Crick pair was chosen from fourfold 38. Springer, M. S., Teeling, E. C., Madsen, O., Stanhope, M. J. & de Jong, W. W.
synonymous sites. Integrated fossil and molecular data reconstruct bat echolocation. Proc. Natl Acad.
To measure the rate of polymorphism in tRNAs we excluded mt-tRNAThr Sci. USA 98, 6241–6246 (2001).
because of its uncharacteristically large rate of polymorphism32. We measured 39. Teeling, E. C. et al. A molecular phylogeny for bats illuminates biogeography and
the fossil record. Science 307, 580–584 (2005).
the average frequency of segregating minor alleles in stem sites that had an AU or
40. Price, S. A., Bininda-Emonds, O. R. & Gittleman, J. L. A complete phylogeny of the
GC Watson–Crick pair in humans and that underwent at least one Watson–
whales, dolphins and even-toed hoofed mammals (Cetartiodactyla). Biol. Rev.
Crick switch in mammalian evolution. To estimate the rate of mutation rate we Camb. Philos. Soc. 80, 445–473 (2005).
used the total mutation rate of 2 3 1025 from ref. 27, an estimate of about 90% of 41. Agnarsson, I. & May-Collado, L. J. The phylogeny of Cetartiodactyla: the
all mutations as transitions from ref. 28 and a combined bias of sixfold increase importance of dense taxon sampling, missing data, and the remarkable promise of
in ARG and CRT in the GT-rich strand and the same bias of reverse mutations cytochrome b to provide reliable species-level phylogenies. Mol. Phylogenet. Evol.
in the AC-rich strand24. We did not use the model of ref. 3 because it assumes a 48, 964–985 (2008).
low rate of mutation, Nem = 1, which is not true of mammalian mitochondria27. 42. Murphy, W. J. et al. Molecular phylogenetics and the origins of placental
Obtaining sequences and phylogenetic reconstruction. We obtained secondary- mammals. Nature 409, 614–618 (2001).
structure-based mt-tRNA alignments from ref. 30. The protein-coding genes were 43. Schmitz, J., Roos, C. & Zischler, H. Primate phylogeny: molecular evidence from
obtained from GenBank33. The phylogeny for each taxon was constructed from retroposons. Cytogenet. Genome Res. 108, 26–37 (2005).
44. Matsui, A., Rakotondraparany, F., Munechika, I., Hasegawa, M. & Horai, S.
muscle34 protein-based codon alignments of 13 mitochondrial protein coding
Molecular phylogeny and evolution of prosimians based on complete sequences
genes by using MrBayes under the HKY model and a gamma-distribution of of mitochondrial DNAs. Gene 441, 53–66 (2009).
substitution rates across sites for 106 iterations35. Our final data set included 12 45. Horner, D. S. et al. Phylogenetic analyses of complete mitochondrial genome
species in the Carnivora, 9 in Chiroptera, 12 in Cetartiodactyla, 17 in Primata, 9 in sequences suggest a basal divergence of the enigmatic rodent Anomalurus. BMC
Rodentia and 12 in Eulipotyphla. Twelve Mysticeti species were placed into an Evol. Biol. 7, 16 (2007).
independent phylogeny because the exact location of whales in the Cetartiodactyla 46. Quérouil, S. et al. Phylogeny and evolution of African shrews (Mammalia:
and the monophyletic status of Odontoceti remain controversial. We used the Soricidae) inferred from 16s rRNA sequences. Mol. Phylogenet. Evol. 20, 185–195
following references to check the consistency and determine the root of each (2001).
phylogenetic tree: Carnivora36,37, Chiroptera38,39, Cetartiodactyla40,41, Primata42–44, 47. Douady, C. J. et al. Molecular phylogenetic evidence confirming the Eulipotyphla
Rodentia45 and Eulipotyphla46,47 and Mysticeti41,48,49. The phylogenetic trees not concept and in support of hedgehogs as the sister group to shrews. Mol.
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displayed in the main text are shown below.
48. Hatch, L. T., Dopman, E. B. & Harrison, R. G. Phylogenetic relationships among the
For polymorphism estimates the sequences of complete human mitochon- baleen whales based on maternally and paternally inherited characters. Mol.
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and complete genome’’ as a query with the Limits option set to mitochondrial 49. Xiong, Y., Brandley, M. C., Xu, S., Zhou, K. & Yang, G. Seven new dolphin
DNA in the Entrez retrieval system33. Genomes with undetermined bases (Ns) in mitochondrial genomes and a time-calibrated phylogeny of whales. BMC Evol.
mt-tRNA sequences were discarded, leaving 5,004 genomes. We also studied Biol. 9, 20 (2009).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08818

LETTERS
Sister chromosome pairing maintains heterozygosity
in parthenogenetic lizards
Aracely A. Lutes1,2, William B. Neaves1,3, Diana P. Baumann1, Winfried Wiegraebe1 & Peter Baumann1,2,4

Although bisexual reproduction has proven to be highly successful, mechanism to produce mature oocytes that carry the complete com-
parthenogenetic all-female populations occur frequently in certain plement of somatic chromosomes and maintain heterozygosity12. To
taxa, including the whiptail lizards of the genus Aspidoscelis. test this hypothesis, we set out to quantify the DNA content in
Allozyme analysis revealed a high degree of fixed heterozygosity oocytes of the diploid parthenogenetic species A. tesselata and the
in these parthenogenetic species1,2, supporting the view that they sexually reproducing control Aspidoscelis gularis. Extant A. gularis
originated from hybridization events between related sexual species. and Aspidoscelis tigris are closely related to the individuals that hybri-
It has remained unclear how the meiotic program is altered to pro- dized to generate the founding specimen of A. tesselata1,2,13,14.
duce diploid eggs while maintaining heterozygosity. Here we show We isolated germinal vesicles (GVs), the oocyte nuclei, and visualized
that meiosis commences with twice the number of chromosomes in 49,6-diamidino-2-phenylindole (DAPI)-stained chromosomes by two-
parthenogenetic versus sexual species, a mechanism that provides photon microscopy. Bivalents were readily observed in GVs from A.
the basis for generating gametes with unreduced chromosome gularis and A. tesselata, and their morphology was consistent with the
content without fundamental deviation from the classic meiotic diplotene stage of prophase I (Fig. 1a, b). Visual inspection of three-
program. Our observation of synaptonemal complexes and chias- dimensional reconstructions of seven gularis and five tesselata GVs
mata demonstrate that a typical meiotic program occurs and that revealed a larger number of bivalents in tesselata GVs compared to
heterozygosity is not maintained by bypassing recombination. gularis. Ambiguities in identifying the boundaries of individual chro-
Instead, fluorescent in situ hybridization probes that distinguish mosomes prevented accurate counting of bivalents at this stage. Instead,
between homologues reveal that bivalents form between sister chro- we quantified the volume occupied by chromosomes in each GV as an
mosomes, the genetically identical products of the first of two pre- indirect measure of DNA content (Fig. 1c; Supplementary Table 1).
meiotic replication cycles. Sister chromosome pairing provides a
mechanism for the maintenance of heterozygosity, which is critical a 1 2 3 4
for offsetting the reduced fitness associated with the lack of genetic
diversity in parthenogenetic species.
True parthenogenesis, characterized by the complete absence of
male contributions, has been described for various species of reptiles,
including whiptail lizards, geckos, blind snakes and rock lizards3.
Whiptail lizards of the genus Aspidoscelis, formerly part of the genus b 1 2 3 4
Cnemidophorus4, are mostly native to the Southwestern United States
and Mexico, and about one-third of the more than 50 species re-
produce by obligate parthenogenesis.
Morphological, karyotypic and biochemical studies provided
strong evidence for hybrid origins of all parthenogenetic Aspidoscelis
species examined1,5,6. While hybridization between individuals from c d 160
Chromosome volume

(arbitrary units)

distinct species can explain the initially high degree of heterozygosity 3,000
DNA content

120

across the genome, allozyme analysis demonstrated surprising per- 2,000 80

(µm3)

sistence of heterozygosity over many generations in several partheno- 40

1,000
genetic lineages of Aspidoscelis, including Aspidoscelis tesselata and 0
ris

ris

Aspidoscelis neomexicana6. The acceptance of skin grafts between indi-

ui
at

an

0
tig

la

ng
el

ic

1 2 3 4 5 6 7 1 2 3 4 5
gu

ss

ex

sa
A.

te

viduals of a parthenogenetic species7–10 and biochemical studies on A. tesselata

A.

A. gularis
om

ex
A.

A.
ne

several laboratory-reared lineages6 further support genetic uniformity.

A.

The mechanism that underlies clonal reproduction and fixed Figure 1 | Oocytes from parthenogenetic A. tesselata contain twice the
heterozygosity has been the topic of much speculation. Most varia- amount of chromosomal DNA compared to sexual A. gularis. a, DAPI-
tions of the meiotic program that would produce diploid oocytes by stained chromosomes in germinal vesicles (GVs) from A. gularis. Three-
skipping a division or by fusion of a haploid oocyte with a polar body dimensional projections of four GVs are shown; details on size and
quantifications are available as Supplementary Table 1. Scale bars, 10 mm.
cannot account for fixed heterozygosity unless recombination b, GVs from A. tesselata. c, Quantification of chromosome volumes.
between homologues is suppressed. On the basis of the exclusion d, Quantification of DNA content in somatic cells by flow cytometry.
of alternative models and the observation of large numbers of chro- Fluorescence intensities from biological triplicates of ,50,000 cells were
mosomes in two oocytes from Aspidoscelis uniparens11, premeiotic averaged and normalized against samples from A. gularis, which was set at
endoreplication of chromosomes was proposed as the most likely 100 to facilitate comparison.
1
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA. 2Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City,
Kansas 66160, USA. 3University of Missouri Kansas City, School of Medicine, Kansas City, Missouri 64110, USA. 4Howard Hughes Medical Institute, Kansas City, Missouri 64110, USA.

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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

Unlike measurements of fluorescence intensity, this approach is robust well-defined lateral and central elements, were observed in all species
against changes in staining efficiency or laser intensity fluctuations. examined, providing further support that a typical meiotic program
A. tesselata chromosomes occupied 2.24 6 0.18 times the volume of is underway (Fig. 2c–f, Supplementary Fig. 2). Based on the presence
the averaged A. gularis samples. While indicative of a twofold increase of synaptonemal complexes in pachytene and chiasmata in diplotene,
in the DNA content of the prophase oocyte in the parthenogenetic we surmise that meiotic chromosome pairing and recombination are
species, differences in genome size could also account for the increased not bypassed in parthenogenetic Aspidoscelis species.
chromosome volume. The premeiotic doubling of chromosomes allows for bivalent
Although somatic cells from A. gularis and A. tesselata both harbour formation to occur either between homologues as in normal meiosis
46 chromosomes15, a direct comparison of genome sizes was needed or between sister chromosomes (Fig. 3). To distinguish between these
to inform our analysis. Taking advantage of the fact that reptilian possibilities, we sought to identify probes that selectively recognize
erythrocytes are nucleated, we subjected blood samples to flow cyto- one particular chromosome in a pair of homologues. We discovered
metry analysis and found that the nuclear DNA content in somatic that 26 of the 46 A. tigris chromosomes, including all 22 macrochro-
cells differed by less than 1% between the two species (Fig. 1d). For mosomes and 4 microchromosomes, harbour large tracks of internal
comparison, samples from sexual diploid A. tigris and parthenogenetic telomeric repeats in addition to the signal at chromosome ends
triploid Aspidoscelis exsanguis (A. exsanguis is the product of two con- (Fig. 4a). In contrast, staining metaphases of A. inornata chromo-
secutive hybridization events involving the sexual species Aspidoscelis somes with a telomeric protein-nucleic acid probe only revealed
inornata, Aspidoscelis burti and A. gularis13) were also analysed, with the signal at the chromosome termini (Fig. 4b). Consistent with its
latter showing an approximately 50% increase in DNA content, as hybrid origin from these two sexual species1,2,5, A. neomexicana chro-
expected (Fig. 1d). Independent confirmation for a doubling in chro- mosomes contained large internal repeats on 13 chromosomes,
mosome number was obtained by examining GVs in late prophase. At allowing us to unambiguously identify 13 chromosomes inherited
this stage, chromosomes are highly condensed and, consistent with a from A. tigris in the original F1 hybrid (Fig. 4c). In the context of a
doubling in chromosome number, we were able to distinguish 46 bivalent, hybridization signals on both sides indicate sister chro-
bivalents in A. tesselata GVs (Supplementary Fig. 1).
mosome pairing, whereas hybridization on only one side supports
Entering meiosis with an 8n chromosome complement would
homologue pairing.
allow parthenogenetic animals to utilize the two normal meiotic
To preserve the three-dimensional arrangements of chromosomes
divisions to generate diploid gametes. However, the long-term main-
in GVs and to provide better spatial resolution than commonly obtained
tenance of heterozygosity across the genome is only ensured if cross-
in chromosome spreads, we adapted the fluorescent in situ hybridiza-
overs between homologues are suppressed. Two-photon imaging of
diplotene chromosomes from A. tesselata and another parthenogenetic tion (FISH) procedure to perform hybridization on intact GVs. At each
species, A. neomexicana, revealed no differences compared to sexual site where chromosome internal hybridization was detected, a signal
controls besides the increased DNA content. Notably, bivalents was observed on both sides of the bivalent (Fig. 4d, e). It is important
appeared to be connected by chiasmata in all samples, indicating that to note that sister chromatids resulting from the most recent round of
crossing-over is not abandoned (Fig. 2a, b). replication appear as one cytologically, as they are closely associated with
To further examine chromosome pairing, thin sections of ovaries each other along their length during this stage of meiosis. The exclusive
from A. tesselata, A. tigris and A. neomexicana were examined by presence of paired hybridization signals therefore strongly suggests that
electron microscopy. Synaptonemal complexes, characterized by bivalents are composed of sister chromosomes, not homologues. On the
basis of this experiment, we concluded that for the 13 chromosomes for
which the telomeric hybridization probe distinguishes sisters and
a c
homologues in A. neomexicana, sister chromosome pairing is the rule.

Replication Pairing Exchange First division Second division

Canonical meiosis

b
e
Premeiotic endoreplication

d
Homologue pairing
or
d e f Sister chromosome pairing

Figure 3 | Meiosis in sexual and parthenogenetic Aspidoscelis species.

Top, in normal (canonical) meiosis, a single round of DNA replication is
Figure 2 | Visualization of chiasmata and synaptonemal complexes in followed by two consecutive divisions that result in a haploid gamete and
parthenogenetic A. tesselata and sexual A. tigris. a, b, Projection of three polar bodies. Homologues are shown in red and blue. Recombination
bivalents from A. tigris (a) and A. tesselata (b) GVs in diplotene. c, Electron generates chimaeric chromosomes. Bottom, premeiotic doubling of
microscopy image of A. tesselata GV in pachytene. Several sections of chromosomes allows for pairing of homologous or sister chromosomes.
synaptonemal complexes are visible. Close-up views of the two boxed areas Homologue pairing and recombination result in some loss of heterozygosity
are shown in d and e. f, Close-up view of a synaptonemal complex from A. in the mature oocyte. Recombination between pairs of sister chromosomes
tigris. Scale bars: 10 mm (a, b); 2 mm (c); 200 nm (d–f). maintains heterozygosity at all loci.
284
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a b c molecular markers in these studies precludes definitive conclusions,

the striking parallels with whiptail lizards strongly indicate that a
common mechanism enables parthenogenetic reproduction in diverse
groups of animals. It seems likely that a relatively simple deviation
from the established program of oogenesis is sufficient to permit par-
thenogenesis. However, loss of heterozygosity, paternal inheritance of
centrosomes, and a requirement for fertilization in triggering comple-
tion of female meiosis are seemingly unconnected obstacles to par-
d e thenogenetic reproduction. A better understanding of the changes that
permit a small but diverse group of animals to reproduce without
males is clearly needed, and may well shed light on the overwhelming
success of sexuality.

METHODS SUMMARY
Laboratory colonies of Aspidoscelis species were from animals collected in Texas
and New Mexico under a permit from the New Mexico Department of Game and
Fish (permit numbers 3199 and 3395).
GV isolation and quantification of chromosome volume. Ovaries from adult
and sub-adult lizards were isolated, stained with 4’,6-diamidino-2-phenylindole
(DAPI) and imaged using a Zeiss LSM 510 system in two-photon excitation
Figure 4 | Internal telomeric repeats distinguish homologues in A. mode. A non-parametric and unsupervised, automatic threshold selection
neomexicana and demonstrate sister chromosome pairing. a, A method developed by Otsu (see Methods) was used to obtain an unbiased
CCCTAA(3) peptide nucleic acid probe (red) identifies chromosome termini measurement of chromosome volumes.
and large internal telomere repeat regions on metaphase spreads of A. tigris Fluorescent in situ hybridization (FISH). Colcemid-treated embryonic fibro-
chromosomes prepared from fibroblast cultures. DAPI-stained blasts were harvested, fixed and used to prepare metaphase spreads. FISH was
chromosomes are shown in blue. b, Chromosome terminal signals, but no performed on dried coverslips using AlexaFluor-labelled peptide-nucleic acid
internal telomeric repeats, are detected in A. inornata. c, Chromosomes (PNA) and locked-nucleic acid (LNA) probes. Samples were imaged on a fluor-
inherited from A. tigris but not from A. inornata are identified by internal escence microscope and images were analysed with AxioVision software. FISH on
telomeric repeats in A. neomexicana. Boxed areas in a–c are magnified in the meiotic chromosomes was performed after embedding GVs in an acrylamide gel.
bottom left corners of each panel. d, Projection of a subset of images from an Transmission electron microscopy. Ultrathin sections of epoxy-embedded
A. neomexicana GV visualized by confocal microscopy. DAPI-stained ovaries were collected on copper grids, stained with 2% uranyl acetate in 50%
chromosomes are shown in white and the telomeric probe in red. e, Close-up methanol for 10 min, followed by 1% lead citrate for 7 min. Sections were photo-
of four representative areas showing paired fluorescence signals. The graphed using a FEI transmission electron microscope at 80 kV.
differences in resolution stem from differences in projection angles.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Screening of several tri-, tetra- and hexanucleotide repeat probes
identified (CCAAGG)n as an additional marker for at least nine Received 16 November 2009; accepted 6 January 2010.
chromosomes in A. neomexicana that are of A. tigris origin Published online 21 February 2010.
(Supplementary Fig. 3a–c). When hybridized to diplotene chromo- 1. Neaves, W. B. Adenosine deaminase phenotypes among sexual and
somes in acrylamide-embedded GVs, only paired signals were parthenogenetic lizards in the genus Cnemidophorus (Teiidae). J. Exp. Zool. 171,
observed (Supplementary Fig. 3d, e). In summary, two independent 175–183 (1969).
probes enabled us to distinguish sister chromosomes from homolo- 2. Neaves, W. B. & Gerald, P. S. Lactate dehydrogenase isozymes in parthenogenetic
teiid lizards (Cnemidophorus). Science 160, 1004–1005 (1968).
gues, and for over 20 bivalents examined, pairing occurred exclu- 3. Vrijenhoek, R. C., Dawley, R. M., Cole, C. J. & Bogart, J. P. in Evolution and Ecology of
sively between sister chromosomes. Unisexual Vertebrates Vol. 466 (eds Dawley, R. M. & Bogart, J. P.) 19–23 (New
Entering meiosis with twice the usual number of chromosomes York State Museum Bulletin, 1989).
allows parthenogenetic species to produce oocytes carrying the 4. Reeder, T. W., Cole, C. J. & Dessauer, H. C. Phylogenetic relationships of whiptail
complete somatic chromosome complement while preserving the lizards of the genus Cnemidophorus (Squamata: Teiidae): a test of monophyly,
reevaluation of karyotypic evolution, and review of hybrid origins. Am. Mus. Novit.
established meiotic program. There are two principal pathways by 3365, 1–61 (2002).
which the premeiotic oocytes of a diploid species may acquire eight 5. Lowe, C. H. & Wright, J. W. Evolution of parthenogenetic species of
rather than four sets of chromosomes. One is the process in which Cnemidophorus (whiptail lizards) in western North America. J. Ariz. Acad. Sci. 4,
chromosome duplication occurs without cytokinesis; this has been 81–87 (1966).
6. Dessauer, H. C. & Cole, C. J. Clonal inheritance in parthenogenetic whiptail lizards:
termed endomitosis or endoreplication16. Alternatively, 8n germ cells biochemical evidence. J. Hered. 77, 8–12 (1986).
may arise by fusion of two cells either before or after the final pre- 7. Cordes, J. E. & Walker, J. M. Skin histocompatibility between syntopic pattern
meiotic doubling of chromosomes. There is ample precedent for classes C and D of parthenogenetic Cnemidophorus tesselatus in New Mexico. J.
either mode of genome amplification in plants and animals, but Herpetol. 37, 185–188 (2003).
the regulatory mechanisms are largely unclear. 8. Cordes, J. E. & Walker, J. M. Evolutionary and systematic implications of skin
histocompatibility among parthenogenetic teiid lizards: three color pattern
In sexual species, homologous chromosomes form bivalents, and classes of Aspidoscelis dixoni and one of Aspidoscelis tesselata. Copeia14–26
meiotic recombination promotes genetic diversity while ensuring (2006).
orderly segregation of chromosomes during the first meiotic division. 9. Maslin, T. P. Skin grafting in the bisexual teiid lizard Cnemidophorus sexlineatus and
The same mechanism would result in loss of heterozygosity in par- in the unisexual C. tesselatus. J. Exp. Zool. 166, 137–149 (1967).
10. Cuellar, O. & Smart, C. Analysis of histoincompatibility in a natural population of
thenogenetic species, whereas formation of bivalents from genetically the bisexual whiptail lizard Cnemidophorus tigris. Transplantation 24, 127–133
identical sister chromosomes preserves heterozygosity. Interestingly, (1977).
this same variation of the meiotic program appears to enable par- 11. Cuellar, O. Reproduction and the mechanism of meiotic restitution in the
thenogenetic reproduction in widely diverged species. Premeiotic parthenogenetic lizard Cnemidophorus uniparens. J. Morphol. 133, 139–165 (1971).
doubling of chromosomes has been documented in triploid ambysto- 12. Neaves, W. B. Tetraploidy in a hybrid lizard of the genus Cnemidophorus (Teiidae).
Brev. Mus. Comp. Zool. 381, 1–25 (1971).
matid salamanders17 as well as a parthenogenetic grasshopper 13. Dessauer, H. C. & Cole, C. J. in Evolution and Ecology of Unisexual Vertebrates Vol.
(Warramaba virgo)18. In both cases, sister chromosome pairing was 466 (eds Dawley, R. M. & Bogard, J. P.) 49–71 (New York State Museum Bulletin,
suggested on the basis of bivalent morphology. Although the lack of 1989).
285
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

14. Parker, E. D. Jr & Selander, R. K. The organization of genetic diversity in the Game and Fish for scientific collection permits; F. Li for electron microscopy; the
parthenogenetic lizard Cnemidophorus tesselatus. Genetics 84, 791–805 (1976). Stowers Institute Microscopy, Cytometry and Molecular Biology Facilities for
15. Wright, J. W. & Lowe, C. H. Evolution of the alloploid parthenospecies support; and S. Hawley, J. Cole, C. Townsend and members of the Baumann and
Cnemidophorus tesselatus (Say). Mamm. Chromosome Newsl. 8, 95–96 (1967). Blanchette laboratories for discussions and comments. We also thank S. Hawley
16. Edgar, B. A. & Orr-Weaver, T. L. Endoreplication cell cycles: more for less. Cell 105, for critical reading of the manuscript. This work was funded by the Stowers
297–306 (2001). Institute for Medical Research, and the Pew Scholars Program in the Biological
17. Macgregor, H. C. & Uzzell, T. M. Jr. Gynogenesis in salamanders related to Sciences sponsored by the Pew Charitable Trusts. P.B. is an Early Career Scientist
Ambystoma jeffersonianum. Science 143, 1043–1045 (1964). with the Howard Hughes Medical Institute.
18. White, M. J. D., Cheney, J. & Key, K. H. L. A parthenogenetic species of
grasshopper with complex structural heterozygosity (Orthoptera: Acridoidea). Author Contributions A.A.L., W.B.N., D.P.B. and P.B. contributed extensively to the
Aust. J. Zool. 11, 1–19 (1963). work presented in this paper. W.W. performed, and provided training in,
microscopy and image data quantification.
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature. Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Acknowledgements We thank the staff of the Reptile Facility for their dedication to Correspondence and requests for materials should be addressed to P.B.
animal husbandry; C. Painter and his colleagues at the New Mexico Department of (peb@stowers.org).

286
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08818

METHODS ether solution and cells were dropped onto the coverslips and immediately washed
Animals. Laboratory colonies of A. tigris, A. inornata, A. neomexicana, A. exsanguis liberally with fixative. Cover slips were then incubated on a heat block at 75 uC for
and A. tesselata were established from animals collected in Socorro, Sierra and 1 min. FISH was performed on dried coverslips as previously described25 using
Otero Counties, New Mexico, under a permit from the New Mexico Department either an AlexaFluor 543-labelled peptide-nucleic acid probe composed of
of Game and Fish (permit numbers 3199 and 3395). A. gularis were collected in 59-(CCCTAA)3-39or an AlexaFluor 488-labelled locked-nucleic acid (LNA) probe
Dickens County, Texas. Animals were propagated in our Reptile and Aquatics composed of 59-(CCAAGG)2CC-39. Washes were modified for the LNA probe as
Facility under conditions similar to a previously published description of captive follows: three 2-min washes in 2X SSC, 0.05% Tween 20 at 37 uC, two 6-min washes
lizard husbandry19. with 0.1X SSC at 55 uC, three 2-min washes with 2X SSC, 0.05% Tween 20 at room
GV isolation and quantification of chromosome volume. Ovaries from adult temperature, followed by two 2-min washes with PBS. Samples were imaged on a
and sub-adult lizards were placed in PBS and GVs were isolated using jeweller’s fluorescence microscope with a 1003, 1.4 NA Plan-Apochromat objective and
forceps. GVs were transferred with a pipette to glass-bottom dishes (MatTek) images were analysed with AxioVision software (Carl Zeiss Jena).
containing 40 ng ml21 4’,6-diamidino-2-phenylindole (DAPI) in PBS, and Ovaries were isolated from A. neomexicana and placed in Buffer A (15 mM
allowed to incubate at 4 uC overnight. The following day the GVs were imaged PIPES, pH 6.8, 20 mM NaCl, 60 mM KCl, 0.5 mM EGTA, 2 mM EDTA, 0.5 mM
using a LSM 510 META (Carl Zeiss Jena) system in two-photon excitation spermidine, 1 mM DTT). GVs were then embedded in an acrylamide mixture
mode20 equipped with a C-Apochromat 403, NA 1.2 water immersion lens consisting of Buffer A, 5% acrylamide, 1 mM DTT, 15 mM sodium sulphite, and
(Carl Zeiss Jena) optimized for ultraviolet and infrared transmission. Two- 11.5 mM sodium persulphate. Embedding chambers consisted of 22 3 55 mm
photon excitation at 735 nm was used to avoid out-of-focus bleaching and all coverslips that had been siliconed to stainless steel washers. After the GV was
fluorescence emission below 650 nm was collected. Images were cropped in added to the washer, half of a 22 3 22 mm coverslip was dropped onto the
Adobe Photoshop to digitally remove the nuclear envelope. washer to seal the top and promote polymerization of the acrylamide. After
To obtain an unbiased measurement of chromosome volumes, we used the 30–60 min, the coverslip was carefully lifted and the gel was washed 4 times
non-parametric and unsupervised, automatic threshold selection developed by for 20 min with Buffer A on a shaking platform. Prehybridization was carried
Otsu21. In this method, the grey-level histogram of an image suffices to find a grey out in 50% deionized formamide, 2X SSC for 80 min with three changes of the
level threshold, which yields the optimal separation of the chromosomes from prehybridization solution. Hybridization mixtures were as described for somatic
the background without other a priori input. The Otsu method treats the nor- cells, and samples were incubated in a sealed chamber to minimize evaporation.
malized histogram as a probability distribution of possible pixel values. Pixels are Slides/washers were placed on a PCR machine with the following program: non-
dichotomized into two classes C0 and C1 (background and objects, in our case heated lid, 1 h at room temp, 30 min at 40 uC, 6 min at 94 uC and overnight at
chromosomes) by a threshold at level k. C0 denotes the class of pixels with values 37 uC. The following day, samples were washed four times in PBS with 0.1%
[0, …, k], and C1 denotes the class of pixels with values [k 1 1, …, L], 0 being the Tween 20, and three times in PBS and stained with DAPI (40 ng ml21) for at least
smallest and L the largest pixel value in the image. The goal is to determine the 1 h at room temperature. Samples were imaged on an inverted confocal micro-
threshold value k corresponding to the best class separation. Otsu showed that k scope with a C-apochromat 403/1.20 W objective (Carl Zeiss Jena). The Alexa
can be found by maximizing the between-class variance s2B ðk Þ for all k between 0 Fluor 543-labelled peptide-nucleic acid probe (59-(CCCTAA)3-39) was excited at
and L. s2B ðk Þ is defined as: 561 nm and fluorescence emission was collected above 575 nm. The Alexa Fluor
488-labelled locked-nucleic acid probe (59-(CCAAGG)2CC-39) was excited at
s2B ðk Þ~P0 (k)ðm0 (k){mG Þ2 zP1 (k)ðm1 (k){mG Þ2 488 nm and fluorescence emission from 505 to 550 nm was collected. DAPI-
stained chromosomes were visualized by excitation at 405 nm and collection
Here P0,1 (k) is the probability that a pixel k is assigned to class C0 or C1, respec-
of fluorescence emission between 420 and 480 nm. To avoid emission cross-talk,
tively, m0,1 (k) represents the mean intensity of all the pixels assigned to class C0
we switched the excitation between the different laser lines and collected data in
or C1 (ref. 22), and mG is the average intensity of the whole image.
the appropriate detection channel only (multi-tracking).
To calculate the threshold, we used the implementation of Otsu’s method by
Transmission electron microscopy. Ovaries were isolated from A. tigris and
C. Mei, A. Joshua and T. Collins as a plug-in for the open-source image proces-
A. neomexicana in PBS then transferred to a tube containing 70% hexane, 0.75%
sing package ImageJ23. The volume was determined with the commercial 3D
paraformaldehyde (Electron Microscopy Sciences), and 0.125% Nonidet (US
image processing software IMARIS 6.3 (Bitplane). This software bases its volume
Biological) in PBS. The tube was gently inverted 10 times then placed in a 20 uC
calculation on an iso-surface rendered at an intensity value k, using the threshold
waterbath for 2.5 h, gently inverting the tube five times every 20 min. The ovaries
determined as described above. This protocol ensures an objective, reproducible,
were washed three times with 0.2% Tween 20 in PBS and three times with PBS
and unbiased comparison of chromosome volumes measured for independent
(20 min each), followed by overnight fixation in 2.5% glutaraldehyde (Electron
samples.
Microscopy Sciences) in PBS at 4 uC. Fixed tissues were washed three times in PBS
Embryonic fibroblast cell culture. Cell cultures were modified from ref. 24.
and water, then post-fixed in aqueous 1% OsO4, 1% K3Fe(CN)6 for 10 min at
Briefly, Aspidoscelis eggs were incubated at 29 uC for 30–40 days, sterilized in 90%
room temperature. Following 3 PBS washes, the tissue was dehydrated through a
ethanol, 120 mM potassium iodide and 39 mM iodine, and opened under sterile
graded series of 30–100% ethanol 100% propylene oxide and then infiltrated in
conditions in a laminar flow hood. Embryos were immediately decapitated and
1:1 mixture of propylene oxide:Polybed 812 epoxy resin (Polysciences) for 1 h.
cut into 1 cm or smaller pieces, rinsed with cold PBS and incubated with trypsin-
After two to three changes of 100% resin over 24 h, tissues were embedded in
EDTA solution (T4049, Sigma) on a stir plate for 5 min at room temperature.
moulds, cured at 37 uC overnight, followed by additional hardening at 65 uC for
The supernatant was discarded and replaced with fresh PBS/trypsin, and incuba-
two more days. Ultrathin (60 nm) sections were collected on copper grids, stained
tion was continued for 15 min. The supernatant was decanted through sterile
with 2% uranyl acetate in 50% methanol for 10 min, followed by 1% lead citrate
cheesecloth into a 50-ml Falcon tube containing 2 ml ice-cold M199 cell culture
for 7 min. Sections were photographed using a FEI transmission electron micro-
media (Sigma) supplemented with 20% fetal bovine serum, 50 mg ml21 gentamycin
scope at 80 kV.
(Sigma), glutamax (Invitrogen), MEM non-essential amino acids (Invitrogen),
MEM vitamin solution (Invitrogen), 56 U ml21 nystatin (Sigma), 100 U ml21 19. Townsend, C. R. Establishment and maintenance of colonies of parthenogenetic
penicillin and 100 mg ml21 streptomycin (Sigma). Cell suspensions were kept on whiptail lizards: Cnemidophorus spp. Int. Zoo. Yb. 19, 80–86 (1979).
ice while the remaining tissue was trypsin-treated for another 15 min and the 20. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence
supernatants were combined. Cells were then pelleted, washed in M199 media plus microscopy. Science 248, 73–76 (1990).
supplements and finally resuspended in 2–6 ml M199 media plus supplements and 21. Otsu, N. Threshold selection method from gray-level-histograms. IEEE Trans. Syst.
seeded in 6-well dishes (Falcon, 353046). Cells were cultured at 30 uC, 5% O2 and Man Cybern. SMC-9, 62–66 (1979).
2% CO2, and split once they had reached 85–100% confluency. 22. Gonzalez, R. C. & Woods, R. E. Digital Image Processing 3rd edn, (Pearson/Prentice
Hall, 2008).
Fluorescent in situ hybridization (FISH). Embryonic fibroblasts were treated
23. ImageJ (US National Institutes of Health, Bethesda, 1997–2009).
with 0.5 mg ml21 Karyomax colcemid (Invitrogen) at 50–70% confluency and 24. Moore, M. K., Work, T. M., Balazs, G. H. & Docherty, D. E. Preparation,
incubated for 3 h at 30 uC, 5% O2 and 2% CO2. The cells were harvested by cryopreservation, and growth of cells prepared from the green turtle (Chelonia
trypsin treatment and subjected to hypotonic swelling in 0.075 M KCl at 37 uC mydas). Methods Cell Sci. 19, 161–168 (1997).
for 10 min. The cells were then pelleted, washed twice and finally resuspended in 25. Sarthy, J., Bae, N. S., Scrafford, J. & Baumann, P. Human RAP1 inhibits non-
methanol:acetic acid fixative (3:1). Coverslips were cleaned with a 1:1 ethanol:ethyl homologous end joining at telomeres. EMBO J. 28, 3390–3399 (2009).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08799

LETTERS
Systematic genetic analysis of muscle morphogenesis
and function in Drosophila
Frank Schnorrer1,2, Cornelia Schönbauer1, Christoph C. H. Langer1, Georg Dietzl2, Maria Novatchkova2,
Katharina Schernhuber2, Michaela Fellner2, Anna Azaryan2, Martin Radolf2, Alexander Stark2, Krystyna Keleman2
& Barry J. Dickson2

Systematic genetic approaches have provided deep insight into the screen (excluding for technical reasons those lines that were not
molecular and cellular mechanisms that operate in simple unicel- viable as homozygotes). For each of these lines, muscle organization
lular organisms. For multicellular organisms, however, the pleio- was visualized in live animals using a green fluorescent protein (GFP)
tropy of gene function has largely restricted such approaches to the marker that specifically labels the sarcomeric Z-line7. Defects were
study of early embryogenesis. With the availability of genome-wide readily observed for 190 genes, either in overall muscle morphology
transgenic RNA interference (RNAi) libraries in Drosophila1,2, it is (Fig. 2a and Supplementary Table 6) or sarcomeric organization
now possible to perform a systematic genetic dissection of any cell (Fig. 2b and Supplementary Table 7).
or tissue type at any stage of the lifespan. Here we apply these We distinguished three classes of defect in muscle morphology
methods to define the genetic basis for formation and function of (Fig. 2c–k): ‘split myofibril’ (53 genes), ‘missing muscles’ (8 genes)
the Drosophila muscle. We identify a role in muscle for 2,785 genes, and ‘rounded muscles’ (13 genes), in which muscles are either split
many of which we assign to specific functions in the organization of into thinner myofibrils, are missing or have the rounded appearance
muscles, myofibrils or sarcomeres. Many of these genes are phylo- characteristic of muscles that undergo normal morphogenesis but fail
genetically conserved, including genes implicated in mammalian to form stable attachments. The ‘split myofibril’ class includes several
sarcomere organization and human muscle diseases. signalling molecules, such as the FGF receptor heartless, Anxb11, the
Muscle biology is an attractive target for analysis by genome-wide actin regulator a-actinin and several RNA-binding proteins (for
transgenic RNAi. The basic cell and developmental biology of muscles example, CG5800; Fig. 2g). In these mutants, the muscles still generally
is largely conserved from insects to mammals3–6, and their multinuc- attach to the appropriate tendon cells. The ‘missing muscles’ class
lear architecture renders them inaccessible to conventional genetic includes splicing factors and His2A (Fig. 2f). The ‘rounded muscles’
mosaic strategies. To systematically disrupt gene functions exclusively class includes known muscle attachment factors such as integrins, ILK
in the muscles of intact animals, we crossed the muscle-specific Mef2-
GAL4 driver to each of the UAS-IR transgenic RNAi lines in our
genome-wide library1. Progeny from these crosses were assayed for a Phenotypic distribution
viability, posture, locomotion and flight. We screened a total of 17,759
RNAi lines, representing 10,461 distinct genes. A total of 3,909 lines
Lethal (1,963; 18.8%)
and 2,785 genes were scored as defective in one or more of these assays Adult lethal day 7 (69; 0.7%)
(Fig. 1, Supplementary Fig. 1 and Supplementary Tables 1 and 2). Flightless (316; 3.0%)
This screen identified 73 of 77 positive control genes (Supplemen- Locomotion (78; 0.7%)
tary Table 3), suggesting a false-negative rate of just 5%. To assess the Wing posture (13; 0.1%)
false-positive rate, we compiled a negative control list of 79 genes Weak flyer (276; 2.6%)
shown by classical genetic studies to have no function in muscles. Of Semi-lethal (70; 0.7%)
the 121 RNAi lines available for these genes, only one scored positive Wild type (7,676; 73.4%)
in our assays (Supplementary Table 4). We thus estimate that our n = 10,461
screen has a false-positive rate of 1.3% of genes (a false-discovery rate
of 5%). b Lethality stages
We selected 1,004 of the positive genes at random for validation
using a second, independent hairpin construct. These lines were Embryonic lethal (31; 1.6%)
obtained from a second RNAi library currently under construction Early larval lethal (213; 10.9%)
using the phiC31 site-specific integrase system (K.K. and B.J.D., Late larval lethal (343; 17.5%)
unpublished observations). A total of 874 genes (87.1%) were again Early pupal lethal (488; 24.9%)
positive (Supplementary Table 5), consistent with the estimated 5% Late pupal lethal (613; 31.2%)
false-discovery rate in our primary screen and a false-negative rate Pharate / adult lethal (211; 10.7%)
comparable to that observed with the first library (,5%). Undefined (64; 3.3%)
We began the detailed analysis of these muscle phenotypes by
examining the morphology of the larval body wall muscles n = 1,963
(Supplementary Fig. 1). Reasoning that defects in these muscles are Figure 1 | Phenotypic classification of primary screen. a, Distribution of
most likely to result in embryonic or larval lethality, we focused on genes into phenotypic classes in the primary muscle screen. b, Distribution
the 436 genes that fell into these phenotypic classes in the primary of the lethal stages for all essential genes.
1
Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. 2Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.
287
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

a Muscle morphology b Sarcomere morphology

Fading Z (94; 21.6%)

Split (53; 12.2%) Spotty Z (50; 11.5%)
Missing (8; 1.8%) Clumpy Z (18; 4.1%)
Rounded (13; 3.0%) Undefined (8; 1.8%)
Wild type (362; 83.0%) Wild type (266; 61.0%)

n = 436 n = 436
c Wild type d Rounded

+ UAS-rhea-IR
e Wild type f Missing g Split

+ UAS-His2A:CG33865-IR UAS-CG5800-IR
h i j k

Wild type Rounded Missing Split

l Wild type m Spotty Z n Wild type o Spotty Z

+ UAS-bt-IR + UAS-how-IR
p Fading Z q Clumpy Z r Clumpy Z

UAS-Actn-IR UAS-flare-IR UAS-Bsg-IR

s t u v

Wild type Spotty Z Fading Z Clumpy Z

Figure 2 | Larval muscle screen. a, b, Muscle (a) and sarcomere phenotypes: wild-type embryonic sarcomeres (l, s) compared with UAS-bt-
(b) morphology classes of the larval body muscles. c–k, Wild-type and IR (TF46253) displaying spotty Z sarcomeres (m, t), and wild-type L3
mutant phenotypes: normal muscles in wild-type embryos (c, h) compared sarcomeres (n) compared with spotty Z in UAS-how-IR (TF13756)
with rounded muscles in UAS-rhea-IR (TF40400) (d, i), wild-type L3 larval (o), fading Z in UAS-Actn-IR (TF7760) (p, u), and clumpy Z in UAS-flare
muscles (e), missing muscles in UAS-CG33865-IR (TF39116) (f, j) and split (TF22851) (q, v) and UAS-Bsg-IR (TF43307) (r). Scale bars, 100 mm (c, d) and
muscles in UAS-CG5800 (TF27519) (g, k). l–r, Sarcomere morphology 50 mm (e–g, i–r).
and rhea, the fly Talin orthologue (Fig. 2d), as well as the fly Parvin Z class. The ‘clumpy Z’ class includes flare (CG10724), the recently
orthologue. identified fly orthologue of actin interacting protein 1 (AIP1), a
We also defined three classes of defect in sarcomeric organization regulator of F-actin disassembly10 and Basigin (Bsg), a muscle trans-
(Fig. 2l–v): ‘fading Z’ (94 genes), ‘spotty Z’ (50 genes) and ‘clumpy Z’ membrane protein required for formation of the neuromuscular
(18 genes), in which the Z-lines are either reduced, discontinuous or junction11 (Fig. 2q, r). We confirmed efficient knockdown of several
appear as large irregular aggregates, respectively. The ‘fading Z’ class of these proteins by antibody staining (Supplementary Fig. 2).
is exemplified by a-actinin (Actn; Fig. 2p), and the ‘spotty Z’ class by We performed a similar analysis of muscle morphology in adults,
how (Fig. 2o), which encodes an RNA-binding protein required for using phalloidin staining to visualize the indirect flight muscles
muscle development8, and bent (Fig. 2m), which encodes a titin- or (IFMs) for all RNAi lines that scored as flightless in the primary
projectin-like protein9. Most of the known sarcomeric components screen (Supplementary Fig. 1 and Fig. 1a). The large regular structure
(bt, sls, Mhc, actin, Tm2, TpnC47D, and TpnC73F) fell into the spotty of the IFMs and their critical role in flight make them ideal models for
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NATURE | Vol 464 | 11 March 2010 LETTERS

studying muscle structure and function12,13. Moreover, unlike the of which are severely disrupted upon knockdown of fliI or CG8578
larval body wall muscles, IFMs resemble vertebrate muscles in their (Fig. 3o and Supplementary Fig. 3a, b), and in fliI mutants (Fig. 3n).
construction from multiple fibres, each composed of many myofi- In the ‘frayed’ class, myofibrils are unusually thin and often frayed at
brils14. Defects in IFMs were observed in 196 of the 328 flightless the edges, as for example upon knockdown of the actin regulator
genes tested. We assigned each of these genes to one or more of nine coronin (Fig. 3i), and the M- and Z-lines are also thinner and appear
distinct phenotypic categories, based on defects in overall muscle bent at the frayed edges of the myofibril (Supplementary Fig. 4d). The
morphology (Fig. 3a and Supplementary Table 8), myofibril mor- ‘trapezoid’ class is characterized by myofibrils with a zigzag-like
phology (Fig. 3b and Supplementary Table 9) or sarcomeric organi- structure, usually with thick Z-lines and thinner actin filaments
zation (Fig. 3c and Supplementary Table 10). towards the M-lines. One gene in this class is the adducin homologue
The two classes of defect in overall IFM morphology were ‘missing hts (Fig. 3k and Supplementary Fig. 4f, g). For 49 genes, mostly of the
IFMs’ (55 genes) and ‘irregular IFMs’ (12 genes). The ‘missing IFM’ ‘trapezoid’ and ‘frayed’ classes, we also observed large actin aggre-
class includes parkin (park) and flightin (fln) (Fig. 3e), both previously gates (Fig. 3m and Supplementary Table 9) that are reminiscent of so-
associated with IFM degeneration15,16, as well as several transcription called zebra bodies21, possibly collapsed Z-lines, that are commonly
factors that may contribute to the specification of individual muscles observed in human nemaline myopathies22.
(Supplementary Table 8). The ‘irregular IFM’ class includes flightless I Three further phenotypic classes were based on sarcomere organi-
(fliI; Fig. 3f, g) and MICAL, which serve as positive controls17,18, as well zation: ‘no sarcomere’ (39 genes), ‘no M’ (12 genes) and ‘fuzzy Z’ (99
as CG8578, the mouse orthologue of which (LRRFIP2) interacts with genes). The ‘no sarcomere’ set generally coincides with the degenerate
the leucine-rich repeats of mouse FliI19. myofibril and missing IFM classes (Fig. 3s, w and Supplementary
We defined four classes of myofibril defect: ‘degenerate’ (39 Fig. 3e). The ‘no M’ class is characterized by sarcomeres with an
genes), ‘irregular’ (7 genes), ‘frayed’ (97 genes) and ‘trapezoid’ (29 apparently normal Z-line, but no discernable M-line in phalloidin
genes). Most genes in the ‘degenerate’ myofibril class were also clas- stainings (Fig. 3q, u). This is seen upon knockdown of the obscurin
sified as ‘missing IFMs’. Distinct myofibrils are difficult to discern in homologue unc-89 (Fig. 3q), and in unc-89 mutants (B. Bullard,
these lines, as is observed upon knockdown of the band 4.1 septate personal communication). Staining unc-89 knockdown IFMs with
junction protein encoded by coracle20 (Fig. 3l). The ‘irregular’ myofi- the M-line marker anti-Mhc revealed that the M-line is indeed pre-
bril class is characterized by misoriented and disorganized myofibrils, sent, but is significantly broadened and invaded by thin actin filaments
as seen with fliI (Fig. 3j and Supplementary Fig. 3) or its interactor (Supplementary Fig. 5a, b). Knockdown of the potassium channel eag
CG8578 (Fig. 3o). FliI protein localizes to M- as well as Z-lines, both leads to a more severe M-line phenotype with complete absence of

a Muscle morphology b Myofibril morphology c Sarcomere morphology

Degenerate (39; 12.0%)

No sarcomere (39; 12.0%)
Missing (55; 16.8%) Irregular (7; 2.1%)
No M (12; 3.7%)
Irregular (12; 3.7%) Trapezoid (29; 8.9%)
Fuzzy Z (99; 30.5%)
Wild type (261; 79.6%) Frayed (97; 29.9%)
Wild type (175; 53.9%)
Wild type (153; 47.1%)

n = 328 n = 325 n = 325

d Wild type e Missing IFM f Irregular IFM g Irregular IFM

UAS-CG1623-IR UAS-fln-IR UAS-fliI-IR fliI[3]/fliI[14]

h Wild type i Frayed j Trapezoid k Irregular

+ UAS-coro-IR UAS-hts-IR UAS-fliI-IR

l Degenerate IFM m Actin blob n Irregular o Irregular

UAS-cora-IR UAS-CG14260-IR fliI[3]/fliI[14] UAS-CG8578-IR

p Wild type q No M–line r Fuzzy Z s No sarcomeres

+ UAS-unc89-IR UAS-hts-IR UAS-CG13366-IR

t Z Z Z u Z Z Z v Z Z Z w

M M M M
Wild type No M–line Fuzzy Z No sarcomeres

Figure 3 | Adult muscle screen. a–c, Distribution of muscle (a), myofibril mutants (n) and UAS-CG8578-IR (TF35968) (o), degenerated IFMs in UAS-
(b) and sarcomere (c) morphology of the adult IFMs. d–g, Normal IFMs in cora-IR (TF9787) (l) and actin blobs in UAS-CG14260 (TF17452)
wild type (d), missing IFMs in UAS-fln-IR (TF46153) (e), irregular IFMs in (m). p–w, Normal sarcomere morphology in wild type (p), no visible M-line
UAS-fliI-IR (TF39528) (f) and fliI mutant (g). h–o, Normal myofibril in UAS-unc89-IR (TF29412) (q), fuzzy Z in UAS-hts-IR (TF29101) (r) and
morphology in wild type (h), frayed in UAS-coro-IR (TF44671) (i), trapezoid no sarcomeres in UAS-CG13366 (TF29606) (s), and the respective
in UAS-hts-IR (29101) (j), irregular in UAS-fliI-IR (TF39528) (k), fliI schematics (t–w). Scale bars, 100 mm (d–g), 10 mm (h–o), 5 mm (p–s).
289
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

a Protein Transcription, Metabolism,

G-protein coupled receptor protein signaling p.

Muscle synthesis splicing mitochondria

Monovalent inorganic cation transporter a.

DNA-directed RNA polymerase activity

Mitochondrial electron transport chain

Proteasome complex (sensu Eukarya)

Structural constituent of cytoskeleton

Transmembrane receptor activity

Transcription, DNA dependent

Transcription factor activity

Exosome (RNase complex)

Oxidative phosphorylation
Mitochondrial ribosome
Arp2/3 protein complex

Spliceosome complex
Translational initiation
Muscle development
Cell-matrix adhesion

Nucleic acid binding

Protein biosynthesis

Cell communication
Sensory perception
Muscle attachment

Muscle contraction
Actin cytoskeleton

Defence response
Lipid metabolism
Integrin complex

ATPase activity
Mitochondrion
RNA binding

Extracellular
Metabolism
Intracellular
Sarcomere

Translation

Ribosome
Myofibril

Nucleus
Mef2 wild type
Mef2 all positive
Mef2 all lethals
Mef2 embryo
Mef2 early larval
Mef2 late larval
Mef2 early pupal
Mef2 late pupal
Mef2 adult
Mef2 day7
Mef2 flightless
Larval muscle wild type
Larval muscle missing
Larval muscle split
Larval muscle rounded
Larval sarcomere wild type
Larval sarcomere clumpy Z
Larval sarcomere fading Z
Larval sarcomere spotty Z

–10

10
–9
–8
–7
–6
–5
–4
–3
–2
–1
0
1
2
3
4
5
6
7
8
9
Legend

Immunology

Metabolic
b c d

Skeletal
Muscle
Mef2 positives

Neuro
M. m.

A. m.

Eyes
C. e.

A. g.
H. s.
S. c.

Skin
A. t.

RNAi Muscle
phenotype expression Mef2 wildtype Mef2 wild type
n = 2,785 255 n = 504 Mef2 all positives Mef2 all positives
2,482 193
30 Mef2 all lethals Mef2 all lethals
18 26 Mef2 flightless Mef2 flightless
Larval sarcomere clumpy Z Larval sarcomere clumpy Z
33
Larval sarcomere fading Z Larval sarcomere fading Z
Larval sarcomere spotty Z Larval sarcomere spotty Z
Mef2
target
n = 107
–10 0 10 –5 0 5

Figure 4 | Bioinformatic analyses. a, GO-term enrichment in the various c, d, Conservation across species (c) and link to clinical diseases (d) in Mef2
phenotypic classes. b, Overlap of genes expressed in muscles, predicted Mef2 lethal as well as the sarcomere phenotypic sets. A. t., Arabidopsis thaliana; S.
targets and positives in the Mef2 RNAi screen. Only genes tested in the RNAi c., Saccharomyces cerevisiae; M. m., Mus musculus; H. s., Homo sapiens; C. e.,
screen are included (n 5 10,460 for Mef2), and only Mef2 high-confidence Caenorhabditis elegans; A. m., Apis mellifera; A. g., Anopheles gambiae.
targets for which expression data were available are included (n 5 107).

Mhc at the M-line (Supplementary Fig. 5c). This may explain why eag Previous studies have sought to identify muscle genes systematically
mutants display severe flight defects23. Surprisingly, we find upon either by expression profiling (http://www.fruitfly.org/cgi-bin/ex/insi-
knockdown of the Par domain transcription factor pdp1 an overlap tu.pl) or chromatin immunoprecipitation of Mef2-binding sites25, both
of Z- and M-lines (Supplementary Fig. 5d), suggesting that mesoder- of which have been focused on embryos. More than half of the muscle-
mal pdp1 (ref. 24) regulates correct sarcomere assembly. The ‘fuzzy Z’ expressed genes (285 of 504, P , 10247, Fig. 4b) and almost half of the
class is characterized by a broadening of the Z-line (Fig. 3r, v), and Mef2 targets (48 of 107, P , 1024, Fig. 4b) were functionally validated in
generally coincides with the frayed and trapezoidal classes of myofibril our screen. A total of 30 genes are positive in all three data sets, repre-
defect. We analysed RNA levels in isolated flight muscles for selected senting a set of Mef2 target genes with confirmed muscle expression and
genes of the ‘irregular’ myofibril class and the ‘no M’ class. This ana- function (Supplementary Table 11). Almost half of these had no func-
lysis confirmed a significant knockdown of the target messenger RNA. tional assignment before this study. Similar large-scale gene expression
In contrast, mRNA levels of predicted off-target genes (defined as and Mef2 target data are not yet available for later developmental stages,
having at least one perfect 16 nucleotide match to the hairpin) were but we note that our RNAi screen has assigned functions to 10 of 12
not altered any more than mRNA levels of other randomly selected genes known to be differentially expressed in adult muscle precursors26
genes (Supplementary Fig. 6). (Supplementary Table 11), and to 10 of 14 genes predicted to function in
We examined the gene ontology (GO) database (www.geneontology. terminally differentiated muscle27 (Supplementary Table 12).
org) to assess the representation of various gene classes in each of our Finally, we found that the genes with a muscle RNAi phenotype
phenotypic categories (Fig. 4a). GO terms related to muscles are all are, on average, much better conserved than those with no phenotype
significantly enriched in the set of Mef2-positive genes, particularly in (Fig. 4c). Moreover, examination of the OMIM database (http://
the embryonic lethals. We also note an enrichment of the GO terms www.ncbi.nlm.nih.gov/omim) revealed that these conserved genes
‘muscle attachment’ and ‘cell-matrix adhesion’ in the ‘rounded’ pheno- are enriched for genes implicated in human muscle diseases, but
type class, consistent with the failure to form a stable muscle attachment not diseases that affect other tissues (Fig. 4d). Thus, our work not
in these lines. Another noticeable enrichment is the GO term ‘muscle only lays a foundation for a comprehensive analysis of muscle biology
contraction’ within the ‘spotty Z’ class of sarcomeric defects, suggesting in Drosophila, but also for the systematic analysis of gene function in
that the actin defects in these lines correlate with impaired contractility. vertebrate muscles.
290
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

METHODS SUMMARY 18. Beuchle, D., Schwarz, H., Langegger, M., Koch, I. & Aberle, H. Drosophila MICAL
regulates myofilament organization and synaptic structure. Mech. Dev. 124,
All RNAi crosses to Mef2-GAL4 were performed at 27 uC and males were assayed
390–406 (2007).
at day 7 of adult life blind to the genotype. Positives were also retested blind, 19. Fong, K. S. & de Couet, H. G. Novel proteins interacting with the leucine-rich
along with previously untested lines in the primary screen. Where multiple RNAi repeat domain of human flightless-I identified by the yeast two-hybrid system.
lines for the same gene resulted in different phenotypes, the gene was assigned the Genomics 58, 146–157 (1999).
strongest of these phenotypes. Larval muscles were visualized with ZCL0663, a 20. Lamb, R. S., Ward, R. E., Schweizer, L. & Fehon, R. G. Drosophila coracle, a member
GFP trap in CG6416 labelling the Z-line7; adult flight muscles were bisected and of the protein 4.1 superfamily, has essential structural functions in the septate
stained with phalloidin. junctions and developmental functions in embryonic and adult epithelial cells.
Mol. Biol. Cell 9, 3505–3519 (1998).
Received 14 January; accepted 30 December 2009. 21. Nongthomba, U., Ansari, M., Thimmaiya, D., Stark, M. & Sparrow, J. Aberrant
splicing of an alternative exon in the Drosophila troponin-T gene affects flight
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vertebrates. Curr. Opin. Genet. Dev. 16, 525–532 (2006). regulates target genes at all stages of muscle development. Dev. Cell 10, 797–807
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Genetics 176, 2223–2234 (2007). Acknowledgements We thank the Vienna Drosophila RNAi Center for transgenic
11. Besse, F. et al. The Ig cell adhesion molecule Basigin controls RNAi lines, and H. Bellen, B. Bullard, J. Saide and T. Volk for their gifts of various
compartmentalization and vesicle release at Drosophila melanogaster synapses. J. antibodies. This work was supported by Boehringer Ingelheim GmbH, the
Cell Biol. 177, 843–855 (2007). Max-Planck Society and a postdoctoral fellowship to F.S. from the Human Frontier
12. Vigoreaux, J. O. in Muscle Development in Drosophila (ed. Sink, H.) 143–156 Science Program. Generation of a second set of RNAi lines was supported by a
(Landes Biosciences, 2006). grant from the European Union 7th Framework Programme. A.S. and M.N. were
13. Vigoreaux, J. O. Genetics of the Drosophila flight muscle myofibril: a window into supported by the Austrian Ministry for Science and Research (GEN-AU
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14. Bate, M. in The development of Drosophila melanogaster, Vol. 2 (eds Bate, M. & Author Contributions F.S. designed and performed the screen and other
Martinez-Arias, A.) 1013–1090 (Cold Spring Harbor Press, 1993). experiments, analysed the data and wrote the manuscript with B.J.D. C.S. and
15. Park, J. et al. Mitochondrial dysfunction in Drosophila PINK1 mutants is C.C.H.L. analysed selected muscle phenotypes in detail. G.D., K.S., M.F. and A.A.
complemented by parkin. Nature 441, 1157–1161 (2006). assisted in the primary screen, and G.D. also in the initial data analysis. M.R.
16. Reedy, M. C., Bullard, B. & Vigoreaux, J. O. Flightin is essential for thick filament performed the RNA microarrays. M.N. and A.S. performed the bioinformatic
assembly and sarcomere stability in Drosophila flight muscles. J. Cell Biol. 151, analyses. K.K. led the team that generated the second RNAi hairpin lines.
1483–1500 (2000).
17. Campbell, H. D. et al. The Drosophila melanogaster flightless-I gene involved in Author Information Reprints and permissions information is available at
gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich www.nature.com/reprints. The authors declare no competing financial interests.
repeat domains and is conserved in Caenorhabditis elegans and humans. Proc. Natl Correspondence and requests for materials should be addressed to F.S.
Acad. Sci. USA 90, 11386–11390 (1993). (schnorrer@biochem.mpg.de) or B.J.D. (dickson@imp.ac.at).

291
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 Vol 464 | 11 March 2010 | doi:10.1038/nature08792

LETTERS
Telomere elongation in induced pluripotent stem cells
from dyskeratosis congenita patients
Suneet Agarwal1, Yuin-Han Loh1, Erin M. McLoughlin1, Junjiu Huang2,3, In-Hyun Park1, Justine D. Miller1,
Hongguang Huo1, Maja Okuka2, Rosana Maria dos Reis2, Sabine Loewer1, Huck-Hui Ng4, David L. Keefe2,
Frederick D. Goldman5, Aloysius J. Klingelhutz6, Lin Liu2,7 & George Q. Daley1,8

Patients with dyskeratosis congenita (DC), a disorder of telomere X-linked DC is caused by mutations in the dyskerin gene (DKC1)9
maintenance, suffer degeneration of multiple tissues1–3. Patient- which encodes an RNA binding protein whose inactivation destabilizes
specific induced pluripotent stem (iPS) cells4 represent invaluable the levels of TERC, resulting in shortened telomeres and premature
in vitro models for human degenerative disorders like DC. A cardinal senescence in patient cell lines10,11. We asked whether a DKC1 mutant
feature of iPS cells is acquisition of indefinite self-renewal capacity, fibroblast line (del37L, refs 9–11) could be reprogrammed and pro-
which is accompanied by induction of the telomerase reverse tran- pagated in a pluripotent state. Compared to normal cells, the repro-
scriptase gene (TERT)5–7. We investigated whether defects in telo- gramming efficiency of del37L cells was poor, yielding only 2–5 colonies
merase function would limit derivation and maintenance of iPS cells from 105 input cells with a delayed latency (Supplementary Table 1).
from patients with DC. Here we show that reprogrammed DC cells Nevertheless, DKC1 mutant iPS colonies showed all the hallmarks of
overcome a critical limitation in telomerase RNA component (TERC) pluripotency, including characteristic morphology (Fig. 1a), gene
levels to restore telomere maintenance and self-renewal. We dis- expression (Fig. 1b and Supplementary Fig. 2a), and formation of
covered that TERC upregulation is a feature of the pluripotent state, teratomas comprising all three embryonic germ layers (Fig. 1c). PCR
that several telomerase components are targeted by pluripotency- restriction fragment length polymorphism (RFLP) analysis for the
associated transcription factors, and that in autosomal dominant DKC1 mutation confirmed the del37L mutation in iPS lines, and karyo-
DC, transcriptional silencing accompanies a 39 deletion at the type analysis was normal (Fig. 1d and Supplementary Fig. 2b, c). These
TERC locus. Our results demonstrate that reprogramming restores data show that the somatic cells from patients with a genetic impair-
telomere elongation in DC cells despite genetic lesions affecting ment in telomere elongation can be reprogrammed to pluripotency.
telomerase, and show that strategies to increase TERC expression Despite induction of endogenous TERT and telomerase activity
may be therapeutically beneficial in DC patients. (Figs 1b, e), early passage del37L iPS cell lines displayed shorter
Reprogramming of somatic cells to a state of pluripotency is char- telomeres relative to the starting fibroblast population (Fig. 1f).
acterized by prolonged self-renewal, implying induction of telomere Addition of TERT to the reprogramming factors did not result in
maintenance mechanisms. Recently, it was reported that reprogram- telomere elongation in del37L mutant cells (Fig. 1f), unlike in normal
ming of mouse cells was accompanied by elongation of telomeres8, but cells (Supplementary Fig. 3), but did increase reprogramming effi-
given the significant differences in telomere length and telomerase regu- ciency (Supplementary Table 1; Supplementary Information). We
lation in mouse and human cells, we asked whether normal human cells obtained similar results with reprogramming of an independent
displayed telomere elongation after reprogramming. We generated iPS DKC1 mutant line (A386T, ref. 11) (Fig. 1g, Supplementary Fig. 4
cell lines by retroviral transduction of primary human fibroblasts with and Supplementary Table 1). Given the telomerase dysfunction and
the factors OCT4 (also known as POU5F1), SOX2, KLF4 and c-MYC shortened telomeres, we expected to observe limited passage of DKC1
(also known as MYC), confirmed the pluripotent phenotype by gene mutant iPS cells in culture. However, unlike the parental DKC1
expression and functional analysis4, and determined the mean terminal mutant fibroblasts, which senesced after 3–4 passages, we were able
restriction fragment (TRF) length of the donor fibroblast lines and to continuously culture the DKC1 mutant iPS cell lines. Compared to
corresponding iPS lines by Southern blot. We found that mean TRF the early passage cells, we found by TRF analysis that telomere length
length and total telomeric DNA was increased in iPS cell lines relative to in del37L iPS lines increased with continued passage (Fig. 2a). Con-
the parental fibroblasts (Supplementary Fig. 1a). Similar findings using sistent with this, despite numerous interval population doublings,
retrovirally-marked single-cell fibroblast sub-clones5 contradict the late passage del37L iPS lines had telomere lengths comparable to the
notion that cells with long telomeres are uniquely susceptible to repro- original fibroblast population by quantitative PCR12 (Fig. 2b). In a
gramming (Supplementary Fig. 1b). Induction of TERT expression and blinded assessment by the complementary method of quantita-
telomerase activity correlated with reprogramming to pluripotency as tive telomere fluorescence in situ hybridization, we confirmed that
shown previously5–7 (Supplementary Figs 1c–e). These data establish telomere length was shortened immediately after derivation but
that direct factor-based reprogramming of human somatic cells results increased over time (Fig. 2c). Late passage del37L iPS cells main-
in net telomere elongation. tained a characteristic morphology, normal karyotype, and the same
1
Division of Hematology/Oncology, Children’s Hospital Boston; Pediatric Oncology, Dana Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School; Division of Hematology, Brigham and Women’s Hospital; Harvard Stem Cell Institute; Manton Center for Orphan Disease Research; Boston, Massachusetts
02115, USA. 2Department of Obstetrics and Gynecology, MDC 3125, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612, USA.
3
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. 4Gene Regulatory Laboratory, Genome Institute of Singapore;
Department of Biological Sciences, National University of Singapore; 60 Biopolis Street, #02-01, Genome, Singapore 138672. 5Department of Pediatrics, The Children’s Hospital of
Alabama, 1600 7th Avenue South, ACC 512, Birmingham, Alabama 35233, USA. 6Department of Microbiology, University of Iowa, 2202 MERF, 375 Newton Road, Iowa City, Iowa
52242, USA. 7College of Life Sciences, Nankai University, Tianjin 300071, China. 8Howard Hughes Medical Institute, Children’s Hospital Boston, Karp Family Research Building 07214,
1 Blackfan Circle, Boston, Massachusetts 02115, USA.

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©2010 Macmillan Publishers Limited. All rights reserved
 NATURE | Vol 464 | 11 March 2010 LETTERS

a b RT–PCR for pluripotency-associated Figure 1 | Derivation and characterization of

gene expression DKC1 mutant iPS cells. a, DKC1 del37L
fibroblasts and iPS colony. b, RT–PCR for
Endo OCT4
endogenous (Endo) OCT4, SOX2, TERT and
Endo SOX2 b-actin transcripts in del37L fibroblasts (Fib) and
Endo TERT
iPS cells derived with and without exogenous
Tert. c, Histology of del37L iPS1 teratomas.
β-Actin
d, PCR RFLP verification of del37L mutation in
DKC1 del37L fibroblast DKC1 del37L iPS Fib 1 2 3 4 5 6 7 iPS clones. e, Telomerase activity assay in DKC1
With TERT
mutant iPS lines (dash 5 background).
iPS
f, Telomere Southern blot in DKC1 del37L
c DKC1 del37L iPS 1 teratoma histology d PCR RFLP verification of DKC1 del37L fibroblasts and early passage iPS clones.
mutation g, Telomere Southern blot in DKC1 A386T
Wild type del37L fibroblasts and early passage iPS clones. kb,
Fib1 Fib2 Fib3 iPS1 iPS2 kilobases.
Xmnl – + – + – + – + – +

Retinal epithelium Muscle/bone Glandular epithelium

(ectoderm) (mesoderm) (endoderm)

e Telomerase activity assay f Telomere length analysis g

DKC1 del37L DKC1 A386T
2.5 kb 21
kb 21
2.0
A450 nm–690 nm

8.6
1.5 8.6
6.1
1.0 5.0 6.1
0.5 3.6 5.0

0 2.7 3.6
del37L del37L del37L A386T A386T
Fib iPS1 iPS2 Fib iPS2
Fib iPS1 iPS2 iPS4 iPS5 iPS6 iPS7 Fib iPS1 iPS2 iPS3
With TERT With TERT

clonal fingerprint as early passage cells, and reversion of the genetic of TERT alone results in telomere elongation in wild-type fibro-
mutation in DKC1 was excluded (Supplementary Fig. 5). These blasts13,14, whereas expression of both TERC and TERT is required
data show that even in cells carrying genetic lesions that reduce to restore telomere elongation in DKC1 mutant fibroblasts11 (Sup-
telomerase function, reprogramming restores telomere elongation plementary Fig. 6). We therefore investigated TERC levels in DC
and self-renewal. fibroblasts and iPS cells. By quantitative RT–PCR, we found that
Previous studies have shown that reduced TERC levels compromise TERC levels in DKC1 mutant fibroblasts were 10–15% of TERC levels
telomerase activity in DKC1 mutant fibroblasts11. Ectopic expression found in wild-type fibroblasts, consistent with previous reports10,11
a b Figure 2 | Telomere elongation in DKC1 mutant
MM Fib del37L iPS1 del37L iPS2 del37L cell line telomere
kb iPS cells. a, Telomere Southern blot of del37L
length by qPCR fibroblasts (Fib) and iPS clones 1 and 2 as a
8.6 6
7.4 function of passage. MM, molecular mass
5
Relative length

marker. b, Quantitative real-time PCR (qPCR)

6.1 4
analysis of telomere length in del37L fibroblasts
3 and iPS clones at indicated passages (p). Error
5.0
2 bars represent s.e.m. c, Quantitative fluorescence
4.2
1 in situ hybridization (qFISH) for telomere length
3.6 0 in del37L cells. a.f.u., arbitrary fluorescence units.
Fib p9 iPS1 p19 iPS2 p20 Inset, mean relative length values shown 6s.d.
2.7
Passage 6 12 14 16 19 5 11 13 15 18 20

c qFISH telomere length analysis 600

500 500
400 iPS1 p8 400 iPS1 p26
300 3.31 ± 1.76 5.37 ± 2.35
300
200 200
100 100
400
Frequency

Fib p9 0 0
300 0 1 3 5 7 9 11 13 15 17 0 1 3 5 7 9 11 13 15 17 19
5.18 ± 3.85
200
100
600
0 400
0 1 3 5 7 9 1113 15 17 19 500
Relative length (a.f.u.) 400 300 iPS2 p25
iPS2 p7
300 3.82 ± 2.53 6.75 ± 2.91
200
200
100 100

0 0
0 1 3 5 7 9 11 13 15 17 0 1 3 5 7 9 11 13 15 17 19

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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

(Fig. 3a). Relative to parental fibroblasts from two patients with of endogenous TERC levels, and provide a mechanism for telomere
different DKC1 mutations, we found TERC levels increased 6–8-fold elongation and indefinite self-renewal in DC iPS cell lines (Sup-
in the reprogrammed derivatives, approaching levels in normal fibro- plementary Fig. 6).
blasts (Fig. 3a). In normal iPS cells, we found that TERC levels were We found that human embryonic stem (ES) cells maintain elevated
approximately threefold higher than the fibroblasts from which they TERC levels similar to those found in wild-type iPS cells (Fig. 3a), and
were derived (Fig. 3a). We next examined a cell line from a patient that TERC levels attained in iPS cell lines derived from DKC1 mutant,
with autosomal dominant DC carrying a heterozygous 821 base pairs TERC1/2 and wild-type cells correlated with their respective telo-
(bp) deletion in the 39 region of the TERC locus (DCHSF1, refs 15, 16). merase activity (Fig. 3b). Furthermore, TERC knockdown in DC iPS
In these TERC1/2 fibroblasts TERC is limiting for telomere elonga- lines compromised telomere maintenance (Supplementary Fig. 8a).
tion, even in the presence of exogenous TERT16. We reprogrammed We found that the TERC locus is not amplified in DC iPS cells, and that
the TERC1/2 patient fibroblasts and obtained several independent iPS on differentiation of DC iPS cells, TERC expression reverted to the
lines that all showed characteristics of pluripotency and maintained pathologically low levels found in patient fibroblasts, and telomere
the mutant genotype (Supplementary Fig. 7). TERC1/2 iPS cells also length decreased (Supplementary Fig. 8b–d). Collectively, these results
showed a threefold induction of TERC relative to fibroblasts (Fig. 3a), show that TERC levels are increased by reprogramming, and are a
and displayed continuous self-renewal in contrast to the early dynamically regulated and reversible property of the pluripotent state.
senescence seen in the parental fibroblasts16. These data demonstrate TERC abundance is tightly regulated by several transcriptional and
that reprogramming of somatic cells is accompanied by upregulation post-transcriptional mechanisms17,18. To investigate transcriptional

a Quantitative RT–PCR for TERC b TRAP assay of iPS cells

3
Relative TERC level

Relative telomerase
2

activity ×103
2

1
1

0 0
Fib iPS Fib iPS Fib iPS Fib iPS hES del37L A386T TERC+/– Wild type
del37L A386T TERC+/– Wild type DKC1 mutant
c ChIP on TERC locus
BJ1 iPS 8 Anti-OCT4 hFib2 iPS
Anti-OCT4
Fold enrichment

4 Anti-GFP Anti-GFP
6

4
2
2

0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Anti-NANOG BJ1 iPS Anti-NANOG hFib2 iPS
Fold enrichment

8 Anti-GFP 16 Anti-GFP
6 12
4 8
2 4
0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
TERC
ARPM1
1 2 3 4 5 6 7 8
Human chromosome 3 821 bp deletion 1 kb

d Quantitative RT–PCR for DKC1 e

Dyskerin
4
Relative DKC1 level

Actin
Fib iPS1 iPS2 Fib iPS10 iPS12 Fib iPS1
3
DKC1 del37L TERC+/– Wild type
2 f
6 ChIP on DKC1 locus Anti-OCT4
Fold enrichment

5 Anti-NANOG
1 4
3
0 2
Fib iPS Fib iPS Fib iPS Fib iPS hES 1
del37L A386T TERC+/– Wild type 0
1 2 3 4
DKC1 mutant DKC1
Human chromosome X
1 23 4 1 kb

Figure 3 | Upregulation of TERC and DKC1 in iPS cells. a, qPCR deletion in TERC1/2 cells is indicated. GFP, green fluorescent protein.
measurement of TERC transcripts in DKC1 mutant, TERC1/2, and wild- d, qPCR measurement of DKC1 transcripts (as in a). e, Western blot of
type fibroblasts (Fib) and iPS cells, and human ES (hES) cells, normalized to dyskerin and actin in wild-type and DC fibroblasts and iPS clones. f, ChIP of
GAPDH and relative to wild-type fibroblasts. b, Quantitative TRAP assay in the DKC1 locus (see map) in wild-type iPS cells. All error bars represent
iPS cells. c, ChIP of the human TERC locus in WT iPS cells. The 821 bp s.e.m.
294
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

mechanisms of TERC upregulation, we performed chromatin immu- reprogramming, thereby accounting for increased telomerase activity
noprecipitation (ChIP) in human iPS cells, and detected enhanced in the pluripotent state and explaining the restoration of telomere
binding of OCT4 and NANOG in the TERC locus (Fig. 3c). These maintenance in DC iPS cells.
data were corroborated in murine ES cells, which likewise showed In an autosomal dominant DC patient carrying an 821 bp deletion
enhanced binding of OCT4 and NANOG at the TERC locus in the 39 region of TERC, earlier studies have concluded that the hap-
(Supplementary Fig. 9). However, we were unable to detect increased loinsufficient phenotype is explained by impaired post-transcriptional
levels of nascent TERC transcription by nuclear run-off assay in iPS accumulation of TERC RNA and reduced assembly of the truncated
cells versus fibroblasts (Supplementary Fig. 10), indicating that TERC RNA into telomerase holoenzyme20–22 (Supplementary Fig. 12).
OCT4 and NANOG may affect transcriptional competence rather Our ChIP data indicated OCT4 and NANOG binding in a region
than transcriptional rate of the TERC locus in pluripotent cells. downstream of TERC that overlaps the 821 bp deletion (Fig. 3c).
Additional mechanisms are required to explain the increased Moreover, we found that part of this region is predicted to have regu-
steady-state levels of TERC. To investigate post-transcriptional latory potential on the basis of comparative genomic sequence align-
mechanisms of TERC regulation, we determined DKC1 levels, which ment (Supplementary Fig. 13a). We proposed that loss of this region
correlate with TERC levels in human cancer cells19. In all normal and might compromise transcriptional activity of the deleted allele. We
DC iPS lines tested, we found an increase in DKC1 transcript levels used two distinct allele-specific methods to analyse the chromatin
and dyskerin protein relative to the fibroblasts (Figs 3d and e). configuration in TERC1/2 iPS cells, and in both cases found the
Moreover, we found that human ES cells, like iPS cells, maintain mutant TERC allele to be transcriptionally inactive (Fig. 4). First,
higher levels of DKC1 (Fig. 3d), that DKC1 levels decreased with allele-specific ChIP showed a striking abrogation of H3K4me3 marks
differentiation of iPS cells into embryoid bodies (Supplementary and RNA polymerase II binding on the deleted allele (Fig. 4a and
Fig. 11a), and that DKC1 knockdown caused a reduction in TERC Supplementary Fig. 13b, c). Second, allele-specific DNase I hypersensi-
levels and compromised cellular viability (Supplementary Fig. 11b, c). tivity analysis showed pronounced nuclease accessibility at the TERC
ChIP showed binding of OCT4 and NANOG to the DKC1 promoter promoter in normal cells (Fig. 4b) and on the normal allele in
in pluripotent cells (Fig. 3f), and infection of fibroblasts with retro- TERC1/2 cells, but complete loss of promoter hypersensitivity on
viruses encoding the four reprogramming factors increased DKC1 the mutant TERC allele (Fig. 4c, d). Collectively, these results identify
levels (Supplementary Fig. 11d). These data establish that several telo- a cis-element in the 39 region of the TERC locus that seems essential
merase components (TERT, TERC, DKC1) are upregulated after for formation of a transcriptionally active chromatin structure, and

a Allele-specific ChIP in TERC+/– iPS cells b TERC DNAse I HS analysis in WT

iPS cells
0 DNase
Anti-H3K4me3 Anti-RNA pol II kb
5,000 14 9.0
12 WT allele
Fold enrichment

4,000
10 Mutant allele
3,000 8 4.5
6 II
2,000
4
1,000
2
2.6
0 0 I
Primers F/R1 F/R2 F/R1 F/R2
TERC

R2 R1 F
1.0

821 bp deletion HS sites II I

ARPM1

1 kb TERC Probe
N N

c Allele-specific DNase I HS in TERC+/– iPS cells d Allele-specific DNase I HS

in TERC+/– iPS cells
0 DNase kb 0
kb
3.8 3.6 del 0 DNase
WT kb
3.7
WT
2.9
del

2.0
IWT
1.2 IWT
1.0 X Idel

1.2 X Idel
0.6 WT
Probe 1 Probe 2
HS site I HS site I
TERC ARPM1 TERC
Probe 2 1 1 kb
Probe
1 kb P
B B B P
821 bp deletion 821 bp deletion

Figure 4 | Transcriptional silencing is associated with a 39 deletion in the HS analysis probing TERC locus Bsu36I (B) fragments in TERC1/2 iPS cells
TERC locus. a, ChIP using TERC1/2 iPS cells with allele-specific primers (F/ (probe 1 assesses wild-type (WT) parent allele and HS site I (IWT); probe 2
R1 for wild type; F/R2 for mutant). Error bars represent s.e.m. b, DNase I assesses both parent alleles (WT and del) but HS I only on the deleted allele
hypersensitivity (HS) analysis probing a TERC locus NdeI (N) fragment in (Idel)). d, As in c, with PflMI (P) digestion and indicated probe for
wild-type iPS cells, showing two discrete HS sites. c, Allele-specific DNase I simultaneous assessment of both WT and deleted parent alleles and HS sites.
295
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

indicate that mutations in this region can cause haplo-insufficiency in 10. Mitchell, J. R., Wood, E. & Collins, K. A telomerase component is defective in the
human disease dyskeratosis congenita. Nature 402, 551–555 (1999).
autosomal dominant DC by diminishing TERC transcription (Sup- 11. Wong, J. M. & Collins, K. Telomerase RNA level limits telomere maintenance in
plementary Fig. 12). X-linked dyskeratosis congenita. Genes Dev. 20, 2848–2858 (2006).
By reprogramming somatic cells from patients with the human 12. Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res.
disease dyskeratosis congenita, we have discovered novel mechanisms 30, e47 (2002).
of regulation of telomerase activity in the pluripotent state, thus 13. Bodnar, A. G. et al. Extension of life-span by introduction of telomerase into
normal human cells. Science 279, 349–352 (1998).
illustrating the value of disease-specific iPS cells for basic and trans- 14. Vaziri, H. & Benchimol, S. Reconstitution of telomerase activity in normal human
lational discovery. Moreover, we have shown that the RNA com- cells leads to elongation of telomeres and extended replicative life span. Curr. Biol.
ponent of telomerase is upregulated in the pluripotent state to a degree 8, 279–282 (1998).
sufficient to overcome limitations to telomere maintenance in 15. Vulliamy, T. et al. The RNA component of telomerase is mutated in autosomal
dominant dyskeratosis congenita. Nature 413, 432–435 (2001).
X-linked and autosomal dominant DC. Drugs that activate the 16. Westin, E. R. et al. Telomere restoration and extension of proliferative lifespan in
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might serve as therapeutic agents for bone marrow failure. Moreover, 18. Yi, X., Tesmer, V. M., Savre-Train, I., Shay, J. W. & Wright, W. E. Both
we speculate that in DC patients certain cell types such as germ cells transcriptional and posttranscriptional mechanisms regulate human telomerase
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with pluripotent cells, may also upregulate TERC to permit germline 19. Montanaro, L. et al. Dyskerin expression influences the level of ribosomal RNA
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20. Fu, D. & Collins, K. Distinct biogenesis pathways for human telomerase RNA and
METHODS SUMMARY H/ACA small nucleolar RNAs. Mol. Cell 11, 1361–1372 (2003).
Fibroblast lines included GM01774 (DKC1 del37L)11, AG04646 (DKC1 A386T)11, 21. Marrone, A., Stevens, D., Vulliamy, T., Dokal, I. & Mason, P. J. Heterozygous
DCHSF1 (TERC1/2 821 bp deletion)16 and NHSF2 (wild type)16. The derivation, telomerase RNA mutations found in dyskeratosis congenita and aplastic anemia
characterization, culture and differentiation of iPS cells was performed as reduce telomerase activity via haploinsufficiency. Blood 104, 3936–3942 (2004).
described5. Verification of mutations in GM01774 was as described9. Telomere 22. Trahan, C. & Dragon, F. Dyskeratosis congenita mutations in the H/ACA domain
of human telomerase RNA affect its assembly into a pre-RNP. RNA 15, 235–243
length analysis was performed with the TeloTAGGG Telomere Length Assay kit
(2009).
(Roche). iPS cells were cultured feeder-free on Matrigel (Becton Dickinson) for 23. Broccoli, D., Young, J. W. & de Lange, T. Telomerase activity in normal and
telomerase and telomere quantitative real-time PCR (qPCR) assays. Telomere malignant hematopoietic cells. Proc. Natl Acad. Sci. USA 92, 9082–9086 (1995).
qFISH and qPCR were performed as described12,28. Telomerase activity assays 24. Chiu, C. P. et al. Differential expression of telomerase activity in hematopoietic
were performed using the TeloTAGGG Telomerase PCR ELISA and PCR progenitors from adult human bone marrow. Stem Cells 14, 239–248 (1996).
ELISAPLUS kits (Roche). Detection of TERT, OCT4, SOX2, NANOG, and b-actin 25. Hiyama, K. et al. Activation of telomerase in human lymphocytes and
transcripts by RT–PCR was as described5. TERC and DKC1 transcript levels were hematopoietic progenitor cells. J. Immunol. 155, 3711–3715 (1995).
measured using qPCR with quantification normalized to GAPDH. Primer 26. Allsopp, R. C., Morin, G. B., DePinho, R., Harley, C. B. & Weissman, I. L. Telomerase
sequences are provided in Methods. Chromatin immunoprecipitation was per- is required to slow telomere shortening and extend replicative lifespan of HSCs
during serial transplantation. Blood 102, 517–520 (2003).
formed on BJ1, HFib2, NHSF2, and DCHSF1 iPS cells as described29. Antibodies
27. Kirwan, M. et al. Exogenous TERC alone can enhance proliferative potential,
and primers sequences are provided in Methods. Western blot was performed on telomerase activity and telomere length in lymphocytes from dyskeratosis
total cellular lysates using antibodies against human dyskerin and actin (Santa congenita patients. Br. J. Haematol. 144, 771–781 (2009).
Cruz). DNase I hypersensitivity analysis was performed as described30. Primer 28. Liu, L. et al. Telomere lengthening early in development. Nature Cell Biol. 9,
sequences for probes are provided in Methods. 1436–1441 (2007).
29. Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency
Full Methods and any associated references are available in the online version of in mouse embryonic stem cells. Nature Genet. 38, 431–440 (2006).
the paper at www.nature.com/nature. 30. Agarwal, S. & Rao, A. Modulation of chromatin structure regulates cytokine gene
expression during T cell differentiation. Immunity 9, 765–775 (1998).
Received 5 November; accepted 18 December 2009.
Published online 17 February 2010. Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
1. Kirwan, M. & Dokal, I. Dyskeratosis congenita: a genetic disorder of many faces.
Clin. Genet. 73, 103–112 (2008). Acknowledgements This work was funded by grants from the National Institutes
2. Mason, P. J., Wilson, D. B. & Bessler, M. Dyskeratosis congenita–a disease of of Health (NIH) and the Manton Center for Orphan Disease Research (G.Q.D.);
dysfunctional telomere maintenance. Curr. Mol. Med. 5, 159–170 (2005). NIH K08HL089150, Amy Clare Potter Fellowship and Manton Center for Orphan
3. Calado, R. T. & Young, N. S. Telomere maintenance and human bone marrow Disease Research (S.A.); the Agency of Science, Technology and Research and the
failure. Blood 111, 4446–4455 (2008). Institute of Medical Biology, Singapore (Y.-H.L.); NIH R01AG0227388 (F.D.G. and
4. Park, I. H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 A.J.K.); and the James and Esther King Biomedical Research Program and MOST
(2008). 973 project (2009CB941000) (D.L.K and L.L).
5. Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with Author Contributions S.A. performed project planning, experimental work, data
defined factors. Nature 451, 141–146 (2008). interpretation and preparation of the manuscript. Y.-H.L., I.-H.P., J.H., E.M.M.,
6. Takahashi, K. et al. Induction of pluripotent stem cells from adult human J.D.M., R.M.R., M.O., H.H. and S.L. performed experimental work. H.-H.N., F.D.G.,
fibroblasts by defined factors. Cell 131, 861–872 (2007). D.L.K., A.J.K., L.L. and G.Q.D. participated in project planning, data interpretation
7. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. and preparation of the manuscript.
Science 318, 1917–1920 (2007).
8. Marion, R. M. et al. Telomeres acquire embryonic stem cell characteristics in Author Information Reprints and permissions information is available at
induced pluripotent stem cells. Cell Stem Cell 4, 141–154 (2009). www.nature.com/reprints. The authors declare competing financial interests:
9. Heiss, N. S. et al. X-linked dyskeratosis congenita is caused by mutations in a details accompany the full-text HTML version of the paper at www.nature.com/
highly conserved gene with putative nucleolar functions. Nature Genet. 19, 32–38 nature. Correspondence and requests for materials should be addressed to G.Q.D.
(1998). (george.daley@childrens.harvard.edu).

296
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08792

METHODS 3S (59-AGAGGTACTGTTTACGGAGCGTTCAGC-39), 3AS (59-AATGCGAT

TTGCTGGCTCGGCCAGTA-39), 4S (59-CCGAGTTGCAAGAAAGTTCTAGAGG
Cell lines and iPS derivation, characterization and culture. Fibroblast lines and
CC-39), 4AS (59-GTAAGGCCAAATGTATGGGTCCCATG-39). Primers spanning
iPS lines for BJ1 (ATCC); GM02416, GM04569, AG20446, GM04281, GM01390
the mouse TERC locus, Supplementary Fig. 9: 1S (59-CCTCCCTCCACTCC
(Coriell Cell Repository); and DH1cf subclones 16, 32 and 34 have been
CATACAGAAGG-39), 1AS (59-CCAAGTCTCTTTGTCCCAACTCCTC-39), 2S
described4,5,31. GM01774 (DKC1 del37L)11, AG04646 (DKC1 A386T)11, and
(59-CCAAATGTGACTCAGTCAATGGCACTCC-39), 2AS (59-GGAGGAAGTTT
GM874 (ALT)32 were also obtained from Coriell Cell Repository. DCHSF1
GGGTTGTGCTCTGTA-39), 3S (59-ACCACATGTGCATGTTCCTGGAGCT-39),
(TERC1/2 821 bp deletion) and age-matched wild-type control cells NHSF2
3AS (59-GCCTCTTCAGTAGCCATCATGCCTAATG-39), 4S (59-TATGCCACTA
have been described16. Derivation, characterization, culture, and differentiation
TGTGGCTTCCACAGAAGG-39), 4AS (59-CCTTTCCTTCCCTCCCTTCCGGT
conditions of iPS cells have been described31.
AC-39), 5S (59-CATAGGGAGCTTCATCAGACTCAGTG-39), 5AS (59-GGCGAC
Retroviral and lentiviral vectors. Retroviruses encoding TERT, OCT4, SOX2,
ATTTCTCAACCAGAAGACAG-39), 6S (59-TCCTTGGCTTCGGTGATGTTGAG
KLF4 and c-MYC were as described5. The DKC1 retroviral vector was created by
TTC-39), 6AS (59-CTAAGCCGGCACTCCTTACAAGGGA-39), 7S (59-TAAGACA
cloning a full-length DKC1 cDNA (OriGene) into pMIG-W (Addgene) and
CCGAACACGGGGACCAGT-39), 7AS (59-AACGTCAGCGCAGGAGCTCCAGG
retrovirus was produced as described31. The TERC lentiviral vector was created TT-39), 8S (59-GAGAAAAACAGCGGGCGGAGAACAA-39), 8AS (59-ACTGGC
by replacing GFP pHIV7/SF-GFP33 (a gift from J.-K. Yee) with cherry fluorescent TAGGAAGAGTGGGGAAGCG-39), 9S (59-CCCATCCCTTCCACACGTCAGTT
protein and inserting a U3 promoter plus TERC from pBABE-U3-TER11 (a gift CT-39), 9AS (59-GCAGTAGTATCTCTCGGGTTGTCCTTCA-39), 10S (59-GAACT
from K. Collins) at an upstream site. Virus was produced by the University of TCACAATGACCATGAGCAGTCCC-39), 10AS (59-CCCAGAGGACATTCCTT
Iowa Gene Transfer Vector Core. CTAGGTTCTCTGT-39), 11S (59-CCAAGGCTTTGTCACTGACTGCTCA-39),
Telomere length and telomerase assays. Using the TeloTAGGG Telomere 11AS (59-GTAGCAAGCACAGCCACCGGTGTCT-39), 12S (59-GTTGGTAAGAT
Length Assay kit (Roche), 1–2 mg of DNA for each sample was digested and GAAGCCATCAGCCTC-39), 12AS (59-GGATAGTTGCCAGGCCACAGATCA
subjected to Southern analysis, and determination of mean terminal restriction AT-39), 13S (59-CTCTTTCTTGAATTGGACCGTGCAGG-39), 13AS (59-GCTTC
fragment (TRF) length34 was performed according to manufacturer’s instruc- TGGTCAGGCAGCTCATCTAAT-39).
tions. Briefly, autoradiographs exposed in the linear range of the hybridization Western analysis. Total cellular lysates were subjected to SDS–PAGE and west-
signal for the lane(s) of interest were scanned with a densitometer. A volume ern transfer to PVDF membranes using standard procedures and analysed using
array consisting of 30–40 quadrants was generated across the entire lane for each antibodies against human dyskerin (Santa Cruz; sc-48974) and actin (Santa
sample from 1–24 kb. After background subtraction, absorbance (Ai) was Cruz; sc-1615).
obtained in each of these quadrants corresponding to different DNA lengths DNase I hypersensitivity (HS) analysis. DNase I HS analysis was performed as
(Li). Mean TRF was defined as S(Ai)/S( Ai/Li) for each sample. To determine described30. Briefly, nuclei were isolated from 5 3 107–1 3 108 iPS cells and
relative total telomeric DNA signal34 in iPS cells versus fibroblasts, the S(Ai) was aliquots were subjected to treatment with various concentrations of DNase I
obtained for each lane and normalized by comparison to ethidium bromide (Worthington), followed by lysis and phenol/chloroform extraction of DNA.
staining of the gel or hybridization to a probe targeting a diploid somatic locus. Approximately 25–50 mg of DNA was digested for each lane and subjected
The normalized, summated absorbance value for each iPS sample was divided by to Southern analysis. Hybridization of PCR generated probes labelled with
that of the corresponding fibroblast line, yielding a relative signal ratio corres- 32
P-dCTP (Ready-to-go DNA Labelling kit (-dCTP); G.E. Healthcare) was per-
ponding to the quantitative difference in total telomeric DNA. Telomere quanti- formed using Rapid-Hyb buffer (G.E. Healthcare) according to manufacturer’s
tative fluorescence in situ hybridization (qFISH) and quantitative real-time PCR instructions. Primers for probes were in Figure 4b, TERCNdeIF (59-TGT
(qPCR) were performed as described12,28 in a blinded fashion. For telomere GATACAGGCCTGAGGAA-39) and TERCNdeIR (59-ATGCTTGCCTGGA
qPCR and telomerase assays, iPS cells were cultured feeder-free on Matrigel TATCTGC-39); in Figure 4c TERCBsu1F (59-CAGGACTCGGCTCACACA
(Becton Dickinson). Telomerase activity assays were performed using the T-39), TERCBsu1R (59-CGATGACCATTAAAGGAACACA-39), TERCBsu2F
TeloTAGGG Telomerase PCR ELISA and PCR ELISAPLUS kits (Roche). (59-GATCATCTGGGGGTAGTTGC-39) and TERCBsu2R (59-AAACTGAGGC
Quantitative real-time PCR (RT–PCR). Quantitative RT–PCR was performed TTACTGAAGCTGA-39). TERCBsu2F/2R probe was used in Fig. 4d.
using SYBR Green Master Mix (Stratagene). Several independent biological Verifications of mutations. Verification of mutations was performed by amp-
samples were used where available for RNA isolation using the RNeasy kit lifying genomic DNA: for GM01774 (DKC1 del37L) as described9, using primers
(Qiagen), and cDNA was prepared using the Superscript III First Strand XAP101F25 (59-ATTGCCAGAAGAAGATGTAGCC-39) and XAP101R27
Synthesis kit (Invitrogen). Detection of endogenous TERT, OCT4, SOX2 and (59-TCTCTTCAGAGGATTTGAACC-39) followed by XmnI digestion; for
b-actin transcripts by RT–PCR was as described5. TERC and DKC1 levels were DCHSF1 (family DCR101)15 as described, using primers HTRF2 (59-CCTG
measured using qPCR, with quantification normalized to GAPDH, using primer CCGCCTTCCACCGTTCATT-39) and HTRR3 (59-CATTACCAGCAACAGTG
pairs TRC3F/3R for TERC and GAPDH0.45F/0.45R for GAPDH, as described35, GACTCT-39); for AG04646 (DKC1 A386T)11 using primers 4646F (59-CC
and DKC1F1: (59-ACAGGGTGAAGAGTTCTGGCACAT-39) and DKC1R: AGGGAGGAACCTTGTTCT-39) and 4646R (59-ACCTCCATGCTCACCTG
(59-TGAAGGTGAGGCTTCCCAACTCAA-39) for DKC1. TTC-39) followed by BstNI digestion. The primers amplify a 360 bp product
Chromatin immunoprecipitation (ChIP). ChIP assay was performed on BJ131, flanking the mutation G1156A, resulting in loss of one of two BstNI sites.
HFib231, NHSF2 and DCHSF1 iPS cells as described29, using anti-OCT4 (Santa Karyotype and DNA fingerprinting. Karyotyping and DNA fingerprinting were
Cruz; sc-8628), anti-NANOG (R&D; AF1997), anti-H3K4me3 (Abcam; ab8580), performed in a blinded fashion by Cell Line Genetics.
anti-RNA polymerase II (Abcam; ab817), anti-H3K27me3 (Millipore; 07-449) Clonal integration analysis by Southern blot. OCT4, SOX2, KLF4, and c-MYC-
and anti-GFP (Santa Cruz; sc-9996). ChIP assay was performed on murine E14 ES encoding MSCV-based retroviruses, each containing an IRES-GFP cassette and a
cells using anti-OCT4 and anti-GFP antibodies above, and anti-NANOG (Cosmo single NcoI restriction site, were used for reprogramming31. Genomic DNA from
Bio; REC-RCAB0002P-F). iPS cells (10 mg) was digested with NcoI and subjected to Southern blot analysis
Primers spanning the human TERC locus, Figure 3c: 1S (59-CAGCACTTTGT using a GFP probe to provide a viral integration fingerprint for each clone.
TCTGATGAAGCCATCCC-39), 1AS (59-GGGTCAAGGGTATCAATGCAGAG TERC and DKC1 shRNA knockdown. A duplex oligonucleotide containing an
GCTGAATAC-39), 2S (59-GCCTATGAATAGCACTGGGGTAGGT-39), 2AS shRNA targeting the template sequence (in bold) of human TERC36 (59-
(59-GCACCAAGGAGTTCAACTTGACCTCA-39), 3S (59-GTAGGCCTCAAAC GTCTAACCCTAACTGAGAACTCGAGTTCTCAGTTAGGGTTAGACTTTT-
TGGTAGGATGGT-39), 3AS (59-CCTCCTAGCTTAGGTTGTTGATAGC-39), T-39) was cloned into the pLKO.1-puro vector to create pLKO-SHTR. Several
4S (59-ACCACTCCTCCCTTACACTTTGTATGACGG-39), 4AS (59-GGGCTT pLKO.1-puro-based shRNA lentiviral constructs against DKC1 were obtained
CCTTTGTAAGGTCTGGAGTTGGTT-39), 5S (59-CTGGTCGAGATCTACC from Open Biosystems, with the most effective sequence (59-GCTC
TTGGGAGAA-39), 5AS (59-AGACGTGAAGGCACCTCCAAAGTCG-39), 6S AGTGAAATGCTGTAGAA-39) targeting the 39UTR. pLKO.1-puro-control
(59-AACCCCGGCTCACTGCCCATTCATTTT-39), 6AS (59-ATGCAGTTCGC consisted of scrambled shRNA sequences. Lentivirus containing knockdown
TTTCCTGTTGGTGG-39), 7S (59-TTTCTATCCTCTGCAGACCAGACGC-39), constructs was produced by co-transfection of 293T cells with pLKO.1 con-
7AS (59-CGAGACAAGATTCTGCTGTAGTCAG-39), 8S (59-TTCCTCAGGCCT structs, psPAX2 and pMD2.G using FuGENE. Supernatants were harvested,
GTATCACATTTCA-39), 8AS (59-GGCCAAGAAACCCGATTGTCTAGAGA-39); filtered and frozen in aliquots on days 3–4 following transfection. Titres and
Figure 4a and Supplementary Fig. 13: DCHSF1 F (59-GCGAAGAGTTGG knockdown efficiency was determined by infection of 293T cells for 8–12 h using
GCTCTGTCA-39), DCHSF1 R1 (59-CACCAACAGGAAAGCGAACTGCAT-39), varying quantities of viral supernatant and 10 mg ml21 of protamine sulphate.
DCHSF1 R2 (59-TCATCAGGATTCAGGCTATCACCC-39). Primers spanning the After 24–36 h, infected cells were selected with 2 mg ml21 puromycin. RNA was
human DKC1 locus, Figure 3e: 1S (59-TAAGAGAACTGAGAAGGCTGCG-39), 1AS harvested after 36–48 h of selection and subjected to quantitative RT–PCR to
(59-ATGGCCACTCATGATGGTTTGGGATC-39), 2S (59-AGCAAAGGCCTTTCA determine knockdown efficiency of DKC1 and TERC. Lentivirus constructs for
ACCTCTCCGAGC-39), 2AS (59-TAGTTGCTTCACCTCCGGGTTTAGCTC-39), knockdown of DKC1 or TERC were introduced into iPS cells as follows. iPS

©2010 Macmillan Publishers Limited. All rights reserved

doi:10.1038/nature08792

cultures were harvested as single cells using Accutase (Stem Cell Technologies), containing full-length human TERC cloned into pUC19, a 439 bp fragment
and 5 3 104 cells per well were plated in a 6-well plate on Matrigel (Becton consisting of exon 3 of human b-actin cloned into pUC19, or pUC19 vector
Dickinson) in mTESR medium (Stem Cell Technologies) in the presence of alone. Hybridization was performed using Rapid-Hyb buffer (G.E. Healthcare)
10 mM Y-27632 (Sigma)37. After 24 h viral supernatants were added in various with pre-hybridization for 12–16 h followed by hybridization for 48 h.
quantities in the presence of 10 mg ml21 protamine sulphate in mTESR medium
for 6 h. After 48 h, infected cells were selected in 0.15 mg ml21 puromycin and 31. Park, I. H., Lerou, P. H., Zhao, R., Huo, H. & Daley, G. Q. Generation of human-
maintained thereafter in mTESR medium with Matrigel or in conventional hES induced pluripotent stem cells. Nature Protocols 3, 1180–1186 (2008).
32. Bryan, T. M., Englezou, A., Gupta, J., Bacchetti, S. & Reddel, R. R. Telomere
medium on DR4 puromycin-resistant feeder cells.
elongation in immortal human cells without detectable telomerase activity. EMBO
TERC copy number determination. TERC locus copy number in iPS relative to J. 14, 4240–4248 (1995).
parental fibroblasts was determined by performing qPCR on genomic DNA from 33. Yam, P. Y. et al. Design of HIV vectors for efficient gene delivery into human
DCHSF1 (TERC1/2) and del37L DKC1 mutant fibroblasts and their iPS deriva- hematopoietic cells. Mol. Ther. 5, 479–484 (2002).
tives. Primers for the TERC locus were TRC3F and TRC3R35 and for the b-actin 34. Harley, C. B., Futcher, A. B. & Greider, C. W. Telomeres shorten during ageing of
locus were ActinF (59- AACGGCAGAAGAGAGAACCAGTGA-39) and ActinR human fibroblasts. Nature 345, 458–460 (1990).
(59-TTCTACAATGAGCTGCGTGTGGCT-39). Amplification of the TERC locus 35. Atkinson, S. P., Hoare, S. F., Glasspool, R. M. & Keith, W. N. Lack of telomerase
was normalized to that of the b-actin locus for each sample, and results are gene expression in alternative lengthening of telomere cells is associated with
displayed as a ratio of normalized TERC signal for iPS cells relative to the parent chromatin remodeling of the hTR and hTERT gene promoters. Cancer Res. 65,
fibroblast. Error bars denote standard error from two biological replicates. 7585–7590 (2005).
36. Li, S. et al. Rapid inhibition of cancer cell growth induced by lentiviral delivery and
Nuclear runoff transcription assay. Nuclei from 5 3 107 to 1 3 108 wild-type
expression of mutant-template telomerase RNA and anti-telomerase short-
(NHSF2) fibroblasts or derivative iPS cells (NHSF2 iPS clone 1) were isolated interfering RNA. Cancer Res. 64, 4833–4840 (2004).
and 32P-labelled runoff transcripts were generated as described38. Labelled nas- 37. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human
cent RNA transcripts were purified using TRIzol (Invitrogen) and hybridization embryonic stem cells. Nature Biotechnol. 25, 681–686 (2007).
was performed using 1–2 3 106 c.p.m. ml21. Membranes containing hybridiza- 38. Greenberg, M. E. & Bender, T. P. in Current Protocols in Molecular Biology Ch. 4, Unit
tion targets were prepared by slot-blot of 5 mg linearized plasmid DNA targets 4.10 (Wiley, 2007).

©2010 Macmillan Publishers Limited. All rights reserved

Vol 464 | 11 March 2010 | doi:10.1038/nature08783

LETTERS
The cells and peripheral representation of sodium
taste in mice
Jayaram Chandrashekar1{, Christina Kuhn2*, Yuki Oka1*{, David A. Yarmolinsky1{, Edith Hummler3,
Nicholas J. P. Ryba2 & Charles S. Zuker1{

Salt taste in mammals can trigger two divergent behavioural stimulation. In essence, TRCs in fungiform papillae were loaded
responses. In general, concentrated saline solutions elicit robust with the calcium-sensitive dye calcium green23 in vivo, and then were
behavioural aversion, whereas low concentrations of NaCl are stimulated and imaged, ex vivo, with a regime that either preferentially
typically attractive, particularly after sodium depletion1–5. Notably, activated the low-concentration pathway (100 mM NaCl), or acti-
the attractive salt pathway is selectively responsive to sodium and vated both the high- and low-concentration pathways (500 mM
inhibited by amiloride, whereas the aversive one functions as a non- NaCl). To separate the contribution of each of the two salt-sensing
selective detector for a wide range of salts1–3,6–9. Because amiloride systems at high-stimulus concentrations, we examined the salt res-
is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC ponses in the presence and absence of 10 mM amiloride (Fig. 1).
has been proposed to function as a component of the salt-taste- Receptor cells that are only activated by high concentrations of salt
receptor system1,3,6–14. Previously, we showed that four of the five also respond to a wide range of non-sodium salts (for example, from
basic taste qualities—sweet, sour, bitter and umami—are mediated KCl to N-methyl-D-glucamine (NMDG)-Cl; Fig. 1 and Supplemen-
by separate taste-receptor cells (TRCs) each tuned to a single taste tary Fig. 1), and their activity is unaffected by the presence of amiloride
modality, and wired to elicit stereotypical behavioural responses5,15–18. (Fig. 1b, c). In contrast, low-concentrations of NaCl activate a com-
Here we show that sodium sensing is also mediated by a dedicated pletely separate population of TRCs; these cells do not respond to
population of TRCs. These taste cells express the epithelial sodium non-sodium salts (Fig. 1a, c, see also Supplementary Fig. 1), and their
channel ENaC19,20, and mediate behavioural attraction to NaCl. We responses are blocked by amiloride (Fig. 1c). These results demon-
genetically engineered mice lacking ENaCa in TRCs, and produced strate the presence of two anatomically distinct salt-sensing systems,
animals exhibiting a complete loss of salt attraction and sodium taste and accordingly suggest that the appetitive and aversive behaviours
responses. Together, these studies substantiate independent cellular are mediated by non-overlapping populations of TRCs. As the TRCs
substrates for all five basic taste qualities, and validate the essential activated by low concentrations of NaCl are highly selective for
role of ENaC for sodium taste in mice. sodium salts, we consider them to be the dedicated sodium-sensing
Sodium is the major cation of extracellular fluids and an essential system and thus are the subject of this study.
component of every fluid compartment in the body. It is therefore not Because amiloride is an inhibitor of the epithelial sodium channel
surprising that animals have evolved dedicated salt-sensing systems, (ENaC), ENaC has been proposed to be a potential component of the
including prominent detectors in the taste system1–3. These salt- salt-taste-receptor system1,6,8,10–12,14. The ENaC channel is made up of
sensitive receptors are crucial for the acceptance of low concentrations three subunits (a, b and c), and has an important role in regulating
of sodium (for example, to satisfy the ‘salt appetite’)1,3 while simulta- trans-epithelial transport of Na1 in a wide range of tissues, including
neously serving as a warning mechanism against hyper-salinity2,3, thus kidneys, airway cells of the lung, epithelial skin cells, and the ducts of
helping to maintain ion and water homeostasis. For humans, the ‘taste salivary and sweat glands19,20. Although the knockout of any ENaC
for salt’ also has direct bearing on excessive Na1-consumption, which subunit is sufficient to completely abolish ENaC function20, conven-
is believed to be a significant dietary risk factor in hypertension, par- tional ENaC knockouts die within a few days of birth20, precluding
ticularly in the developed world21. In mice, the low-concentration, their use in physiological and behavioural studies of taste.
and behaviourally ‘attractive’ salt-taste pathway has three salient To examine the role of ENaCs in the taste system, we used a floxed
properties: it is activated at NaCl concentrations as low as 10 mM, it ENaCa conditional knockout strategy (Scnn1aflox)24. In essence, we
is highly selective for sodium versus other cations, and it is blocked by generated animals in which ENaC function was selectively eliminated
lingual application of the ion-channel inhibitor amiloride8,9,14,22. The in all differentiated TRCs by using the cytokeratin19 gene—a marker
high-concentration (aversive) pathway, conversely, begins to be sig- for all mature taste cells (see Supplementary Fig. 2) to drive the
nificant only at concentrations greater than 150 mM NaCl, it is non- expression of Cre-recombinase in the taste system. To investigate
selective for sodium (that is, other salts are equally effective), and it is the taste responses of the conditional ENaCa knockout mice, we
amiloride-insensitive9,14,22. recorded tastant-induced action potentials from nerves innervating
To explore the cellular basis for the taste of NaCl (that is, determine the taste cells of the tongue; this physiological assay monitors the
whether the distinct physiological and behavioural responses are activity of the taste system at the periphery and provides a reliable
mediated by the same or separate TRCs), we developed a new pre- measure of TRC function9,18,25. In wild-type mice, NaCl elicits a dose-
paration that allows functional imaging of TRCs in response to salt dependent increase in action potentials in the chorda tympani nerve,
1
Howard Hughes Medical Institute and Departments of Neurobiology and Neurosciences, University of California at San Diego, La Jolla, California 92093-0649, USA. 2National
Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA. 3Pharmacology and Toxicology Department, Faculty of Biology and
Medicine, University of Lausanne, CH-1005 Lausanne, Switzerland. {Present addresses: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147,
USA (J.C.); Departments of Biochemistry and Molecular Biophysics and of Neuroscience, Howard Hughes Medical Institute, Columbia College of Physicians and Surgeons, Columbia
University, New York, New York 10032, USA (Y.O., D.A.Y., C.S.Z.).
*These authors contributed equally to this work.
297
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

a c 9

NaCl NaCl KCl

ΔF/F (%)
[Low] (sodium selective) 100 mM 500 mM 500 mM
5
[Low]

ΔF/F (%)
0
9

ΔF/F (%)
0

[High]
0
6
1%

ΔF/F (%)
2s
[High]
[Low]

0
NaCl NaCl KCl
500 mM 500 mM 500 mM
b NaCl NaCl KCl + amiloride
100 mM 500 mM 500 mM
[High] (non-selective)

5
ΔF/F (%)

[Low] cells 1%

0
[High] cells

1%
1% 2s
2s

Figure 1 | Two classes of TRCs mediate distinct salt taste responses. pseudo-coloured as depicted. b, A different population of TRCs (labelled as
Fungiform taste buds loaded with the activity sensor calcium green respond [high]) are activated only at increased NaCl concentrations (500 mM) and
with high selectivity and specificity to different concentrations of salt. a, A are also stimulated by KCl; shown are individual traces for three different
unique subset of TRCs (labelled as [low]) respond to low concentrations of TRCs. c, Amiloride selectively blocks [low] responses but has no effect on
sodium chloride (100 mM) as well as higher concentrations (500 mM) but [high] responses; shown are individual traces for four different TRCs; the
not to other salts (KCl). Shown below the imaging data are individual traces duration of tastant application is denoted by black bars. See Supplementary
from four different TRCs depicting the kinetic and amplitude changes in Fig. 1 for a diagram of the preparation, quantifications and responses to
intracellular calcium levels after salt stimulation; calcium changes were other salts.

with a physiological response threshold of approximately 10 mM tastants are indistinguishable from control animals (Fig. 3d). These
(Fig. 2). Loss of ENaCa in the taste system does not affect responses results validate ENaC as the mammalian taste receptor responsible
to four of the five basic taste qualities: sweet, bitter, umami and sour for behavioural acceptance of (and attraction to) NaCl.
stimuli (Fig. 2d and Supplementary Fig. 3). In contrast, ENaCa knock- Our previous studies have shown that sweet, bitter, umami and
outs show a complete loss of the responses to low concentrations of sour tastes are mediated by independent populations of TRCs, each
NaCl (Fig. 2). As would be expected if ENaC was the sodium sensor, tuned to a single taste modality5,15,17,18. If this labelled-line logic of
these animals are also missing all amiloride-sensitivity in their NaCl taste coding at the periphery extends to all five basic taste modalities,
responses (Fig. 2). Importantly, the knockout mice retain all responses then sodium taste should also be mediated by a unique population of
to non-sodium salts (Fig. 2d and Supplementary Fig. 3). These results TRCs. Thus, we examined whether the amiloride-sensitive salt-
demonstrate that taste responses to salts are mediated by genetically sensing cells indeed define a sub-population of TRCs separate from
separable components. sweet, bitter, sour and umami TRCs. We engineered mice expressing
Animals ranging from simple invertebrates to mammals readily Cre-recombinase under the control of the ENaCa gene, and then
consume low to moderate concentrations of Na1, and actively seek it crossed them to a floxed green fluorescent protein (GFP) reporter
under conditions of salt deprivation1,2,4,26. Therefore, we carried out line (Z/EG). To validate the fidelity of Cre expression in ENaCa-
behavioural tests of salt consumption to examine the taste behaviour expressing cells, we analysed progeny from four independent Cre-
of the ENaCa conditional knockout animals both under conditions driver founders and confirmed proper GFP reporter expression in
of salt depletion (to test attraction) and under water deprivation (to the airway cells of the lung as well as in the kidney cortical collecting
test aversion)5. We reasoned that if ENaC encodes the principal duct cells and distal convoluted tubules—well-characterized sites of
sodium taste sensor, it should mediate all attraction to salt, and ENaCa expression19 (see Supplementary Fig. 4).
consequently, the knockout mice should have a total loss of beha- Co-labelling with the sweet/umami/bitter TRC marker, TrpM5 (ref.
vioural attraction to NaCl. Indeed, ENaCa knockout mice show no 17), demonstrated that ENaCa-expressing cells are distinct from
significant attraction to salt, even under conditions in which control sweet-, bitter- or umami-TRCs (Fig. 4a and Supplementary Fig. 6).
animals have an extraordinary appetite for sodium (Fig. 3a). In con- In fungiform and palate taste buds co-localization with a sour cell
trast, the aversive responses to high concentrations of NaCl (and marker, Car4 (ref. 27), showed the presence of two populations of
KCl) are unaltered in the same knockout animals (Fig. 3b, c). TRCs: one exhibiting co-expression of ENaCa and Car4 (Fig. 4b),
Notably, behavioural responses to sweet, sour, umami and bitter and importantly, a second one expressing ENaCa, but not sour, sweet,
298
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a NaCl (mM)
10 30 60 120 250 500 1,000
Amiloride – + – + – + – + – + – + – +
Control
ENaC-KO

45 s

b c d
60 Control Control 500 mM salt Control
ENaC-KO ENaC-KO ENaC-KO
Normalized response

80
80
40

40 40
20

0 0
0

Cs l
Cl

KCl
12 mM l
M l
l
Um eet

Bi i
So r
ur
C

-C

m KC
KC

am
tte
DG 2l
NM gC
Na

Sw
10 100 1,000 10 100 1,000

M
NaCl (mM) NaCl (mM)

0
0
25
Figure 2 | ENaC is necessary for high sensitivity taste responses to sodium responses of control and knockout animals; the coloured boxes are as in
salts. Conditional knockout of ENaCa in TRCs (ENaC-KO) abolishes a (n 5 4, mean 6 s.e.m., P , 0.001 for amiloride-sensitive responses of
responses to low NaCl concentrations, and eliminates amiloride-sensitivity. control and mutant groups at 30–1,000 mM NaCl). d, ENaC-KO mice retain
a, Integrated neural recordings from the chorda tympani nerve of normal normal responses to other salts or other taste qualities (n 5 4,
(control) and ENaC-KO mice in the presence (blue traces) or absence of mean 6 s.e.m.); see Methods for details of calculations, tastants used,
10 mM amiloride. Shaded boxes illustrate the amiloride-sensitive (pink) and concentrations, genotype of strains and abbreviations.
-insensitive (blue) components. b, c, Quantifications of integrated neural

a 15 bitter or umami markers (referred to as ‘ENaC-alone’ cells; Fig. 4b, c).

ENaC-KO
Control We hypothesized that the ENaC-alone cells are the bona-fide sodium
taste sensors, and that the expression of ENaCa in sour cells may just be
Salt-attraction lick ratio

a consequence (that is, non-functional) of a common lineage between

10
the cells mediating ionic tastes. Thus, we carried out further studies.
First, we generated animals lacking ENaCa solely in Car4-expressing
cells by using a sour-cell Cre driver to excise the conditional ENaCa
5 knockout allele. As expected, these mice show wild-type responses to
sweet, bitter, umami and sour stimuli. Notably, they have normal salt
responses that are indistinguishable from wild-type controls
(Supplementary Fig. 5), thus demonstrating that the ENaCa expres-
0 sion in the sour cells is in fact not required for salt taste. In a comple-
60 120 240 480
NaCl (mM) mentary study, we also generated mice entirely lacking sour-sensing
cells15; these animals show a total loss of sour sensing, yet they maintain
i
am

b c d
t

normal salt responses (Supplementary Fig. 5). Most critically, we

ee

r
ur
tte
Um
Sw

So
Bi

1 directly imaged salt and sour responses using our new peeled epithe-
Preference ratio

1
Salt-aversion

1.0 ENaC-KO
lium preparation. Indeed, there is total segregation of the cells respond-
lick ratio

Control
0.5
ing to salt (low and high concentrations) versus those responding to
Aversion ratio

0 acid stimulation (that is, sour cells never respond to salt stimuli13; see
0 0 0.5 Supplementary Fig. 1). Taken together, these results substantiate the
0.5 1.0 0.5 1.0 functional and anatomical segregation of sodium-sensing TRCs, and
1.0
NaCl (M) KCl (M)
prove that all five basic taste modalities are mediated by separate and
Figure 3 | ENaC function in TRCs is required for behavioural attraction to dedicated receptor cells at the periphery.
salt. Conditional knockout of ENaCa in TRCs selectively abolishes the An unusual feature of the physiology of sodium taste in mice has
attractive taste of NaCl. a, After diuretic-induced Na1-depletion, ENaC-KO been the observation that the back of the tongue (circumvallate
mice show little or no preference for NaCl solutions relative to water, whereas papillae) contains no sodium-selective (amiloride-sensitive) res-
littermate controls exhibit very robust attractive responses (P , 0.02 between
control and mutant groups at 120–480 mM NaCl). b, c, In contrast, in salt-
ponses10,22,28, highlighting a strong topographic segregation (front
aversion assays, water-deprived ENaC-KO mice and controls are to back) in salt taste (see later). With the identity of the amiloride-
indistinguishable in their responses to NaCl (b) or KCl (c). d, Behavioural sensitive salt taste receptor at hand, we reasoned that it should now be
responses to other taste qualities are unaffected in the ENaC-KO animals; possible to explore the molecular basis of the absence of sodium
shown are means 6 s.e.m. (n $ 7); see Methods for further details. sensing at the back of the tongue. ENaC channels are composed of
299
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

people subjected to controlled salt intake may help clarify the role, if
a b c
any, of ENaC in human taste.

METHODS SUMMARY
Transgenic animals and mouse strains. ENaCa-IRES-Cre, ENaCb-IRES-tTA and
Fungiform
cytokeratin19-IRES-Cre are bacterial artificial chromosome (BAC)-transgenics
engineered to express Cre-recombinase or the tetracycline-dependent transactiva-
tor (tTA) by inserting an IRES-Cre or IRES-tTA transgene 39 to the Scnn1a, Scnn1b
or Krt19 stop codon. Other strains have been described previously15,24.
ENaCα ENaCα Calcium imaging. Fungiform TRCs were pre-loaded in vivo with Calcium
TrpM5 Car4 Car4 Green-1 dextran 3kD (Invitrogen) by electroporating single taste buds. After
24–36 h, taste epithelium was enzymatically peeled11,18 and placed on a recording
chamber with the apical side of TRCs facing up (Supplementary Fig. 1). Taste
stimuli were delivered in artificial saliva by focal application. Changes in intra-
cellular calcium ([Ca21]i) were imaged using a 5-Live confocal microscope
Palate

(Zeiss) and the relative change in fluorescence (DF/F) from individual TRCs
analysed and pseudo-coloured as described previously23.
Nerve recording, behavioural and localization studies. All procedures were as
described previously5,17,18,25,27.

Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Figure 4 | ENaC defines a novel population of TRCs. Transgenic mice
expressing GFP under the control of ENaCa (see Methods for details) were Received 4 November 2009; accepted 5 January 2010.
Published online 27 January 2010.
immunostained for markers of known classes of TRC. a, No overlap in the
expression of ENaCa (green) and TrpM5 (red; a marker of sweet, bitter and 1. Contreras, R. J. in Neural Mechanisms in Taste (ed. Cagan, R. H.) 119–145 (CRC,
umami TRCs) was observed in fungiform or palate taste buds. b, In contrast, 1989).
Car4-expressing sour cells (red) co-express ENaCa (green; compare red- and 2. Duncan, C. J. Salt preferences of birds and mammals. Physiol. Zool. 35, 120–132
yellow-labelled cells). However, ENaCa is also expressed in a unique subset (1962).
of ‘ENaC-alone’ TRCs (green-only cells in b and c); see Methods for details of 3. Lindemann, B. Receptors and transduction in taste. Nature 413, 219–225 (2001).
mice and the illustration in c. Scale bar, 10 mm. Note that because Car4- 4. Beauchamp, G. K., Bertino, M., Burke, D. & Engelman, K. Experimental sodium
depletion and salt taste in normal human volunteers. Am. J. Clin. Nutr. 51, 881–889
expressing sour cells do not express the essential ENaCb subunit, they do not
(1990).
respond to salt (see Supplementary Fig. 6). 5. Mueller, K. L. et al. The receptors and coding logic for bitter taste. Nature 434,
225–229 (2005).
three essential subunits (a, b and c), thus we anticipated that this 6. Eylam, S. & Spector, A. C. Taste discrimination between NaCl and KCl is disrupted
by amiloride in inbred mice with amiloride-insensitive chorda tympani nerves.
Na1- and amiloride-insensitivity could be easily explained if the Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, R1361–R1368 (2005).
functional ENaC heterotrimeric channel was not found in the cir- 7. Halpern, B. P. Amiloride and vertebrate gustatory responses to NaCl. Neurosci.
cumvallate papillae. Indeed, our results show that at the back of the Biobehav. Rev. 23, 5–47 (1998).
tongue, ENaCa and ENaCb subunits are found in completely non- 8. Heck, G. L., Mierson, S. & DeSimone, J. A. Salt taste transduction occurs through
overlapping populations of TRCs (Supplementary Fig. 6). Therefore, an amiloride-sensitive sodium transport pathway. Science 223, 403–405 (1984).
9. Hettinger, T. P. & Frank, M. E. Specificity of amiloride inhibition of hamster taste
amiloride-insensitivity at the back of the tongue is due to the lack of responses. Brain Res. 513, 24–34 (1990).
functional ENaC channels. 10. Doolin, R. E. & Gilbertson, T. A. Distribution and characterization of functional
In this study we have shown that ENaC, first proposed to have a amiloride-sensitive sodium channels in rat tongue. J. Gen. Physiol. 107, 545–554
role in salt taste more than 25 years ago8,12, functions as the sodium (1996).
11. Kretz, O., Barbry, P., Bock, R. & Lindemann, B. Differential expression of RNA and
taste receptor. We also demonstrated that sodium taste is mediated protein of the three pore-forming subunits of the amiloride-sensitive epithelial
by a dedicated population of TRCs separate from those mediating sodium channel in taste buds of the rat. J. Histochem. Cytochem. 47, 51–64 (1999).
sweet, umami, bitter and sour taste. Notably, the taste of sodium and 12. Schiffman, S. S., Lockhead, E. & Maes, F. W. Amiloride reduces the taste intensity
non-sodium salts are detected by genetically, pharmacologically and of Na1 and Li1 salts and sweeteners. Proc. Natl Acad. Sci. USA 80, 6136–6140
physiologically distinguishable TRCs. The availability of two channels (1983).
13. Vandenbeuch, A., Clapp, T. R. & Kinnamon, S. C. Amiloride-sensitive channels in
(and cellular pathways) for salt-sensing endows animals with the type I fungiform taste cells in mouse. BMC Neurosci. 9, 1 (2008).
ability to distinguish sodium-containing salts from other salts; this 14. Yoshida, R. et al. NaCl responsive taste cells in the mouse fungiform taste buds.
affords mammals with a powerful mechanism to select food sources Neuroscience 159, 795–803 (2009).
containing adequate sodium but at the same time to avoid ingesting 15. Huang, A. L. et al. The cells and logic for mammalian sour taste detection. Nature
442, 934–938 (2006).
excessive amounts of salt.
16. Yarmolinsky, D. A., Zuker, C. S. & Ryba, N. J. Common sense about taste: from
The presence of salt shakers on dinner tables around the world mammals to insects. Cell 139, 234–244 (2009).
attests to the appetitive role of salt taste in the human diet. Indeed, 17. Zhang, Y. et al. Coding of sweet, bitter, and umami tastes: different receptor cells
salt has been a food additive shared by humans for thousands of sharing similar signaling pathways. Cell 112, 293–301 (2003).
years, with empires from the Roman (for salary) to the British (for 18. Zhao, G. Q. et al. The receptors for mammalian sweet and umami taste. Cell 115,
255–266 (2003).
taxes) valuing it as a precious commodity. Does ENaC function in 19. Canessa, C. M. et al. Amiloride-sensitive epithelial Na1 channel is made of three
human salt taste? Physiological recordings in non-human primates homologous subunits. Nature 367, 463–467 (1994).
have clearly demonstrated an amiloride-sensitive component in taste 20. Hummler, E. & Beermann, F. Scnn1 sodium channel gene family in genetically
responses to salt stimuli29,30. However, psychophysical experiments engineered mice. J. Am. Soc. Nephrol. 11, S129–S134 (2000).
21. Strazzullo, P., D’Elia, L., Kandala, N. B. & Cappuccio, F. P. Salt intake, stroke, and
in humans remain inconclusive7, with some reports of amiloride
cardiovascular disease: meta-analysis of prospective studies. BMJ. doi:10.1136/
altering salt taste7,12, and several failing to substantiate a significant bmj.b4567 (2009).
effect for amiloride in the perception of saltiness (reviewed in ref. 7). 22. Ninomiya, Y. Reinnervation of cross-regenerated gustatory nerve fibers into
Given the molecular similarities between mice and humans in all amiloride-sensitive and amiloride-insensitive taste receptor cells. Proc. Natl Acad.
other taste modalities16, a ‘human-specific’ molecular mechanism Sci. USA 95, 5347–5350 (1998).
23. Oka, Y. et al. Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity
for salt taste would be surprising. Perhaps more likely, the contri- and specificity of receptor-defined glomeruli. Neuron 52, 857–869 (2006).
bution of ENaC to human salt taste may be masked as a result of 24. Hummler, E., Merillat, A. M., Rubera, I., Rossier, B. C. & Beermann, F. Conditional
experience, exposure to salt, and diet. Future experiments studying gene targeting of the Scnn1a (aENaC) gene locus. Genesis 32, 169–172 (2002).
300
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

25. Nelson, G. et al. An amino-acid taste receptor. Nature 416, 199–202 (2002). valuable comments. This research was supported in part by the intramural
26. Dethier, V. G. The taste of salt. Am. Sci. 65, 744–751 (1977). research program of the NIH, NIDCR (N.J.P.R.). C.S.Z. is an investigator of the
27. Chandrashekar, J. et al. The taste of carbonation. Science 326, 443–445 Howard Hughes Medical Institute.
(2009).
28. Frank, M. E. Taste-responsive neurons of the glossopharyngeal nerve of the rat. Author Contributions J.C. designed the study, carried out electrophysiological and
J. Neurophysiol. 65, 1452–1463 (1991). expression studies, analysed data and wrote the paper; C.K. designed and carried
29. Hellekant, G., Danilova, V. & Ninomiya, Y. Primate sense of taste: behavioral and out behavioural experiments and analysed expression in engineered and knockout
single chorda tympani and glossopharyngeal nerve fiber recordings in the rhesus mice; Y.O. designed and carried out calcium imaging experiments and analysed
monkey, Macaca mulatta. J. Neurophysiol. 77, 978–993 (1997). data; D.A.Y. carried out molecular studies and helped write the paper; E.H.
30. Hellekant, G. & Ninomiya, Y. Bitter taste in single chorda tympani taste fibers from provided essential reagents; N.J.P.R. and C.S.Z. designed the study, analysed data
chimpanzee. Physiol. Behav. 56, 1185–1188 (1994). and wrote the paper.

Supplementary Information is linked to the online version of the paper at Author Information Reprints and permissions information is available at
www.nature.com/nature. www.nature.com/reprints. The authors declare competing financial interests:
details accompany the full-text HTML version of the paper at www.nature.com/
Acknowledgements We thank W. Guo and A. Becker for generation and nature. Correspondence and requests for materials should be addressed to C.S.Z.
maintenance of mouse lines, and K. Scott and members of our laboratories for (cz2195@columbia.edu).

301
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08783

METHODS glutamate plus 1.0 mM inosine monophosphate (umami); 1–10 mM quinine

Transgenic animals and mouse strains. The PKD2L1-IRES-Cre, ROSA-DTA, hydrochloride (bitter); 1–50 mM citric acid (sour). The responses to 50 mM citric
Z/EG and ENaCa conditional knockout (Scnn1aflox/flox) strains have been acid were used to normalize responses to each experimental series in control and
described previously15,24. ENaCa-IRES-Cre, ENaCb-IRES-tTA and cytokeratin19- ENaCa-KO (Fig. 2). To compute the amiloride-sensitive salt component (Fig. 2b),
IRES-Cre are BAC-transgenics engineered to express Cre-recombinase or the the stimulation regime involved sequential applications of NaCl solutions
tetracycline dependent transactivator (tTA) by inserting an IRES-Cre or IRES- first without and then with amiloride (in the same experimental series). The
tTA transgene 39 to the Scnn1a, Scnn1b or Krt19 stop codon. The ENaCb-IRES- amiloride-insensitive component was defined as the response in the presence of
tTA transgenic mice also carried a TetO-sapphire (modified GFP) reporter in the amiloride (Fig. 2c). The fraction of the response inhibited by amiloride was defined
BAC-transgene. Four independent lines of ENaCa-IRES-Cre expressed the Cre as the amiloride-sensitive component (amiloride-sensitive component 5 response
transgene appropriately in kidney and lung and labelled equivalent populations without amiloride 2 response with amiloride; Fig. 2b). Responses in experiments
of taste cells (Supplementary Figs 4 and 6). Similarly, three founder lines expressing involving PKD2L1-IRES-Cre Rosa-DTA (PKD2L1-DTA) and PKD2L1-IRES-
ENaCb-IRES-tTA showed equivalent tTA-expression in lung, kidney and taste Cre ENaCa flox/flox (PKD2L1-ENaC-KO) were normalized to responses obtained
tissue. Mice were inter-crossed as described in the text to generate appropriate with 30 mM acesulphameK (Supplementary Fig. 5). NaCl solutions used in dose-
genotypes for physiological, behavioural and anatomical experiments. Control response studies for measuring the amiloride-insensitive sodium responses (Fig. 2c)
groups were littermates not carrying the IRES-Cre transgene and/or animals with included 10 mM amiloride. Differences between knockout and control responses
at least one copy of the wild-type Scnn1a allele. Wild type, heterozygous and the were analysed for statistical significance using an unpaired, two-tailed Student’s
conditional ENaCa taste knockouts had no obvious differences in size, weight, t-test and 95% confidence limits.
fertility, life expectancy or food consumption. Behavioural assays. Behavioural assays used a custom-made gustometer to
Calcium imaging. Fungiform TRCs were pre-loaded in vivo with calcium green-1 measure immediate lick responses as described previously17,18. For salt-attraction
dextran 3kD (Invitrogen) by electroporating single taste buds in the tongue of assays, mice were injected with furosemide (50 mg kg21) and were placed on a
anaesthetized mice using a 3.5 mA, 50 pulses s21 3 16 cycles regime. On average, low sodium diet with unrestricted water for 16–20 h to deplete sodium before
8–9 taste buds were loaded per animal. After 24–36 h of recovery, tongues were testing5. For salt-aversion assays, mice were water deprived for 24 h before test-
removed and the taste epithelium enzymatically peeled as described previously11. ing5,17,18. Control tastants were 32 mM acesulphameK (sweet), 100 mM mono-
The epithelium was then placed on a recording chamber with the apical side of sodium glutamate plus 1 mM inosine monophosphate and 0.1 mM amiloride
TRCs facing up (Supplementary Fig. 1), ensuring that the integrity and polarity of (umami), 1 mM quinine sulphate (bitter), and 150 mM citric acid (sour).
taste buds is maintained. The apical surface of the preparation was bathed in Differences between knockout and control responses were analysed for statistical
artificial saliva at a constant flow rate of 4.5 ml min21, and taste stimuli were significance using an unpaired, two-tailed Student’s t-test and 95% confidence
delivered by focal application using a custom made dispensing pipette (800 mm limits; for Supplementary Fig. 5b a one-way analysis of variance (ANOVA) with
diameter). Tastant application was for 1 s, with a minimum of 10 s of artificial Newman–Keuls posterior test was used to compare data sets.
saliva between stimuli. Changes in [Ca21]i were imaged using a 5-Live confocal Immunohistochemistry and cell labelling. Immunostaining, whole-mount
microscope (Zeiss) using a 340 C-Apochromat 1.20W objective; images were imaging (GFP) and in situ hybridization were performed as described previ-
captured at 4 Hz, and DF/F from individual TRCs analysed and pseudo-coloured ously17,18; animals were perfused with 4% paraformaldehyde and tissue was
as described previously23. post-fixed for 6–48 h to allow localization of GFP. Images were obtained using
Nerve recordings. Lingual stimulation and recording procedures were performed a Leica SP2 TSC or a Zeiss 510 LSM meta confocal microscope. Anti-TrpM5 and
as previously described18,25. All data analyses used the integrated response over a 25-s anti-Car4 antibodies were as described previously17,27. The illustration in Fig. 4c
period immediately after the application of the stimulus. Tastants used for nerve was composed by converting the red and green channels in Fig. 4b to greyscale
recordings were: 3–30 mM acesulphameK (sweet); 1–100 mM monopotassium and overlaying with the ENaC-alone (green only) cells using Adobe Photoshop.

©2010 Macmillan Publishers Limited. All rights reserved

 Vol 464 | 11 March 2010 | doi:10.1038/nature08782

LETTERS
B-cell-derived lymphotoxin promotes
castration-resistant prostate cancer
Massimo Ammirante1*, Jun-Li Luo2*, Sergei Grivennikov1, Sergei Nedospasov3 & Michael Karin1

Prostate cancer (CaP) progresses from prostatic intraepithelial IKK-b in myc-CaP cells did not affect primary tumour growth or
neoplasia through locally invasive adenocarcinoma to castration- castration-resistant CaP re-growth in castrated FVB mice
resistant metastatic carcinoma1. Although radical prostatec- (Supplementary Fig. 3).
tomy, radiation and androgen ablation are effective therapies for By contrast, deletion of IKK-b in interferon-responsive cells upon
androgen-dependent CaP, metastatic castration-resistant CaP is a induction of Mx1-Cre expression prevented castration-induced
major complication with high mortality2. Androgens stimulate metastatic spread in TRAMP/IkkbF/F/Mx1-Cre mice (Supplementary
growth and survival of prostate epithelium and early CaP. Fig. 4a). To narrow down the role of IKK-b to bone-marrow-derived
Although most patients initially respond to androgen ablation, cells, we reconstituted irradiated FVB mice with bone marrow from
many develop castration-resistant CaP within 12–18 months2. IkkbF/F/Mx1-Cre mice and inoculated the resulting IkkbF/F and IkkbD/D
Despite extensive studies, the mechanisms underlying the emer- (mice injected with poly(IC) to induce Mx1-Cre) chimaeras (Sup-
gence of castration-resistant CaP remain poorly understood and plementary Fig. 4b) with myc-CaP cells. Absence of IKK-b in bone-
their elucidation is critical for developing improved therapies. marrow-derived cells had no impact on primary tumour growth, but
Curiously, castration-resistant CaP remains androgen-receptor delayed castration-resistant CaP emergence after castration (Sup-
dependent, and potent androgen-receptor antagonists induce plementary Fig. 4c). A similar delay in castration-resistant CaP growth
tumour regression in castrated mice3. The role of inflammation was seen in castrated tumour-bearing mice treated with specific IKK-b
in castration-resistant CaP has not been addressed, although it inhibitors14,15 (Supplementary Fig. 4d and data not shown).
was reported that intrinsic NF-kB activation supports its growth4. Dependence of castration-resistant CaP emergence on IKK-b in
Inflammation is a localized protective reaction to injury or infec- bone-marrow-derived cells suggested that androgen deprivation elicits
tion, but it also has a pathogenic role in many diseases, including a tumour-associated inflammatory response. Castration of mice bear-
cancer5. Whereas acute inflammation is critical for host defence, ing myc-CaP tumours resulted in CaP cell death, peaking within
chronic inflammation contributes to tumorigenesis and metastatic 1 week (Fig. 1a). Concurrently, the regressing tumours were infiltrated
progression. The inflammation-responsive IkB kinase (IKK)-b and with T and B lymphocytes, natural killer cells and myeloid cell types
its target NF-kB have important tumour-promoting functions (Fig. 1b and Supplementary Fig. 5). Infiltration was transient, declin-
within malignant cells and inflammatory cells6. The latter, includ- ing by 2 weeks after castration. B- and T-lymphocyte infiltration was
ing macrophages and lymphocytes, are important elements of the also detected in 100% of human CaP samples (untreated patients with
tumour microenvironment7–9, but the mechanisms underlying Gleason scores of 6–8), but B cells were undetectable in normal
their recruitment remain obscure, although they are thought to prostate or benign prostatic hyperplasia (Fig. 1c). The messenger
depend on chemokine and cytokine production10. We found that RNAs for many inflammatory chemokines were also upregulated in
CaP progression is associated with inflammatory infiltration and the myc-CaP allografts, but no changes in androgen-receptor mRNA
activation of IKK-a, which stimulates metastasis by an NF-kB- expression were found (Supplementary Fig. 6a). These chemo-
independent, cell autonomous mechanism11. Here we show that kines may recruit lymphoid and myeloid cells into the regressing
androgen ablation causes infiltration of regressing androgen- tumour. Indeed, antibody-mediated inhibition of CXCL13, a B-cell
dependent tumours with leukocytes, including B cells, in which chemoattractant16, prevented castration-induced B-cell recruitment
IKK-b activation results in production of cytokines that activate (Supplementary Fig. 6b, c). Inflammatory cytokine mRNAs, including
IKK-a and STAT3 in CaP cells to enhance hormone-free survival. interleukin (IL)-6, IL-12, TNF-a and lymphotoxin, were also upregu-
To determine whether IKK-b-driven NF-kB participates in the lated in the regressing myc-CaP allograft, but only lymphotoxin
development of castration-resistant CaP, we conditionally deleted expression was reduced upon CXCL13 inhibition (Supplementary
the Ikkb (also known as Ikbkb) gene in prostate epithelial cells of Fig. 7). Castration resulted in nuclear export of androgen receptor,
TRAMP mice, in which CaP is induced by prostate-specific expres- but after 3 weeks androgen receptor was nuclear again (Supplementary
sion of simian virus 40 T antigen12. Counter to previous expecta- Fig. 8), suggesting it is activated at late phases of castration-resistant
tions4, IKK-b ablation in prostate epithelial cells had no effect on CaP growth despite androgen depletion.
genesis and progression of androgen-dependent CaP (Supplemen- STAT3 was proposed to promote activation of unliganded andro-
tary Fig. 1) or development of castration-resistant CaP after castra- gen receptor17. Indeed, STAT3 was activated during emergence of
tion (Supplementary Fig. 2). To facilitate mechanistic analysis of castration-resistant CaP, faster than androgen receptor (Supplemen-
castration-resistant CaP development we used subcutaneous allo- tary Fig. 9). Mx1-Cre-mediated IKK-b deletion, which was nearly
grafts of the mouse androgen-dependent CaP cell line myc-CaP, complete in mature B and T lymphocytes (Supplementary Fig. 10a),
which is derived from the FVB genetic background13. Silencing of prevented STAT3 activation in regressing tumours, but did not affect
1
Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology and Cancer Center, School of Medicine, University of California, San Diego, 9500 Gilman Drive,
La Jolla, California 92093-0723, USA. 2Scripps Research Institute-Florida, Department of Cancer Biology, 130 Scripps Way, Jupiter, Florida 33458, USA. 3Laboratory of Molecular
Immunology, Engelhardt Institute of Molecular Biology, 119991, Moscow, Russia.
*These authors contributed equally to this work.

302
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a Weeks after castration a b

Tumour volume (mm3)

Rag1 + B cells

p-STAT3
p
Normal Sham Week 1 Week 2 Week 3
Rag1 + B cells
1,000 WT + Rag1

TUNEL
500 ** WTWT +Ikkk F/F
W+T Ikk-β
Ikk
kk F//F
F
8 9 128 95 12 Apoptotic
cells per field

p
p-STAT3
b CD2R (T) CCR1 (Th1) CCR3 (Th2) 0
300 0 6 12 18 24 30 36 42 48
Relative mRNA

2,000 120
Days after castration
200
80
c
*** ** **
1,000 100 WT + Rag1
40

p-STAT3
0 0 0

p
p-STAT3
Normal Sham C1 C2 C3 Normal Sham C1 C2 C3 Normal Sham C1 C2 C3

CD8a (CTL) B220 (B) F4/80 (MAC)

12
Relative mRNA

3,000 10 3000
8 2000
2,000
*** 6 ** *** Rag1 + B Rag1 + T Rag1 + WT /
WT + Ikk-β∆∆
1,000 4 1000
2
0 0 0 Figure 2 | Role of B cells and IKK-b in STAT3 activation and castration-
Normal Sham C1 C2 C3 Normal Sham C1 C2 C3 Normal Sham C1 C2 C3
resistant CaP emergence. a, myc-CaP tumours were established in wild-
CD208 (DC) NK1.1 (NK) CXCR1 (Neut)
3.0 type mice reconstituted with bone marrow from Rag1–/– males (n 5 10) or in
800
Relative mRNA

2,000 2.5 Rag1–/– males. When tumours reached 1,000 mm3, mice were castrated.
1,600 600 2.0
1,200 *** 1.5 **
Three days before castration, Rag1–/– mice (n 5 10 per group) received
400
800 *** 1.0 purified splenic B or T cells through the tail vein. Tumour volume was
200 0.5
400 measured. Results are averages 6 s.e.m. P values were determined and are
0 0 0
Normal Sham C1 C2 C3 Normal Sham C1 C2 C3 Normal Sham C1 C2 C3 indicated as above. b, Tumours were removed from radiation chimaeras
c reconstituted with IkkbF/F, IkkbD/D or Rag1–/– bone marrow, 1 week after
castration. STAT3 phosphorylation was analysed by
Number of cells per field
CD20

600 CD4
immunohistochemistry. c, Tumour-bearing Rag1–/– males were injected
**
500 CD8 ** with wild-type splenocytes (Rag1 1 WT), or purified splenic B (Rag1 1 B) or
400 F4/80
CD20 T (Rag1 1 T) lymphocytes. One day later, mice were castrated and after
300
* 1 week tumours were removed and analysed for STAT3 phosphorylation.
DAPI

200
100 ** Original magnifications, 320 (b) and 340 (c).
0
Normal Benign Malignant Normal Benign Malignant
which lack only mature T lymphocytes. Castration-resistant CaP
Figure 1 | Androgen ablation induces tumour inflammatory infiltration. growth was delayed in mice reconstituted with bone marrow from
Six-week-old FVB males (n 5 10) were inoculated with myc-CaP cells. When JH–/– mice (Supplementary Fig. 14c), which lack mature B cells, or upon
tumours reached 1,000 mm3, mice were left untreated, castrated or sham-
B-cell depletion with CD20 antibody19 (Supplementary Fig. 14d).
operated. Tumours were collected when indicated for analysis. a, Paraffin-
embedded tumour sections were stained by TdT-mediated dUTP nick end
Reconstitution of Rag1–/– FVB mice with splenic B cells, but not T cells
labelling (TUNEL) to determine apoptotic cell frequency (results are of FVB mice, restored rapid castration-resistant CaP re-growth
averages, n 5 3). b, Total RNA was isolated from tumour samples and (Fig. 2a). Primary tumours, isolated from Rag1–/– chimaeric mice
expression of indicated cell-marker mRNAs was quantified and normalized 1 week after castration, did not show STAT3 activation (Fig. 2b), but
to that of cyclophilin A (norm, normal; C1, 2, 3, mice analysed 1, 2 or 3 weeks reconstitution with B cells, rather than T cells, restored castration-
after castration; sham, sham-operated). Results are averages 6 s.d. (n 5 10). induced STAT3 phosphorylation (Fig. 2c).
c, Frozen human prostate sections (normal tissue (n 5 3), prostatic CaP allografts from castrated, but not sham-operated, mice exhibited
hyperplasia (n 5 3) and malignant CaP with Gleason scores 6–8 (n 5 10)) IKK-a nuclear translocation (Fig. 3a, b). Silencing of IKK-a in myc-CaP
were stained with CD4, CD8 and CD20 antibodies and 49,6-diamidino-2-
phenylindole (DAPI) and analysed by immunofluorescent microscopy. The
cells using short interfering RNA (siRNA) (Supplementary Fig. 15a)
histogram denotes average frequencies of indicated cell types (n 5 3 per had little effect on primary tumour growth, but delayed emergence of
sample). P values were determined and are depicted as insignificant, castration-resistant CaP (Fig. 3c). Nuclear translocation of IKK-a was
significant (*), very significant (**) or highly significant (***). Original dependent on IKK-b in bone-marrow-derived cells and on B cells, but
magnifications, 320 (a, c). not on T cells (Fig. 3d). IKK-a nuclear translocation parallels progres-
sion of human and murine CaP and coincides with primary tumour
extracellular signal-regulated kinase (ERK) and AKT activation (Sup- infiltration with cells expressing IKK-a-activating cytokines, RANK
plementary Fig. 10b). Immunohistochemical analysis confirmed ligand and lymphotoxin-a11. Castration induced lymphotoxin-a and
STAT3 activation in CaP cells, which was inhibited by A490 (Sup- lymphotoxin-b in regressing myc-CaP allografts, but did not alter
plementary Fig. 10c), an inhibitor of STAT3 phosphorylation18 that RANK ligand expression (Supplementary Fig. 7). Lymphotoxin expres-
delayed appearance of castration-resistant CaP (Supplementary Fig. sion in regressing tumours was absent in Rag1–/– mice (Supplementary
10d) but did not inhibit IKK activation (Supplementary Fig. 11a). Fig. 16a), and flow cytometry localized it to tumour-infiltrating B cells
Conversely, ML120B did not inhibit STAT3 activation (Supplemen- (TIBCs; Supplementary Fig. 16b). We characterized TIBCs by seven-
tary Fig. 11b). colour flow cytometry with several markers and a lymphotoxin-b
Ablation of bone-marrow-derived cell IKK-b did not prevent leu- receptor-immunoglobulin fusion protein (LTbR-Ig) to detect lympho-
kocyte recruitment into regressing tumours (Supplementary Fig. 12) toxin. The typical TIBC was a conventional, mature B2 cell that
but did inhibit cytokine induction (Supplementary Fig. 13). To investi- expressed lymphotoxin on its surface and was negative for B1 markers
gate the role of lymphocytes further, we used chimaeric mice generated (Supplementary Fig. 17). IKK-b deletion abolished lymphotoxin
by transplantation of bone marrow from lymphocyte-deficient Rag1–/– expression by B cells (Fig. 4a), supporting the previously suggested20
mice. Although primary tumour growth was identical in mice receiv- role of NF-kB in lymphotoxin-a/b induction. To examine whether
ing wild-type or Rag1–/– bone marrow (Supplementary Fig. 14a), lymphotoxin production by tumour-infiltrating lymphocytes stimu-
castration-resistant CaP growth was significantly delayed in mice lates castration-resistant CaP growth, we transplanted bone marrow
receiving Rag1–/– bone marrow (Fig. 2a), but not in mice reconstituted from B-Ltb–/– or T-Ltb–/– mice, which lack lymphotoxin-b in either
with bone marrow from Tcrb2/2/d2/2 mice (Supplementary Fig. 14b), B or T cells21, into lethally irradiated mice. Lymphotoxin-b ablation in B
303
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

activation (Supplementary Fig. 19). Silencing of LTbR in Myc-CaP cells

14 d
7 d

y ate
a b

y te
da stra
(Supplementary Fig. 15b) also delayed growth of castration-resistant

am

da str
a
a
Sh

C
C
CaP (Fig. 4d). Exogenous lymphotoxin maintained myc-CaP growth

IKK-α
IKK-α
in the presence of flutamide, a clinically used androgen-receptor anta-
H3
C N C N C N
gonist3, in a manner dependent on IKK-a (Fig. 4e), whose nuclear
Sham Castrated translocation was lymphotoxin inducible (Fig. 4f).
c d Castration-resistant CaP is a major complication that limits the
success of androgen ablation therapy and is responsible for most

IKK-α
1,500 sc
mortality from prostate cancer2. Castration-resistant CaP was
Tumour volume (mm3)

IKK-α siRNA
1,000
studied mainly at the level of androgen-receptor function, the central
WT + IkkβF/F WT + Ikkβ∆ /∆ player in this process4. Our results suggest that an inflammatory
500
** response triggered by death of androgen-deprived primary cancer is

IKK-α
another important contributor to emergence of castration-resistant
0 CaP. In addition to dying CaP cells, critical participants in this res-
0 3 6 9 12 15 18 21 24 27 30 33 36 39
Days after castration ponse are tumour-infiltrating B cells, which produce lymphotoxin-a:b
Rag1 + B cells Rag1 + T cells
heterotrimers that stimulate LTbR on CaP cells to induce IKK-a nuc-
Figure 3 | Role of IKK-a in emergence of castration-resistant CaP. lear translocation and STAT3 activation, thereby enhancing androgen-
Tumour-bearing mice were castrated or sham operated as above. a, Tumours independent growth (Supplementary Fig. 20). Interference with any
were analysed 1 week later for nuclear IKK-a by immunohistochemistry. component of this response results in a significant and reproducible
b, Tumours removed at indicated times were divided into cytosolic (C) and
nuclear (N) fractions, and IKK-a and histone H3 distributions determined.
3- to 4-week delay in appearance of castration-resistant CaP. Although
c, Tumours were established using myc-CaP cells transduced with these inhibitory effects are not absolute, extrapolation from ‘mouse
lentiviruses expressing scrambled siRNA (sc) or IKK-a-specific siRNA. Mice time’ to ‘human time’ suggests that interventions that prevent lym-
were castrated as above and tumour volume was measured. Results are photoxin production or signalling may delay appearance of castration-
averages 6 s.e.m. (n 5 10). P values were determined and are indicated as resistant CaP in patients undergoing androgen ablation therapy by 2.3
above. d, Tumours were established in lethally irradiated FVB males to 3.1 years. Importantly, our results suggest that, at least for CaP, the
reconstituted with IkkbF/For IkkbD/D bone marrow or in Rag1–/– males inflammatory response elicited by the dying primary tumour contri-
reconstituted with either B or T cells. IkkbF/F and IkkbD/D chimaeras were butes to the failure rather than the previously proposed success of anti-
intraperitoneally injected three times with poly(IC) (250 mg) before
castration to delete IKK-b. One week after castration, tumour samples were
cancer therapy23. Although we have not determined how death of
analysed for IKK-a distribution by immunohistochemistry. Nuclear IKK-a androgen-deprived CaP triggers the inflammatory response described
results in punctate staining, whereas cytoplasmic IKK-a results in diffuse above, necrotic cell death releases mediators, such as HMGB1 (ref. 24)
staining. Original magnifications, 320 (a, d). and IL-1a (ref. 25), that activate IKK-b and NF-kB and stimulate
production of chemokines, one of which, CXCL13, recruits B cells
cells, but not in T cells, delayed growth of castration-resistant CaP into the regressing tumour. Notably, TIBCs were detected not only
(Fig. 4b) and abolished lymphotoxin-b expression within tumours in androgen-deprived mouse CaP, but also in human CaP. Although
but did not prevent B-cell or macrophage infiltration (Supplemen- B cells were reported to promote progression of skin carcinomas9 and
tary Fig. 18). Treatment of mice with the LTbR-Ig decoy22 was as effec- exert immunosuppressive effects through activation of inhibitory Fc
tive as B-cell-specific lymphotoxin-b ablation in delaying growth of receptors on myeloid cells26, the critical tumour-promoting B-cell
castration-resistant CaP (Fig. 4c) and prevented IKK-a and STAT3 function in our experimental model is production of lymphotoxin,

a b c
WT + T-Ltβ–/– hlgG
Tumour volume (mm3)

Tumour volume (mm3)

30 Ikkβ F/F WT + B-Ltβ–/– LTβR lg

1,000 1,000
Relative mRNA

∆ /∆
Ikkβ
20 **
500 ** **
500
10
*
0 0 0
LT-α LT-β 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Days after castration Days after castration

d e f
sc sc siRNA + flutamide
Tumour volume (mm3)

50 sc siRNA + flutamide +
Number of cells ×10–5

LTβR siRNA
LT-α2β1
1,000 40 LT-α2β1 0 5 10 15 20 ng ml–1
IKK-α siRNA + flutamide
30 IKK-α siRNA + flutamide IKK-α
** + LT-α2β1
500 20
** IKK-γ
Histone H3
C N C N C N C N C N
10
0 0
0 6 12 18 24 30 36 42 48 0 5 10
Days after castration Days

Figure 4 | IKK-b-dependent lymphotoxin production by tumour-infiltrating expressing scrambled (sc) siRNA or lymphotoxin-b-specific siRNA. Mice
B cells stimulates IKK-a-dependent androgen-free survival. a, RNA from were castrated and tumour volume was measured. Results are
splenic B cells of IkkbF/Fand IkkbD/D mice was analysed for lymphotoxin averages 6 s.e.m. (n 5 10). e, Myc-CaP cells (previously infected with
(LT)-a and lymphotoxin-b expression as above. Results are averages 6 s.d. lentiviruses expressing scrambled or IKK-a siRNAs) were plated at 40%
(n 5 3). b, Lethally irradiated FVB males were reconstituted with bone confluency. After 6 h, the cells were cultured with or without flutamide
marrow from B-Ltb2/2 or T-Ltb2/2 mice (n 5 6 per group). After 8 weeks, (10 mM) in the absence or presence of lymphotoxin-a2b1, (LT-a2b1) and the
myc-CaP tumours were established, mice were castrated and tumour volume cell number determined. f, Myc-CaP cells were plated at 60% confluency.
was measured as above. Results are averages 6 s.e.m. c, FVB mice (n 5 6 After 12 h, cells were stimulated for 1 h with lymphotoxin-a2b1, collected,
each group) bearing myc-CaP tumours were castrated and given hIgG or divided into cytosolic (C) and nuclear (N) fractions and IKK-a and histone
LTbR-Ig (100 mg) every 5 days, starting 4 days before castration. Tumour H3 distributions determined. In a–e, P values were determined and are
volume was measured as above. Results are averages 6 s.e.m. d, Tumours indicated as above.
were established using myc-CaP cells transduced with lentiviruses
304
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

an IKK-a-activating cytokine27, which promotes survival of androgen- 14. Park, B. K. et al. NF-kB in breast cancer cells promotes osteolytic bone metastasis
by inducing osteoclastogenesis via GM-CSF. Nature Med. 13, 62–69 (2007).
deprived CaP. Another important function of TIBCs is activation of
15. Wen, D. et al. A selective small molecule IkB kinase b inhibitor blocks nuclear
STAT3, an anti-apoptotic and pro-tumorigenic transcription factor28. factor kB-mediated inflammatory responses in human fibroblast-like
Although the critical STAT3-activating cytokine in this system remains synoviocytes, chondrocytes, and mast cells. J. Pharmacol. Exp. Ther. 317, 989–1001
to be identified, castration induces expression of STAT3-activating (2006).
IL-6 and IL-12 family members. Furthermore, CaP cells use autocrine 16. Legler, D. F. et al. B cell-attracting chemokine 1, a human CXC chemokine
expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/
IL-6 to stimulate their progression29 and activated STAT3 promotes CXCR5. J. Exp. Med. 187, 655–660 (1998).
ligand-independent androgen-receptor activation29. Lymphotoxin is 17. Chen, T., Wang, L. H. & Farrar, W. L. Interleukin 6 activates androgen receptor-
also involved in the aetiology of human CaP. An epidemiological study mediated gene expression through a signal transducer and activator of
revealed that reduced CaP risk due to consumption of non-steroidal transcription 3-dependent pathway in LNCaP prostate cancer cells. Cancer Res.
60, 2132–2135 (2000).
anti-inflammatory drugs, such as aspirin, is limited to men who
18. Eriksen, K. W. et al. Constitutive STAT3-activation in Sezary syndrome: tyrphostin
express a common polymorphic LTa allele that specifies high lympho- AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth
toxin production30. Further work should examine the effect of LTa of leukemic Sezary cells. Leukemia 15, 787–793 (2001).
polymorphism on the response to androgen ablation. Our results pre- 19. Hamel, K. et al. Suppression of proteoglycan-induced arthritis by anti-CD20 B cell
dict that individuals who produce high levels of lymphotoxin are more depletion therapy is mediated by reduction in autoantibodies and CD41 T cell
reactivity. J. Immunol. 180, 4994–5003 (2008).
likely to develop castration-resistant CaP and should therefore be the 20. Worm, M. M., Tsytsykova, A. & Geha, R. S. CD40 ligation and IL-4 use different
main beneficiaries of anti-lymphotoxin therapy. mechanisms of transcriptional activation of the human lymphotoxin a promoter
in B cells. Eur. J. Immunol. 28, 901–906 (1998).
METHODS SUMMARY 21. Tumanov, A. V. et al. Dissecting the role of lymphotoxin in lymphoid organs by
Mice were handled according to institutional and National Institutes of Health conditional targeting. Immunol. Rev. 195, 106–116 (2003).
22. Lee, Y. et al. Recruitment and activation of naive T cells in the islets by
guidelines. Tumours were grown in FVB mice. Where indicated, lethally irra-
lymphotoxin b receptor-dependent tertiary lymphoid structure. Immunity 25,
diated FVB mice were reconstituted with bone marrow from different strains 499–509 (2006).
that were backcrossed into the FVB background for at least two generations. Ltb 23. Ghiringhelli, F. et al. Activation of the NLRP3 inflammasome in dendritic cells
knockout strains were, however, in the BL6 background, which does not elicit a induces IL-1b-dependent adaptive immunity against tumors. Nature Med. 15,
graft versus host response in FVB mice. Conditions for antibody used were 1170–1178 (2009).
posted to http://biorating.com. Human material was obtained from the 24. Scaffidi, P., Misteli, T. & Bianchi, M. E. Release of chromatin protein HMGB1 by
Cooperative Human Tissue Network, along with pathology reports. Histology, necrotic cells triggers inflammation. Nature 418, 191–195 (2002).
gene expression and cell signalling were analysed as described11,25. 25. Sakurai, T. et al. Hepatocyte necrosis induced by oxidative stress and IL-1 a
release mediate carcinogen-induced compensatory proliferation and liver
Full Methods and any associated references are available in the online version of tumorigenesis. Cancer Cell 14, 156–165 (2008).
the paper at www.nature.com/nature. 26. Nimmerjahn, F. & Ravetch, J. V. Fc-receptors as regulators of immunity. Adv.
Immunol. 96, 179–204 (2007).
Received 1 May; accepted 29 December 2009. 27. Bonizzi, G. et al. Activation of IKKa target genes depends on recognition of specific
kB binding sites by RelB:p52 dimers. EMBO J. 23, 4202–4210 (2004).
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independent prostate cancer. Clin. Adv. Hematol. Oncol. 1, 49–57 (2003). prostate cancer progression. Cytokine Growth Factor Rev. 12, 33–40 (2001).
3. Tran, C. et al. Development of a second-generation antiandrogen for treatment of 30. Liu, X., Plummer, S. J., Nock, N. L., Casey, G. & Witte, J. S. Nonsteroidal
advanced prostate cancer. Science 324, 787–790 (2009). antiinflammatory drugs and decreased risk of advanced prostate cancer:
4. Jin, R. J. et al. The nuclear factor-kB pathway controls the progression of prostate modification by lymphotoxin alpha. Am. J. Epidemiol. 164, 984–989 (2006).
cancer to androgen-independent growth. Cancer Res. 68, 6762–6769 (2008).
5. Karin, M., Lawrence, T. & Nizet, V. Innate immunity gone awry: linking microbial Supplementary Information is linked to the online version of the paper at
infections to chronic inflammation and cancer. Cell 124, 823–835 (2006). www.nature.com/nature.
6. Karin, M. Nuclear factor-kB in cancer development and progression. Nature 441,
431–436 (2006). Acknowledgements We thank C. Sawyers for myc-CaP cells, L. Coussens for JH–/–
7. Balkwill, F., Charles, K. A. & Mantovani, A. Smoldering and polarized inflammation (FVB) mice, Y. X. Fu for LTbR-Ig fusion protein, R. Rickert for help with B-cell
in the initiation and promotion of malignant disease. Cancer Cell 7, 211–217 phenotyping, H. Cheroutre for flow cytometer use and C. Ware for bone marrow.
(2005). M.A. was supported by Fondazione Italiana per la Ricerca sul Cancro and
8. Coussens, L. M. & Werb, Z. Inflammation and cancer. Nature 420, 860–867 American-Italian Cancer Foundation fellowships. J.-L.L. was supported by a Life
(2002). Science Research Fellowship. Work in M.K.’s laboratory was supported by grants
9. de Visser, K. E., Korets, L. V. & Coussens, L. M. De novo carcinogenesis promoted by from the National Institutes of Health, the US Army Medical Research and Material
chronic inflammation is B lymphocyte dependent. Cancer Cell 7, 411–423 (2005). Command and Prostate Cancer Foundation. M.K. is an American Cancer Society
10. Mantovani, A., Allavena, P., Sica, A. & Balkwill, F. Cancer-related inflammation. Research Professor.
Nature 454, 436–444 (2008). Author Contributions M.A., J.-L.L. and M.K. designed the study; M.A., J.-L.L. and
11. Luo, J. L. et al. Nuclear cytokine activated IKKa controls prostate cancer S.G. performed research; S.N. provided lymphotoxin-deficient mice; M.A. and M.K.
metastasis by repressing maspin. Nature 446, 690–694 (2007). analysed data and wrote the paper.
12. Bai, A., Higham, E., Eisen, H. N., Wittrup, K. D. & Chen, J. Rapid tolerization of
virus-activated tumor-specific CD81 T cells in prostate tumors of TRAMP mice. Author Information Reprints and permissions information is available at
Proc. Natl Acad. Sci. USA 105, 13003–13008 (2008). www.nature.com/reprints. The authors declare no competing financial interests.
13. Ellwood-Yen, K. et al. Myc-driven murine prostate cancer shares molecular Correspondence and requests for materials should be addressed to M.K.
features with human prostate tumors. Cancer Cell 4, 223–238 (2003). (karinoffice@ucsd.edu).

305
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08782

METHODS and processed as described11, using antibodies for androgen receptor (Santa
Mice and cell culture. IkkbF/F (BL6) mice were crossed to TRAMP (BL63129) Cruz), B220 (BD Biosciences), CD20 (BD Biosciences), CD4 (BD Biosciences)
mice31 and PB-Cre4 (BL6)32 or Mx1-Cre (BL6) mice33 to generate TRAMP1/1/ and CD8 (BD Biosciences).
IkkbF/F and IkkbF/F/PB-Cre41/2 and IkkbF/F/Mx1-Cre1/2 progeny that were Analysis of RNA and protein expression. Total tissue RNA was prepared using
intercrossed with each other for six generations. After that, TRAMP1/1/ RNAeasy (Qiagen). Quantitative PCR was performed as described38. Cells and
IkkbF/F mice were crossed to IkkbF/F/PB-Cre41/2 to generate TRAMP1/2/ tumours were lysed and analysed by SDS–PAGE and immunoblotting38 with
IkkbF/F/PB-Cre41/2 and TRAMP1/2/IkkbF/F mice. TRAMP1/1/IkkbF/F mice antibodies to histone H3, a-tubulin, STAT3 (Santa Cruz Biotechnology), ERK,
were crossed to IkkbF/F/Mx1-Cre1/2 to generate TRAMP1/–/IkkbF/F/Mx1- phospho-ERK, AKT, phospho-AKT and phospho-STAT3 (Cell Signaling).
Cre1/2 and TRAMP1/2/IkkbF/F mice. Only male littermates were used. FVB, Nuclear extracts were prepared and analysed for NF-kB DNA binding as
Tcrb–/–d–/– (BL6), Mx1-Cre and Rag1–/– (BL63129) mice were from the Jackson described39.
Laboratory. JH–/– mice (FVB) were provided by L. Coussens (Cancer Research Lentiviral and retroviral transduction. siRNAs to mouse IKK-a, IKK-b and
Institute and Anatomic Pathology, University of California, San Francisco). LTbR mRNAs were cloned into pLSLPw, provided by I. Verma (the Salk
Bone marrow from B-Ltb–/– or T-Ltb–/– mice21 was provided by C. F. Ware Institute), and lentivirus stocks were prepared as described11. Virus-containing
(La Jolla Institute for Allergy and Immunology). PB-Cre4 and TRAMP mice supernatants were added to myc-CaP cells for 2 days with polybrene, and trans-
were from the Mouse Models of Human Cancer Consortium. Mice were main- duced cells were selected in 5 mg ml21 puromycin (Invitrogen).
tained under specific pathogen-free conditions, and experimental protocols were Leukocyte purification and flow cytometry. Peripheral blood mononuclear cells
approved by the University of California, San Diego Animal Care Program, were isolated by centrifugation on a double-layered Histopaque-Ficoll (GE
following National Institutes of Health guidelines. Radiation chimaeras were Lifescience) gradient. Splenic B and T lymphocytes were isolated by magnetic cell
generated as described34. In general, irradiated FVB mice were reconstituted with sorting with CD4, CD8 or CD19 antibodies conjugated to magnetic beads. Tumour-
bone marrow from different strains that had been backcrossed to the FVB back- infiltrating leukocytes were stained with CD45, B220, LTbR-Ig, TCRb, Gr1, CD4 and
ground for at least two generations. However, in the case of B-Ltb–/– and T-Ltb–/– CD8 fluorescent antibodies, as well as Aqua LIVE/DEAD dye (Molecular Probes)
mice, bone marrow donors were of the BL6 background, whose bone marrow did and analysed on a flow cytometer (Accuri C6 or Becton Dickinson LSR II).
not lead to a graft versus host response in irradiated FVB mice. Myc-CaP cells Statistical analyses. Results are expressed as means 6 s.e.m. or s.d. Data were
derived from the FVB background were provided by C. Sawyers (University of analysed by Student’s t-test and Kaplan–Meier survival analysis using the
California, Los Angeles and Memorial Sloan Kettering Cancer Center)35 and GraphPad Prism statistical program. Error bars depict the s.e.m. or s.d.
were cultured under standard conditions and confirmed to be mycoplasma free. P values . 0.05 were considered insignificant, 0.01–0.05 were considered
myc-CaP cells were injected subcutaneously into the flank of male FVB mice as significant, 0.001–0.01 were considered very significant and ,0.001 were con-
described35. Tumour growth was measured with a calliper. Surgical procedures sidered highly significant.
were as described35.
Human specimens. Anonymous human prostate, benign prostatic hyperplasia 31. Greenberg, N. M. et al. Prostate cancer in a transgenic mouse. Proc. Natl Acad. Sci.
USA 92, 3439–3443 (1995).
and prostate cancer frozen sections were provided by the Cooperative Human
32. Wu, X. et al. Generation of a prostate epithelial cell-specific Cre transgenic mouse
Tissue Network. Pathology reports were provided by Network for each sample. model for tissue-specific gene ablation. Mech. Dev. 101, 61–69 (2001).
CXCL13 and B-cell depletion and lymphotoxin inhibition. CXCL13 neutralizing 33. Kuhn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice.
antibody was purchased from R&D and administered intraperitoneally at 200 mg Science 269, 1427–1429 (1995).
per mouse as described36. Anti-CD20 was provided by Genentech and was admi- 34. Kim, S. et al. Carcinoma-produced factors activate myeloid cells through TLR2 to
nistered intraperitoneally at 250 mg per mouse. LTbR-Ig fusion protein was a gift stimulate metastasis. Nature 457, 102–106 (2009).
from Y.-X. Fu (University of Chicago) and was administered as described22. hIgG 35. Watson, P. A. et al. Context-dependent hormone-refractory progression revealed
and mouse IgG2a were purchased from Sigma-Aldrich and were used as controls. through characterization of a novel murine prostate cancer cell line. Cancer Res.
IKK-b inhibitors. ML120 was provided by Millennium and administered orally 65, 11565–11571 (2005).
as described37. IKK-b inhibitor IV was purchased from Calbiochem and was 36. Zheng, B. et al. CXCL13 neutralization reduces the severity of collagen-induced
arthritis. Arthritis Rheum. 52, 620–626 (2005).
injected through the tail vein as described14.
37. Izmailova, E. S. et al. Use of molecular imaging to quantify response to IKK-2
Histological procedures. Mouse prostate and CaP tissues and dissected meta-
inhibitor treatment in murine arthritis. Arthritis Rheum. 56, 117–128 (2007).
static tumours were immersed in 10% neutral buffered formalin before section- 38. Luo, J. L., Maeda, S., Hsu, L. C., Yagita, H. & Karin, M. Inhibition of NF-kB in cancer
ing and embedding in paraffin. Sections were stained and processed as cells converts inflammation-induced tumor growth mediated by TNFa to TRAIL-
described11, using haematoxylin and eosin stain, TUNEL assay kit or antibodies mediated tumor regression. Cancer Cell 6, 297–305 (2004).
for IKK-a (Imgenex), phospho-STAT3 (Cell Signaling) and CD19 (eBioscience) 39. Senftleben, U. et al. Activation by IKKa of a second, evolutionary conserved, NF-
as described38. Frozen sections of human and mouse origins were fixed in acetone kB signaling pathway. Science 293, 1495–1499 (2001).

©2010 Macmillan Publishers Limited. All rights reserved

 Vol 464 | 11 March 2010 | doi:10.1038/nature08788

LETTERS
JARID2 regulates binding of the Polycomb repressive
complex 2 to target genes in ES cells
Diego Pasini1,2{, Paul A. C. Cloos1,2*, Julian Walfridsson1,2*, Linda Olsson1,2, John-Paul Bukowski1,2,
Jens V. Johansen1, Mads Bak3, Niels Tommerup3, Juri Rappsilber4 & Kristian Helin1,2

The Polycomb group (PcG) proteins have an important role in nuclear extracts demonstrated that all components of the PRC2 com-
controlling the expression of genes essential for development, plex co-purified with JARID2 (Fig. 1b). The specificity of these results
differentiation and maintenance of cell fates1,2. The Polycomb was validated by co-immunoprecipitations using the same cells as in
repressive complex 2 (PRC2) is believed to regulate transcriptional Fig. 1b (Supplementary Fig. 1b) or in independent experiments (Sup-
repression by catalysing the di- and tri-methylation of lysine 27 on plementary Fig. 1c, d). Moreover, several other findings supported
histone H3 (H3K27me2/3)2. At present, it is unknown how the PcG the interaction between JARID2 and the PRC2 members. For
proteins are recruited to their target promoters in mammalian cells3. example, (1) Jarid2 expressed at sub-physiological levels in mouse
Here we show that PRC2 forms a stable complex with the Jumonji- ES cells (Supplementary Fig. 1e) associates with components of the
and ARID-domain-containing protein, JARID2 (ref. 4). Using PRC2 complex (Fig. 1c). (2) Endogenous Jarid2 associates with the
genome-wide location analysis, we show that JARID2 binds to more PRC2 complex (Fig. 1d). (3) The association between JARID2 and
than 90% of previously mapped PcG target genes. Notably, we show PRC2 is stable at high salt concentrations (Supplementary Fig. 1f).
that JARID2 is sufficient to recruit PcG proteins to a heterologous (4) Jarid2, Ezh2 and Suz12 co-elute in high molecular mass frac-
promoter, and that inhibition of JARID2 expression leads to a major tions in embryonic stem (ES) cells (Supplementary Fig. 2a). (5)
loss of PcG binding and to a reduction of H3K27me3 levels on target Recombinant Flag–HA–JARID2 binds to EZH2, EED and SUZ12
genes. Consistent with an essential role for PcG proteins in early in insect cells (Supplementary Fig. 2b, c). (6) Epitope-tagged JARID2
development5–8, we demonstrate that JARID2 is required for the co-localizes with the PRC2 components EZH2 and SUZ12, and with
differentiation of mouse embryonic stem cells. Thus, these results the PRC1 components RING1B and CBX8 in Polycomb bodies
demonstrate that JARID2 is essential for the binding of PcG proteins (Supplementary Fig. 3a). Taken together, these results strongly indicate
to target genes and, consistent with this, for the proper differenti- that JARID2 interacts with the core of the PRC2 complex, and that
ation of embryonic stem cells and normal development. JARID2 is found in a stable complex with PRC2 in vivo.
PcG proteins are essential for embryonic development5–8. They We generated several JARID2 mutants (Fig. 2a), and identified
execute their repressive functions in two distinct multiprotein com- that a small region of JARID2 (amino acids 147–165) is required
plexes named PRC1 and PRC2 (refs 2, 3) and share a large number of for the interaction with PRC2 (Supplementary Fig. 3b). Notably, this
common target genes9–11. The recruitment of the PRC1 complex is to region is part of a previously mapped transrepression domain of
a large extent dependent on the activity of the PRC2 complex12–14. In JARID2 (ref. 17) (Fig. 2a), suggesting that PRC2 binding could be
contrast, the mechanism by which mammalian PRC2 is recruited to important for the repression function of JARID2.
target genes is still enigmatic3. Stable expression of wild-type or D147–165 Gal4–JARID2 fusion
To identify proteins involved in PRC2 binding to DNA we decided proteins (Fig. 2b) in 293T cells that contain an integrated heterologous
to purify proteins that associate with the PRC2 complex. We per- Gal4/luciferase reporter construct, showed that wild-type JARID2, but
formed a Flag-immunopurification on nuclear extracts prepared not the D147–165 mutant, induces efficient repression of luciferase
from a HeLa cell line that stably expresses physiological levels of a activity (Fig. 2c and Supplementary Fig. 3c). Notably, chromatin
Flag–haemagglutinin (HA)–SUZ12 fusion protein (Supplementary immunoprecipitation (ChIP) analysis of the same cells showed that
Fig. 1a) and identified proteins that co-purify with SUZ12 by mass the recruitment to the luciferase promoter of wild-type but not of
spectrometry. This analysis identified JARID2 (Fig. 1a) and the proteins D147–165 Gal4–JARID2 is sufficient for the recruitment of SUZ12
previously reported to be components of PRC2 (refs 12, 14–16). and EZH2, and induces a strong enrichment of H3K27me3 and a loss
JARID2 is a transcriptional repressor17 and a member of the Jumonji of H3K4me3 (Fig. 2d). Moreover, the expression of wild-type Gal4–
C (JmjC) and ARID domain protein family18, which is essential for early JARID2 but not D147–165, also led to the recruitment of RING1B to
embryonic development4,19. Because the ARID domain of JARID2 can the same promoter, supporting the model of PRC2 dependency for
bind directly to DNA17,20, we speculated that JARID2 could be involved PRC1 association to target genes (Fig. 2d)13. Together, these data
in the recruitment of PRC2 to target genes. strongly indicate that JARID2 can actively recruit PRC2 and PRC1
While purifying PRC2-associated proteins, simultaneous studies activities to promoters, and that JARID2 association with the PRC2
in our laboratory were aimed at understanding the functional role of complex is essential for the transcriptional repression mediated by
JARID2. To this extent, we had generated a 293T cell line that stably JARID2.
expresses a Flag–HA–JARID2 fusion protein to purify JARID2- To determine whether Jarid2 could have a role in regulating PcG
associated proteins. HA-Flag tandem immunopurification from association with target genes, we determined the DNA-binding sites
1
Biotech Research and Innovation Centre (BRIC), 2Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark. 3Wilhelm Johannsen Centre
For Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark. 4Wellcome Trust Centre
for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK. {Present address: European Institute of Oncology, Department of Experimental Oncology, Via Adamello 16, 20141
Milan, Italy.
*These authors contributed equally to this work.

306
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a Protein Mass (Da) Peptides Score c Inputs Flag-IP a b

PRC2 Repression Δ147–
SUZ12 83,744 58 3,490 E14 E14c6 E14 E14c6 binding domain Gal4–JARID2 – WT 165
EZH2 87,959 45 2,896 JmjN
JARID2 140,301 24 1,015 Jarid2 + 1 ARID JmjC zf 1246 Gal4
EZH1 87,325 21 991 (HA/Flag) JmjN
EED 50,907 17 701 + 1 JmjC zf 1246
Δ618–728 β-Tubulin
AEBP2 53,242 10 421
JmjN
RBBP4 47,780 9 406 Ezh2
RBBP7 48,132 7 299
+ 1 ARID zf 1246
Δ916–1031
HDAC2 66,294 6 207 JmjN
+ 84 ARID JmjC zf 1246
b Suz12 c
Protein Mass (Da) Peptides Score JmjN

Relative luciferase activity

JARID2 140,301 103 5,436 – 161 ARID JmjC zf 1246 1.2

SUZ12 83,744 41 2,172 1.0

EZH2 87,959 37 2,041 + 1 352
JmjN 0.8
EED 50,907 24 1,117
AEBP2 53,242 13 804
– 1 ARID JmjC zf 1246 0.6
Eed Δ84–160 0.4
EZH1 87,325 3 142 JmjN
RBBP7 48,132 6 306 + 1 ARID JmjC zf 1246 0.2
HDAC2 66,294 3 116 Δ106–120
JmjN
RBBP4 47,780 2 69 – WT Δ147–165
– 1 ARID JmjC zf 1246
Gal4–JARID2
IPs Δ147–165
d Ezh1
Input Ctrl Suz12 Ezh2
d 3.5 IgG Gal4 1.6 IgG SUZ12 4.0 IgG EZH2
Jarid2

Percentage of input

Percentage of input

Percentage of input
3.0 1.4 3.5
Ring1b 2.5 1.2 3.0
1.0 2.5
Suz12 2.0
0.8 2.0
1.5
0.6 1.5
1.0 0.4 1.0
Figure 1 | JARID2 is a component of the PRC2 complex. a, b, Summary of 0.5 0.2 0.5
the peptides identified by mass spectrometry from an anti-Flag purification
Gal4–JARID2 – WT Δ147– – WT Δ147– – WT Δ147–
of Flag–HA–SUZ12 stably expressed in HeLa cells (a) and from a Flag–HA 165 165 165
tandem purification of Flag–HA–JARID2 expressed in 293T cells (b). Mass,

Percentage of histone H3

Percentage of histone H3
0.2 IgG RING1B 35 IgG H3K27me3 80 IgG H3K4me3
observed peptides and Mascot score are indicated for each protein.
Percentage of input

30 70
c, Endogenous PRC2 components, but not the PRC1 component Ring1b, is 25 60
associated with ectopically expressed Flag–HA–JARID2. A representative 20
50
0.1 40
western blot, using antibodies to the indicated proteins, of lysates prepared 15 30
from E14 ES cells with stable sub-physiological expression of 10 20
Flag–HA–JARID2 (E14c6) (Supplementary Fig. 1d). Two per cent of input is 5 10
presented as loading control. d, Association of endogenous Jarid2 with the
Gal4–JARID2 – WT Δ147– – WT Δ147– – WT Δ147–
endogenous PRC2 complex. A representative western blot using antibodies 165 165 165
specific for the indicated proteins of cell extracts prepared from mouse ES
cells. Antibodies specific for Suz12, Ezh2 and haemagglutinin (negative Figure 2 | JARID2-mediated transcriptional repression is dependent on
control (ctrl)) were used for immunoprecipitations (IPs); where indicated PRC2 binding. a, JARID2 amino acids 147–165, previously mapped to be
2% of input is presented as loading control. part of the transcriptional repression domain of JARID2, is required for
interaction with the PRC2 complex. Schematic representation of the
different JARID2 mutants used in Supplementary Fig. 3b, highlighting the
of Jarid2 and Suz12 in mouse ES cells by ChIP followed by DNA
overlap between the previously mapped transcriptional repression domain
sequencing (ChIP-seq). This analysis showed that Jarid2 and Suz12 with the amino acids required for PRC2 binding. zf, zinc finger. b, Western
have highly comparable binding profiles at target gene loci (Fig. 3a, e blot analysis of 293T cells with a stably integrated Gal4-luciferase reporter
and Supplementary Fig. 4) and 90% of the nucleotides bound by construct, with or without expression of wild-type (WT) and D147–165
Jarid2 are also bound by Suz12 (Fig. 3b). In agreement with these Gal4–JARID2 fusions. The indicated antibodies were used to detect the
results, Jarid2 and Suz12 also share a large number of target genes, expressed proteins. c, JARID2 amino acids 147–165 are required for JARID2
which are almost identical to previously mapped genes for the PRC2 transcriptional repression. Average luciferase activity of different cell clones
complex (Fig. 3c and Supplementary Fig. 5a)9,21. Consistent with the expressing either wild-type or D147–165 Gal4–JARID2. Luciferase activity
was normalized to the amount of protein and presented as the fold difference
overlap with PcG targets, the Jarid2 target genes also associate with
relative to 293T cells not expressing Gal4–JARID2. d, JARID2 amino acids
H3K27me3 (Fig. 3e and Supplementary Figs 4 and 5b), show some 147–165 are required for the recruitment of PRC2 to DNA. Quantitative
overlap with H3K4me3 as reported for PcG proteins (Supplementary ChIP analysis, by real-time qPCR, using primers specific for the luciferase
Fig. 5c—so-called bivalent genes), but do not overlap with transcription start site of the cells presented in c. ChIP was performed using
H3K36me3 (Supplementary Fig. 5d) or H3K9me3 (data not shown). the indicated antibodies. Purified rabbit IgG was a negative control.
Moreover, in agreement with being PcG target genes, Jarid2 binds to Percentage of input indicates bound/input 3 100. All error bars denote s.d.,
a large number of genes that encode transcription factors and signal- n 5 3.
ling proteins that have essential roles in regulating proper develop-
ment (Supplementary Fig. 5e). (Fig. 3d). ChIP analysis followed by real-time quantitative PCR
To test the specificity of the Jarid2 ChIP-seq and validate the Jarid2 (qPCR) using short interfering RNA (siRNA) and shRNA sequences
target genes, we established ES cells stably expressing Jarid2 short targeting different regions of Jarid2 (Supplementary Fig. 6a, c) further
hairpin RNA (shRNA). The Jarid2 shRNA led to a significant reduc- validated the ChIP-seq results (Supplementary Fig. 6b, d). Notably,
tion of Jarid2 expression without leading to detectable changes in the inhibition of Jarid2 expression also led to a strong reduction of
expression of the PRC2 complex (Supplementary Fig. 5f). As sum- Ezh2 and Suz12 binding to target genes and, consistent with this, to a
marized in Fig. 3d and Supplementary Table 1, and also shown for reduction of H3K27me3 levels at promoters (Fig. 3h and Sup-
selected genes in Fig. 3e and Supplementary Fig. 6, Jarid2 inhibition plementary Fig. 6d). The expression of Ezh2, Suz12 and Eed (Sup-
led to a complete loss of gene-specific enrichment using the Jarid2 plementary Figs 5f, 6a, c and 7a) was not affected by the inhibition of
antibody. Notably, inhibition of Jarid2 expression also led to a more Jarid2 expression; however, consistent with a role for Jarid2 in recruit-
than tenfold reduction of Suz12 binding to 90% of its target genes ing the PRC2 complex to target genes, the global levels of H3K27me3
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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 11 March 2010

a Peaks at b c d f Flag–JARID2 – – + – g 12 12
Fzd1
14
promoters WT IgG IgG IgG
Covered Reduced and/or lost Ezh2
Peaks not at Flag–JARID2 – – – + 10 Jarid2 10 Suz12 12
Not covered Not reduced ΔARID
promoters 10
Suz12 shRNA SCR Jarid2 Jarid2 Jarid2 8 8
100 100 4,568 100 8
90 90 2,697 6 6
169 90 Jarid2 6
70% 73% 89% 69% 90% 96% 98% 100%
80 80 4 4
80
Nucleotide coverage (%)

Percentage of input
4
70 70 2 2
70 2
Total peaks (%)

Flag

Total peaks (%)

60 60 1,054 60
70
50 50 50 Hoxd9
3 666 Ezh2 5 8 8
IgG IgG IgG
40 40 40 7 7
Jarid2 14 4 Jarid2 Suz12 Ezh2
30 30 1,296 6 6
Ezh2 30 Suz12
(ref. 21) 3 5 5
20 20 1,737 20
4 4
10 10 10 2
Eed 3 3
0 2 2
ChIP Jarid2 v Suz12 Not 10-fold 5-fold 3-fold Not 1
6

6
–1

–1
2× 2

2× 2

P<2.2×10–16 detect detect 1 1

2. uz1

2. arid
10

10
P< S

P< J

ChIP Suz12 Jarid2

β-Tubulin + – – – + – – –
SCR v Jarid2 shRNA shSCR + – – –
shJarid2 – + + + – + + + – + + +
HA–Flag–JARID2 WT – – + – – – + – – – + –
e H3K36me3
20 Bmp6 locus 20 Fzd1 locus 20 Fgf4 locus JARID2 ΔARID – – – + – – – + – – – +
(ref. 29) 10 10 10 h Fzd1
0 0 0 120 IgG Jarid2 120 IgG Suz12 120 IgG Ezh2 120 IgG K27me3
20 20 40
H3K4me3 100 100 100 100
(ref. 29) 10 10 20
0 0 0 80 80 80 80
20 20 20

Percentage of fold difference SCR v RNAi

H3K27me3 10
10 10 60 60 60 60
(ref. 29)
0 0 0
40 40 40 40 40 40 40
Ezh2
(ref. 21) 20 20 20
20 20 20 20
0 0 0
20 20 20
Jarid2
(shSCR) 10 10 10 Hoxd9
0 0 0 120 IgG Jarid2 120 IgG Suz12 120 IgG Ezh2 120 IgG K27me3
20 20 20
Jarid2 100 100 100 100
(shJarid2) 10 10 10
0 0 0 80 80 80 80
Suz12
50 50 50
(shSCR) 60 60 60 60
0 0 0
40 40 40 40
Suz12 50 50 50
(shJarid2) 20 20 20 20
0 0 0
Bmp6 Fzd1 Fgf4 shSCR + – + – + – + – + – + – + – + –
shJarid2 – + – – – + – – – + – – – + – –
38430000 38440000 4750000 4755000 4760000 152045000 152050000 152055000 shSuz12 – – – + – – – + – – – + – – – +

Figure 3 | JARID2 and PRC2 associate with a very similar set of genes in ES obtained from previous studies are indicated for comparison21,29. The y-axis
cells. a, Jarid2 and Suz12 preferentially associate with promoter regions, as denotes sequence tags read. f, Western blot analysis, using indicated
shown by the percentage binding obtained using ChIP-seq. False discovery antibodies, of mouse ES cells expressing non-specific (SCR) or Jarid2-
rate (FDR) , 0.1 from ChIP analyses of ES cells using Suz12- and Jarid2- specific shRNAs before and after transient expression of wild-type
specific antibodies. b, Nucleotide coverage of Jarid2 over Suz12 binding Flag–HA–JARID2 and DARID mutant. b-Tubulin is used as a loading
regions, showing a highly significant overlap between Jarid2- and Suz12- control. g, The ARID domain of JARID2 is required for JARID2 binding to
binding sites. c, Venn diagram showing overlap of Jarid2, Suz12 and Ezh2 target genes. ChIP analysis of Fzd1 and Hoxd9 promoters in the ES cells
target genes identified by ChIP-seq analysis in ES cells. d, The percentage of presented in f using indicated antibodies. Purified rabbit IgG was a negative
Suz12 and Jarid2 (right column) peaks that are not detected or reduced in control. h, JARID2 requires the PRC2 complex for efficient association to
intensity after ChIP-seq analysis in Jarid2-shRNA-expressing ES cells. A target genes. ChIP analysis of Fzd1 and Hoxd9 promoters in the ES cells
scrambled shRNA sequence (SCR) was used as negative control for Jarid2 presented in f and in Supplementary Fig 6b using indicated antibodies.
depletion. Different intensity cutoffs for Suz12 peaks are presented. The Purified rabbit IgG was a negative control. The percentage fold difference of
total number of reads present in each individual peak defines peak intensity. SCR versus RNA interference (RNAi) is calculated as the difference in
An equal number of aligned sequences was used to compare ChIP signals. percentage of the ChIP signal in ES cells expressing Jarid2-specific shRNA
e, Examples of ChIP-seq results for the indicated loci in ES cells expressing relative to cells expressing a non-specific (SCR) shRNA. All error bars denote
non-specific and Jarid2-specific shRNAs. Binding profiles of ChIP- s.d., n 5 3.
sequencing results for Ezh2, H3K27me3, H3K4me3 and H3K36me3

and H3K27me2 (Supplementary Fig. 7a) were decreased. In agree- regulates the binding of PRC2 to its target genes and that this is
ment with the notion that PRC2 activity is required for PRC1 asso- dependent on its ARID domain.
ciation to target genes, loss of Jarid2 also led to a major displacement To understand if JARID2 recruitment of PRC2 is a two-step process
of the core subunit of the PRC1 complex Ring1b (Supplementary or if the PRC2–JARID2 complex may be recruited as one entity, we
Fig. 6b, d). Taken together, these data demonstrate that JARID2 is tested whether JARID2 can associate with its target genes in the absence
required for the binding of the PRC2 and PRC1 complexes to their of the PRC2 complex. ChIP-qPCR analyses performed from Suz12 and
target genes. Jarid2 (control) shRNA-expressing ES cells (Supplementary Figs 7a
To test whether the ARID domain of Jarid2 is involved in the and 8a) show that inhibition of Suz12 expression led to a significant
binding of PRC2 to target genes, we transiently expressed wild-type decrease in Jarid2 association with its target genes (Fig. 3h and
and DARID JARID2 (Fig. 2a) in ES cells expressing Jarid2 shRNA Supplementary Fig. 8b). Notably, however, the inhibition of Suz12
(Fig. 3f). As shown in Fig. 3g and Supplementary Fig. 7b, the re- expression led to a significant downregulation of Jarid2 levels in the
expression of wild-type, but not DARID mutant JARID2, was able ES cells (Supplementary Fig. 8a), suggesting that the stability of
to partly rescue both JARID2 and PRC2 binding to target genes. Jarid2—as has been shown for members of the PRC2 complex7—is
Importantly, the loss of Jarid2 does not disrupt the formation of regulated by complex formation. In agreement with this notion, the
the core of the PRC2 complex or affect its enzymatic activity inhibition of Suz12 expression does not lead to a decrease in Jarid2
(Supplementary Fig. 7c). These results demonstrate that JARID2 messenger RNA levels (Supplementary Fig. 8c). Taken together, these
308
©2010 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 464 | 11 March 2010 LETTERS

a SCR shRNA Jarid2 shRNA b shJarid2

– +
c Jarid2 Fgf4 Oct4
(pluripotent) (pluripotent)

Fold difference
Jarid2 1.2 1.4
1.0 1.2 1.4
1.0 shCtrl
Day 2 Ezh2 0.8 1.0
0.6 0.8 shJarid2
0.6 0.6
Suz12 0.4 0.4
0.2 0.2 0.2
β-Tubulin Days 0 10 0 10 0 10
Day 7
Msi1 Nes Olig1 Olig2
(ectoderm) (ectoderm) (ectoderm) (ectoderm)

Fold difference
25 35 20 2,500
20 30 16 2,000
25
15 20 12 1,500
Day 10 10 15 8 1,000
5 10 4 500
5
0
Days 0 10 0 10 0 10 0 10
d 1,019 shCtrl shJarid2
Foxa2 Gata4 (endo/ Pax3 Hoxa4
815 (endoderm) mesoderm) 6,000 (mesoderm) 1,000

Fold difference
498 120 30
100 25 5,000 800
453 80 20 4,000 600
204 60 15 3,000
92 45 40 2,000 400
29 10
296 74 20 5 1,000 200
0 0 0
Upregulated genes Downregulated genes Days 0 10 0 10 0 10 0 10

Figure 4 | Jarid2 is required for ES cell differentiation. a, Jarid2 is required shRNA. qPCR analysis of the indicated genes during ES cell differentiation of
for ES cell differentiation. Pictures of embryoid bodies formed from ES cells the cells presented in a. Oct4 is also known as Pou5f1. Error bars denote s.d.,
expressing non-specific or Jarid2-specific shRNAs. Original magnification, n 5 3. d, Downregulation of Jarid2 changes the gene expression pattern in ES
35. b, Western blot analysis of ES cells expressing non-specific and Jarid2- cells induced to differentiate. Venn diagrams of the genome-wide expression
specific shRNAs used in a and c. The western blots were probed with the profiles of protein-coding genes significantly altered 10 days after induction
indicated antibodies. b-Tubulin is presented as a loading control. of ES cells expressing control or Jarid2 shRNAs.
c, Differentiation markers are not induced in ES cells expressing Jarid2

results indicate that JARID2 is in a stable complex with PRC2, and that is a critical regulator of PRC2 binding to target genes. This is further
the JARID2–PRC2 complex is recruited to the PcG target genes as one supported by the ability of JARID2 to directly recruit PRC2 activity
entity depending on the DNA-binding ARID domain of JARID2. This to an artificial promoter (Fig. 2d). Previous studies have identified
suggestion is supported by the ChIP-seq data demonstrating that PRC2 several DNA sequences binding to the ARID domain of JARID2
is always present at the same genomic positions of JARID2. (ref. 17). The diversity of these sequences suggests that JARID2 might
PRC2 activity is not required for the proliferation of ES cells, but is have a broader specificity for DNA, and they could indicate that
essential for the ability of ES cells to differentiate22–24. To determine the association of PRC2 with specific target genes is not only deter-
whether Jarid2 shares common biological functions with PcG proteins, mined by JARID2, but also in combination with other transcription
we challenged ES cells that stably express Jarid2-specific shRNAs factors and by the affinity of PRC2 for H3K27me2/3 (refs 26, 27).
(Fig. 4b) to undergo differentiation by forming embryoid bodies in Furthermore, the requirement of Jarid2 for ES cell differentiation,
suspension cultures. Although Jarid2-depleted ES cells showed no pro- but not for ES cell self-renewal, is consistent with the biological func-
liferation defects and formed embryoid bodies with similar efficiency tions of PcG proteins8,22–24 and the embryonic lethality observed in
as control cells (Fig. 4a, day 2), the prolonged differentiation of Jarid2- Jarid2 knockout mice4,19, further illustrating the critical role of JARID2
depleted embryoid bodies was severely compromised when compared in regulating PcG function.
to control cells (Fig. 4a, days 7 and 10, and Supplementary Fig. 9a,
day 7). The failure of proper differentiation shown by the Jarid2- METHODS SUMMARY
ES cells. ES cells were grown, manipulated and differentiated as previously
depleted embryoid bodies is consistent with the lack of transcriptional
described22,28. In brief, undifferentiated ES cells were induced to differentiate
activation of differentiation markers in the same cells (Fig. 4c and into embryoid bodies by hanging-drop formation, and were stimulated with
Supplementary Fig. 9b). This result was further confirmed by the 0.5 mM all-trans-retinoic acid (ATRA) between days 2 and 5 of differentiation.
genome-wide expression analyses showing that both the activation Embryoid bodies were analysed at the indicated times.
and the repression of differentiation-regulated genes are strongly Protein purification. For each purification, 100–200 mg of nuclear extracts were
compromised during the differentiation of Jarid2-depleted ES cells used. In brief, nuclei were lysed in high salt buffer (300 mM NaCl) and incubated
(Fig. 4d). Together, these results indicate that Jarid2 and PcG proteins overnight with M2 Flag-agarose beads (Sigma). Beads were washed six times in
share common biological functions, and demonstrate that Jarid2, high salt buffer and purified proteins were eluted with 0.5 mg ml21 of Flag
similar to PcG proteins, is required for correct differentiation of ES peptide. When required, eluted proteins were incubated overnight with protein
A-Sepharose beads cross-linked with haemagglutinin (12CA5)-specific antibodies,
cells.
washed six times in high salt buffer and purified proteins were eluted with
The JARID2 protein is highly conserved throughout evolution 0.5 mg ml21 haemagglutinin peptide. Eluted proteins were identified by mass
(Supplementary Fig. 10a) and belongs to a family of ARID- and spectrometry.
Jumonji-domain containing proteins together with JARID1A–D (also ChIP and ChIP-sequencing. ChIP experiments were performed as described28.
known as KDM5A–D; Supplementary Fig. 11c). However, JARID2 is Chromatin immunoprecipitated DNA was prepared for sequencing following
only distantly related to members of the JARID1 family and shows the Illumina recommended procedure, and sequenced using the Genome
a low degree of similarity (<20%, Supplementary Fig. 11d, e), par- Analyser II (Illumina). Two mismatches within the first 32 bases of a read were
ticularly within the highly conserved PRC2-binding region (<10%, used as cutoff for mapping against the mouse mm9 genome. Enriched regions
were determined using CisGenome software and genome-wide analysis per-
Supplementary Fig. 10a, b). The members of the JARID1 family func-
formed using the Galaxy (http://galaxy.psu.edu/) application.
tion as specific H3K4me3/2 demethylases, and we previously reported
that the activity of one of the members of this family, JARID1A, is Full Methods and any associated references are available in the online version of
involved in PcG-repressive functions but is not required for PcG the paper at www.nature.com/nature.
binding to target genes25. In contrast, JARID2 is probably not acting Received 28 July 2009; accepted 5 January 2010.
as a hydroxylase and hence a histone demethylase, owing to the lack Published online 14 January 2010.
of conservation of two essential histidine residues in the HXD/EXnH
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12. Cao, R. et al. Role of histone H3 lysine 27 methylation in Polycomb-group Acknowledgements We thank K. Hansen for the RING1B antibody, J. Vikesaa for
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14. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. D.P. was supported by a post-doctoral fellowship from the Danish Medical
Histone methyltransferase activity associated with a human multiprotein complex Research Council, P.A.C.C. by a grant from the Benzon Foundation and J.W. by a
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15. Czermin, B. et al. Drosophila enhancer of Zeste/ESC complexes have a histone H3 Fellow of the Wellcome Trust. The Wilhelm Johannsen Center is supported by the
methyltransferase activity that marks chromosomal Polycomb sites. Cell 111, Danish National Research Foundation and the Lundbeck Foundation. The work in
185–196 (2002). the Helin laboratory was supported by grants from the Danish National Research
16. Müller, J. et al. Histone methyltransferase activity of a Drosophila Polycomb group Foundation, the Danish Cancer Society, the Novo Nordisk Foundation, the Danish
repressor complex. Cell 111, 197–208 (2002). Medical Research Council, and the Danish Natural Science Research Council.
17. Kim, T. G., Kraus, J. C., Chen, J. & Lee, Y. JUMONJI, a critical factor for cardiac
development, functions as a transcriptional repressor. J. Biol. Chem. 278, Author Contributions D.P. and K.H. conceived and designed the project. D.P.
42247–42255 (2003). performed experiments in Figs 1a, c, d, 2a, 3 and 4 and Supplementary Figs 1a, c–f,
18. Cloos, P. A., Christensen, J., Agger, K. & Helin, K. Erasing the methyl mark: histone 2a, b, 3b and 4–9. P.A.C.C. designed and performed the experiments in Fig. 1b and
demethylases at the center of cellular differentiation and disease. Genes Dev. 22, Supplementary Figs 1b and 3a. J.W. performed experiments in Fig. 2b–d,
1115–1140 (2008). Supplementary Figs 2c and 3a. J.-P.B. cloned JARID2. L.O. provided technical
19. Takeuchi, T. et al. Gene trap capture of a novel mouse gene, jumonji, required for support. M.B. and N.T. performed the Solexa sequencing. J.R. performed mass
neural tube formation. Genes Dev. 9, 1211–1222 (1995). spectrometry analyses. J.V.J. provided bioinformatics support. D.P. and K.H. wrote
20. Kim, T. G., Chen, J., Sadoshima, J. & Lee, Y. Jumonji represses atrial natriuretic the manuscript.
factor gene expression by inhibiting transcriptional activities of cardiac
transcription factors. Mol. Cell. Biol. 24, 10151–10160 (2004). Author Information ChIP-seq data are available at the Gene Expression Omnibus
21. Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two (GEO) under accession GSE19365. Reprints and permissions information is
classes of bivalent domains. PLoS Genet. 4, e1000242 (2008). available at www.nature.com/reprints. The authors declare competing financial
22. Pasini, D., Bracken, A. P., Hansen, J. B., Capillo, M. & Helin, K. The Polycomb group interests: details accompany the full-text HTML version of the paper at
protein Suz12 is required for embryonic stem cell differentiation. Mol. Cell. Biol. 27, www.nature.com/nature. Correspondence and requests for materials should be
3769–3779 (2007). addressed to K.H. (kristian.helin@bric.ku.dk).

310
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08788

METHODS Protein purifications and immunoprecipitations. Immunoprecipitations were

performed using 1 mg of cell extracts prepared in high salt buffer as described
Cloning and plasmid preparation. Human JARID2 and the different mutations
earlier using the indicated antibodies. Complex purifications were carried out
were PCR-amplified from Open Biosystems image clone 4520786, introduced
from nuclear extracts prepared in high salt buffer as described earlier. Protein
into the Gateway Entry vector pCR8/GW/TOPO (Invitrogen) following the
extracts (100–200 mg) were incubated overnight at 4 uC on a rotating platform
manufacturer’s protocol and sequenced verified. Different constructs were
with 200 ml packed M2-Flag agarose beads. Beads were washed six times in
subcloned in the desired vectors by Gateway technology (Invitrogen). pBABE-
high salt buffer and proteins were eluted with 0.5 mg ml21 of a Flag peptide
Flag-HA SUZ12 was subcloned by EcoRI digestion from a previously described
(DYKDDDDK). When indicated, Flag-eluted proteins were incubated with
pCMV-SUZ12 expression vector30. Three silent mutations in the targeting
200 ml of packed anti-haemagglutinin (12CA5) cross-linked protein-A
sequence of wild-type JARID2 and D147–165 mutant were introduced by site-
Sepharose beads overnight at 4 uC on a rotating platform, washed six times in
directed mutagenesis to obtain an RNAi refractory JARID2 mRNA sequence.
high salt buffer, and eluted with 0.5 mg ml21 of a haemagglutinin peptide
Point mutations correspond to positions 2 and 4 (CGCRAGA, Arg 1050) and
(YPYDVPDYA). Eluted proteins were analysed by nanoelectrospray tandem
position 10 (ATCRATA, Ile 1052) of the Jarid2 shRNA targeting sequence.
mass spectrometry32.
RNA interference. Stable shRNA experiments were performed as described28.
ChIP and ChIP-sequencing. ChIP analyses were performed as described28. For
LKO.1 vectors expressing control (scrambled) or Jarid2-specific shRNA
ChIP-seq, the DNA from three independent ChIP experiments was pooled and
sequences were purchased from Sigma. When required, the puromycin-selection
prepared for sequencing with the Illumina ChIPSeq Sample Prep kit. DNA
cassette of LKO.1 vectors was exchanged with a neomycin-selection cassette by a
libraries were quantified using a fluorometer (Fluoroscan) and diluted to
BamHI-KpnI subcloning. Transient siRNA transfection was obtained with
5–10 pM. Diluted libraries were used directly for cluster generation and sequencing
Lipofectamine 2000 (Invitrogen) with a scrambled sequence (UNC) or Jarid2-
analysis using the Genome Analyser II (Illumina) following the protocol of the
specific siRNA oligonucleotides purchased from Sigma. Jarid2 siRNA oligonu- manufacturer. Base calling and mapping to the mouse genome (mm9, July 2007
cleotide (Sigma catalogue SASI_Mm01_00093112) targets Jarid2 mRNA release) of the 36-bp sequences were done using Illumina’s analysis Pipeline v1.4
sequence (NCBI accession NM_021878) around base pair (bp) position 1085. allowing up to two mismatches in the first 32 bases of a read. The enriched genomic
Jarid2 shRNA LKO.1 lentiviral vector (TRCN0000096642) targets Jarid2 mRNA regions from our ChIP-seq experiments and the sequencing results from previ-
with the following sequence 59-CCGCCATATAGCTAAGCCATT-39 starting ously published ChIP-seq data sets were determined using CisGenome software33
from base-pair position 3332. The Suz12 shRNA letiviral vector has been by two-sample analysis using the IgG ChIP-signal as a negative control.
described28. CisGenome two sample analysis determines regions where the ChIP signals are
Tissue culture. HeLa, HeLa S3, 293T, Flp-In T-REx 293 and U2OS cells were enriched relative to the control ChIP (IgG) using a conditional binomial model
grown in 10% FBS (Hyclone). Stable Flp-In T-REx 293 cell lines were generated that involves 100-bp windows passing a 0.1 FDR cut-off. A detailed description of
following manufacturer’s instructions. Doxycycline (200 ng ml21; Sigma) was CisGenome statistical analysis has been described33.
added for biochemical purification. ES cells (E14 clone) were cultured as We have annotated 22,064 promoter regions as the 65 kb chromosomal regions
described22. Stable ES cell clones were generated by transient transfection from the transcription start site of the refseq gene data set (mm9) extracted from the
(Lipofectamine 2000, Invitrogen) of the expression constructs followed by selec- UCSC database (http://genome.ucsc.edu) using Galaxy application (http://galaxy.
tion with 2 mg ml21 of puromycin and single-cell clone isolation. psu.edu). Target genes were defined as the promoter regions that overlapped with at
ES cells expressing Jarid2 shRNA from a LKO.1-neomycin vector were trans- least one enriched genomic region in the different ChIP-seq data sets. All location
fected with pCAG-iresPRURO vectors expressing wild-type JARID2 and DARID analyses were performed using the Galaxy application (http://galaxy.psu.edu).
using Lipofectamine 2000. Cells were cultured for 48 h after transfection and P values were determined by chi-square test. The intensity of the ChIP enriched
selected for a further 18 h with 5 mg ml21 puromycin. Fifty-six hours after trans- regions is defined by the total number of reads included in each peak.
fection samples were collected for western blot and ChIP analyses. Different ES Real-time qPCR. qPCR analysis for expression and ChIP assays were carried out
cell lines were induced to differentiate as previously described22. using Applied Biosystems Real-Time PCR Systems. Primer sequences are listed
Antibodies. Antibodies are listed in Supplementary Table 3. in Supplementary Table 3.
Size-exclusion chromatography and immunoprecipitations. Nuclear extracts Gene expression analysis. Total RNA was extracted with the Qiagen RNAesy
were prepared as described28, lysed in high salt buffer (50 mM Tris-HCl, pH 7.6, Plus RNA extraction kit. RNA was hybridized on mouse Gene 1.0 ST arrays by
300 mM NaCl, 10% glycerol, 0.2% (v/v) Igepal CA 630) and fractionated on a the RH Microarray Center at Rigshospitalet, Copenhagen, following Affymetrix
Superose 6 PC 3.2/30 (GE Healthcare). To test the stability of the Jarid2–PRC2 procedures and analysis. RNA normalized signals at day 0 were used as a baseline
interaction, the salt concentration of the nuclear extracts was adjusted to the for comparison.
indicated concentration and equilibrated for 2 h at 4 uC on a rotating platform.
Equilibrated extracts were incubated for 2 h with antibodies pre-coupled to 30. Villa, R. et al. Role of the polycomb repressive complex 2 in acute promyelocytic
protein G Sepharose at 4 uC on a rotating platform. After six washes the immu- leukemia. Cancer Cell 11, 513–525 (2007).
31. Christensen, J. et al. RBP2 belongs to a family of demethylases, specific for tri-and
noprecipitated proteins were analysed by western blots.
dimethylated lysine 4 on histone 3. Cell 128, 1063–1076 (2007).
Recombinant proteins. Recombinant baculoviruses were generated as 32. Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by
described31. EZH2, EED and SUZ12 baculoviruses have been described7. Flag/ nano-electrospray mass spectrometry. Nature 379, 466–469 (1996).
histidine-tagged JARID2 was expressed and purified by Flag-affinity chromato- 33. Ji, H. et al. An integrated software system for analyzing ChIP-chip and ChIP-seq
graphy essentially as described31. data. Nature Biotechnol. 26, 1293–1300 (2008).

©2010 Macmillan Publishers Limited. All rights reserved

 CAREERS NATURE|Vol 464|11 March 2010

PROSPECTS

Speak up
Peter Fiske argues that too many young scientists adopt a passive voice, to the detriment of their careers.
When I was in graduate school studying pattern of passivity among young scientists. Scientists must communicate about
geology and environmental sciences, many As a result, few of them pursue such their work — to other scientists, sponsors
of my professors insisted that we students collaborations on their own. of their research and the general public.
write our manuscripts in the passive voice: This ‘passive voice’ also arises when Active communication means more than
“This was done” rather than “I did this”. They engaging in professional networking. Most merely accepting invitations to give talks
reasoned that removing the agent from the scientists recognize that networking with at other institutions. An ‘active voice’
description of the action lent an objective colleagues has a crucial role in career in communication means searching for
tone. As scientists, we stood apart from our development. But rather than proactively opportunities to give talks and lectures
work and encouraged others to critique it reaching out to others, many wait to — and seeking audiences that are outside
(rather than us). receive such gestures. Networking is both one’s immediate sphere of scientific influence
Today, teachers are much more likely to acceptable and valued in the business world, at, for example, science museums or local
advocate use of the active voice civic organizations.
A. RUGGIERI/IMAGES.COM/CORBIS

in manuscripts. In general, the

active voice is more readable Public perceptions
and engaging, which can be Scientists may also have passive
particularly helpful in light of attitudes towards political
the sometimes turgid prose of advocacy. Many scientists are
scientific papers. incredulous at how little the
In the years since I moved from general public knows about
academic science to business, science and technology.
young scientists have started to A survey carried out by the Pew
adopt a more active tone in their Research Center for the People
manuscripts. Still, the culture of and the Press in Washington DC
science still seems to encourage last July found that 85% of US
a ‘passive voice’ in much of the scientists interviewed saw the
rest of their careers. Peers and public’s lack of knowledge as a
mentors often imply that they major problem for science. Half of
should remain at the bench them fault the public for having a
rather than actively reach out to poor understanding of the pace of
potential sponsors, supporters scientific discovery.
and collaborators. Young But scientists do little to
scientists think that doing good address the gap in understanding.
science will be enough to advance Most think that their successes
their careers. “If you’re the best, in the lab are manifestly evident,
you’ll get a job” is the assurance making education about the
they receive. value of their work unnecessary.
Few ever communicate with
Modesty or arrogance? their elected officials. With the
In life, as in writing, a passive public footing most of the bill, this
approach may be intended to misguided belief seems naive and
suggest a degree of objectivity. undermines those who campaign
Avoiding the appearance of ‘self- for more funding.
promotion’ may seem desirable In a global recession, taking a
for establishing credibility. But failure to make but many scientists and engineers view it passive career approach poses an increasing
any attempt to advocate for oneself and one’s as ‘schmoozing’. Those who try to forge disadvantage. Stronger competition for
abilities can be misinterpreted as indifference, close ties to their administration or financial a finite number of research positions will
even arrogance. sponsors may earn a reputation for being favour those who combine outstanding work
Collaboration is a case in point. Working ‘political’ among jealous colleagues. Younger with an ability to engage their communities
with researchers in other departments scientists witness this pathological behaviour and constituencies. Excellent work is a
and institutions is by far the most effective and quickly absorb the lesson: ‘true’ scientists prerequisite for career progress, but is
way for young scientists to advance their are above networking. not sufficient by itself. Broadcasting one’s
careers. Yet few PhD and postdoc advisers Although scientists may increasingly accomplishments and exercising the ‘active
encourage their charges to pursue any such write their papers in the active voice, the voice’ in all aspects of one’s work is the best
partnerships. Understandably, PhD advisers way they promote their work often remains way to earn notice, gain recognition and make
may worry that their students will lose focus passive. Scientists expect their publications the public at large aware of the value of the
by working outside their research group. But to communicate for them. A large body of scientific enterprise. ■
some resist simply because letting students scholarly work certainly confers a degree of Peter Fiske is chief technology officer of PAX
engage in outside work would mean fewer authority and knowledge. But even the best- Water Technologies in San Rafael, California,
hands in the lab. PhD and postdoc advisers written papers never completely capture the and author of Put Your Science to Work
have lots of influence, and this can lead to a passion and insight that led to their creation. (American Geophysical Union, 2001).
312
© 2010 Macmillan Publishers Limited. All rights reserved
NATURE|Vol 464|11 March 2010 CAREERS

Lidia Brito, Mozambique’s former science minister,

Q&A now heads the science-policy division at the

United Nations Educational, Scientific and Cultural
Organization (UNESCO) in Paris.
IN BRIEF
What made you decide to of forestry. I learned that to Research Fund, a funding
pursue science? be a good manager I should agency, and programmes
Better prospects
When I was 14 years old, always approach my goals such as the annual innovation Demand for both permanent and contract
Mozambique became an with a critical eye, I should competitions or Science Fair, researchers is expected to increase this
independent country. It was ask the hard questions to are now flourishing pieces of year in the United Kingdom across
a life-changing moment that determine whether goals a much larger ministry. several sectors that employ scientists,
motivated me to contribute are being reached and, if according to a survey of employers.
by pursuing my interests in necessary, redefine a policy What research is most Slough-based recruitment company SRG
chemistry — at the time, mission. That led me to a job needed to inform future found that 29.5% of respondents expect
I thought in industry. But as deputy rector of academic science policy? to boost numbers of permanent scientific
when I was 15, a teacher affairs at the university and a We need to better understand staff in 2010. Fields surveyed included
shortage led to the closing completely different path. how policy drives social pharmaceuticals and biotechnology, oil
of the last years of high change. For example, we need and petrochemicals, health care, research
school in all provinces. I was Why do you think you are to know whether and how institutes and government agencies.
fortunate to be able to go to a effective in policy roles? certain policies really promote About half of the employers in the oil, gas
special pre-university school I have had the great fortune basic values in society, such as and petrochemicals sector expect to have
created by the government of having worked with, and equality, inclusivity and access open permanent research positions, and
in Maputo, the capital city. learned from, wonderful to resources. none is forecasting cutbacks. More than
At first I was encouraged to teams of people. I’ve 550 employers completed the survey.
study agricultural sciences, combined my life experiences What is your top scientific
but I did not like that at to sharpen a critical eye. Early goal at UNESCO?
all because I couldn’t see in my career I did field work UNESCO has an important
Salary freeze
a way to combine it with with industry while I was mandate to use its focus on In 2009, some two-thirds of private and
chemical engineering. In my still linked to the university science, communication public US universities gave no salary
second year, I jumped at the doing teaching and culture to increases to their chief executives,
opportunity to be among the and research. As offer relevant academic deans and other senior
first class of foresters trained a result, I got a policy advice. executives, and 2.1% cut salaries, according
in Mozambique under a new more practical Our challenge to a survey released on 22 February. The
forestry degree programme. understanding of is to identify College and University Professional
what is at stake at the triggers Association for Human Resources,
Did you immediately follow the production and drivers of based in Knoxville, Tennessee, polled
an academic track? level. I developed development 1,280 institutions for its Administrative
No. After I finished my the capacity to — for example, Compensation Survey Report.
undergraduate degree simply ask the right questions, education initiatives — that Nevertheless, the median base salary
at Eduardo Mondlane which is very important can really make a difference. for the chief executive of a doctorate-
University in Maputo, I was when you work in policy. In Clearly, the scientific granting institution was up 7% from 2008
recruited to stay and teach the end, policy-makers must challenge for UNESCO is to to US$375,000 in 2009. Respondents
there, but I was required create feedback mechanisms, do more research on science- predicted restricted hiring for the fiscal
to work in forestry before I be open to change and look policy development. For year 2010, with fewer than 1% expecting
could begin my academic at things with a critical eye. I example, we were recently “significantly more” positions to be filled.
career. After getting some took all those lessons with me involved in a large research
field experience introducing
new sawmilling techniques to
when, along with colleagues,
I helped to establish
project in Turkey and Malta
to study the effects of science
Wellcome translation
improve the quality of wood Mozambique’s Ministry of policies on equality in society. The Wellcome Trust is planning to
products, I took some time Higher Education, Science We need to promote more provide funding for 100 new PhD
off for my husband to focus and Technology. of this kind of ‘think tank’ positions in biomedical translational
on his career before I finished approach to science-policy research in UK and Irish universities.
my MSc in wood sciences What job did you find the development — preferably The charitable trust, based in the United
and PhD in forest sciences in most rewarding? through partnerships Kingdom, aims to create four PhD
the United States. I returned It’s difficult to choose. I with organizations that do programmes, each lasting five years and
to Mozambique and slowly enjoyed life as an academic complementary work such as funding five new graduate students per
built an academic career working with students, but the United States Agency for year, for research in areas highlighted by
conducting research related working in the Mozambique International Development. the trust this year as key to its ten-year
to biomass production and science ministry was very strategic plan — applying genomics
forestry management. special because it was a new What was the best piece of and genetics to understanding disease,
ministry. The team was small advice you were given? brain and mental disorders, infectious
Did you have a career but we were able to develop People who are not afraid of disease, ageing and chronic disease, and
turning point? new policies and mobilize thinking differently from the links among environment, nutrition and
Yes. The turning point in resources. It is very gratifying rest are the ones that come up health. The closing date for preliminary
my professional life was to see that many of our with alternative solutions to applications to create and direct one
developing leadership skills initial projects, such as the old problems. ■ of the four programmes is 11 May and
as head of the department establishment of the National Interview by Virginia Gewin winners will be announced in December.
313
© 2010 Macmillan Publishers Limited. All rights reserved
FUTURES NATURE|Vol 464|11 March 2010

The Omniplus Ultra

You can’t live without it.

Paul Di Filippo generally extends no further than their TV skins and ergonomically perfect controls,
remotes. and the more I lust to own one. Although
Everyone wants an Omniplus Ultra, and I So I must content myself with studying nothing in my condition has really changed,
am not immune to the urge. But of course the advertisements and the gadget-porn and although I have enough money, love
they are almost impossible to purchase — reviews. These can’t say enough about the and security, my life feels incomplete and
for love or money. life-changing capabilities of the Omniplus empty without an Omniplus Ultra.
Since their debut nine months ago at Ultra, its potential to shatter all old para- But there was just no way for me to get
the annual Consumer Electronics Show, digms and to literally remake the world. my hands on one.
more than 40 million units have been sold Publisher’s Weekly: After five centuries, Until I saw my boss’s boss’s boss walk
worldwide, exhausting the initial stockpile the printed book has found its worthi- through the warehouse carrying one.
but barely sating a fraction of consumer est successor in the Omniplus Ultra. The Then and there, I knew what I had to do.
demand. The Chinese factories that pro- future of reading is safely triumphant. As a low-level employee, I certainly
duce the Omniplus Ultra are tooling up as The Huffington Post: Opens new chan- could not jump several levels of manage-
fast as possible to make more, but retail- nels for the spread of democracy. ment and directly approach my boss’s
ers cannot guarantee delivery any sooner Boing-Boing: Coolest gadget since the boss’s boss and ask to fondle his Omniplus
than six months. On eBay, each available iPhone! The cold-laser picoprojector alone Ultra. But I had a scheme.
Omniplus Ultra, with a MSRP of $749.99, is worth the cost. It took me six frustrating weeks, but
sells for upwards of $5,000. Car and Driver: Jack the Omniplus Ultra at last I managed it. In a series of furtive
OmninfoPotent Corporation, the enig- unauthorized forays into executive terri-

JACEY
matic firm behind the Omniplus Ultra, has tory, I caught the lucky Omniplus Ultra
leapt to the top of the NASDAQ exchange. owner in a lavatory break with his prized
Its reclusive founders, Pine Martin and possession carelessly left behind on
Sheeda Waxwing, have vaulted into his desk.
the lower ranks of the Forbes 400. That’s when I pulled the fire alarm.
Sales of the device are being cred- While everyone else rushed outside,
ited with jump-starting the ailing I darted into the guy’s office, snatched
economy almost single-handedly. his Omniplus Ultra off the desk, and
The ad campaign for the Omni- sank down behind the furniture in the
plus Ultra has already won six knee well, out of sight.
Clios. The catchy theme music, O U With trembling hands I sought to
Kidz by the Black Eyed Peas, and the shuffle aside the protective wings of
images of average people of every the device, utilizing all the instructions
race, age, gender, nationality and creed I had lovingly memorized, and expose
using their Omniplus Ultras to navigate its intimate control and display surfaces
a plethora of life situations ranging from to my wanton gaze and lewd touch.
sweetly comic to upliftingly tragic have But I was doing something wrong! The
generated their own fan clubs, YouTube expected blossoming failed to happen.
mash-ups and punchlines for late-night into your dash’s USB port and driving will Instead, after some fumbling, the unit
comedians. Allusions to the Omniplus never be the same! split open like a simple styrofoam clam-
Ultra, as well as its invocation in meta- Entertainment Weekly: If you can’t shell container full of leftovers.
phors, similes, rants, raves, jeremiads and download your favourite show onto your The interior gaped utterly vacant, except
paeans, fill watercooler conversations and Omniplus Ultra, it’s not worth watching. for a simple piece of printed cardboard.
the printed pages of the world’s magazines, Variety: First flicks helmed with the Dumbfounded, I removed the card-
newspapers and blogs. The first instant Omniplus Ultra to hit big screens soon! board and read the message.
book on the Omniplus Ultra — Uber- Aerospace & Defense Industry Review: Dear Consumer: the Omniplus Ultra
power! by Thomas Friedman and Charles Guaranteed to be standard equipment for is not what you need. You are already
Stross — is due out any day. all future warriors. everything you thought it could do. Pass
I myself do not know anyone who actually Mother Jones: The Omniplus Ultra is the this message on as widely as you see fit.
FUTURES

owns an Omniplus Ultra, but I’m dying to greenest invention since the Whole Earth Or not. Hopefully yours, Pine Martin and
see and handle one. But even 40 million Catalog. Sheeda Waxwing, for the OmninfoPotent
units, distributed across 7 billion people, BusinessWeek: Every chief executive will Corporation.
means that there is only one Omniplus benefit from having an Omniplus Ultra to I put the card back inside, resealed the
Ultra for every 175 citizens. Of course, the hand — and anyone without one will watch Omniplus Ultra, dropped it with a dull
gadgets are not seeded evenly around the competitors eat their lunch. thud on the desk, and joined all my peers
planet. They are concentrated in the hands Rolling Stone: Elvis. The Beatles. The Sex outside, waiting to resume our lives. ■
of relatively wealthy and elite consumers Pistols. The Omniplus Ultra. The sequence Paul Di Filippo’s new novel, Roadside
and early adopters: circles I do not really is complete at last. Bodhisattva, will be available in spring 2010.
travel in, given my job in a Staples ware- The more such talk I read, the longer He continues to review for various venues.
house and a set of friends whose familiarity I drool over pictures of the sleekly tactile Join the discussion of Futures in Nature at
with the latest products of Silicon Valley Omniplus Ultra, with its customizable sexy go.nature.com/QMAm2a

316
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