Eukaryotic Chemotaxis: A Network of Signaling Pathways Controls Motility, Directional Sensing, and Polarity

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Eukaryotic Chemotaxis:
A Network of Signaling
Pathways Controls Motility,
Annu. Rev. Biophys. 2010.39:265-289. Downloaded from arjournals.annualreviews.org
Directional Sensing,
and Polarity
by OSAKA UNIVERSITY on 07/17/10. For personal use only.
Kristen F. Swaney, Chuan-Hsiang Huang,

and Peter N. Devreotes
Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore,
Maryland 21205; email: pnd@jhmi.edu
Annu. Rev. Biophys. 2010. 39:265–89 Key Words

First published online as a Review in Advance on Dictyostelium, adaptation, Local Excitation Global Inhibition (LEGI)
February 2, 2010
model, protein localization
The Annual Review of Biophysics is online at
biophys.annualreviews.org Abstract
This article’s doi: Chemotaxis, the directed migration of cells in chemical gradients, is
10.1146/annurev.biophys.093008.131228
a vital process in normal physiology and in the pathogenesis of many
Copyright ⃝ c 2010 by Annual Reviews. diseases. Chemotactic cells display motility, directional sensing, and po-
All rights reserved
larity. Motility refers to the random extension of pseudopodia, which
1936-122X/10/0609-0265$20.00 may be driven by spontaneous actin waves that propagate through the
cytoskeleton. Directional sensing is mediated by a system that detects
temporal and spatial stimuli and biases motility toward the gradient.
Polarity gives cells morphologically and functionally distinct leading
and lagging edges by relocating proteins or their activities selectively to
the poles. By exploiting the genetic advantages of Dictyostelium, inves-
tigators are working out the complex network of interactions between
the proteins that have been implicated in the chemotactic processes of
motility, directional sensing, and polarity.
265
ANRV411-BB39-14 ARI 12 April 2010 18:44
examples of the importance of chemotaxis in

Contents disease (follow the Supplemental Material
link from the Annual Reviews home page at
INTRODUCTION: CHEMOTAXIS
http://www.annualreviews.org).
OCCURS IN MANY CELL
Although an increasing number of cell types
TYPES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
that carry out chemotaxis are being discovered,
MOTILITY, DIRECTIONAL
the signal transduction events mediating di-
SENSING, AND POLARITY . . . . . 267
rected migration have been most thoroughly
Motility: Pseudopod Extension
studied in Dictyostelium, neutrophils, and a vari-
in Migrating Cells . . . . . . . . . . . . . . 268
ety of transformed mammalian cells (100, 105,
Directional Sensing: Temporal and
119; see also Related Resources). The amoe-
Spatial Sensing of
boid movements of Dictyostelium toward 3′ ,5′ -
Chemoattractants . . . . . . . . . . . . . . . 268
cyclic adenosine monophosphate (cAMP) and
Polarity: Selective Localization
of neutrophils toward a variety of chemokines

of Molecules and Reactions to the
are based on the extension of pseudopodia.
Front or Back of Cells . . . . . . . . . . . 272
In both cell types, chemoattractants activate
A NETWORK OF SIGNALING
G-protein coupled receptors (GPCRs), result-

PATHWAYS CONTROLS
ing in the localized accumulation of signaling
CHEMOTAXIS . . . . . . . . . . . . . . . . . . . 275
molecules, such as phosphatidylinositol 3,4,5-
Receptor and G-Protein Module . . . 277
trisphosphate (PIP3 ), toward the high side of
RasC and RasG Modules . . . . . . . . . . . 278
the gradient. PIP3 accumulation leads to pseu-
PIP3 and PIP2 Modules . . . . . . . . . . . . 278
dopodia extension at the leading edge of cells,
PKB and PKB Substrate Modules . . . 279
which is thought to be driven by localized
cAMP Module . . . . . . . . . . . . . . . . . . . . . 280
Rac-mediated actin polymerization. Primordial
cGMP/Myosin II Module . . . . . . . . . . 280
germ cells (PGCs) also use GPCRs to sense
RasB/Rap1/MHCK Module . . . . . . . . 281
chemoattractants, but these cells maintain uni-
Linking the Signaling Network
form PIP3 levels throughout the membrane and
to the Cytoskeleton . . . . . . . . . . . . . 281
migrate by extending actin-free blebs (3, 30).
These blebs may be generated by myosin-based
Chemotaxis: the contraction, which is also important at the lag-
directed migration of
ging edge for migration in Dictyostelium and
cells toward higher or INTRODUCTION: CHEMOTAXIS
lower concentrations neutrophils. These contractions are generally
OCCURS IN MANY CELL TYPES directed by Rho, and in Dictyostelium, myosin
of chemical stimuli
cAMP: 3′ ,5′ -cyclic Many cells have an internal compass that al- regulation also involves 3′ ,5′ -cyclic guanosine
adenosine lows them to detect extracellular chemical gra- monophosphate (cGMP). The combination of
monophosphate dients and move toward or away from higher forces at the leading and lagging edges gener-
GPCR: G-protein concentrations. This process is referred to as ates rapid movement. Adaptation to persistent
coupled receptor chemotaxis or directed cell migration. During stimulation in Dictyostelium and neutrophils al-
PIP3 : embryogenesis, chemotaxis is important for in- lows for enhanced sensitivity to differences in
phosphatidylinositol dividual and group cell migration events, or- chemoattractant concentrations across the cell.
3,4,5-trisphosphate gan formation, and wiring of the nervous sys- Stimulation of fibroblasts or breast carci-
cGMP: 3′ 5′ -cyclic tem. In the adult, chemotaxis is critical for noma cells with growth factors that bind to
guanosine the trafficking of immune cells and in inflam- Receptor Tyrosine Kinases (RTKs) also results
monophosphate
mation, regenerative processes such as wound in PIP3 accumulation and Rac-mediated actin
Adaptation: the healing, and maintenance of tissue architec- polymerization at the leading edge (reviewed
tendency of responses
ture. Evidence suggests that chemotaxis al- in References 64, 100, and 119). In carcinoma
to subside when
receptor occupancy is lows stem cells to target to and persist in cells, the activation of Cofilin through its re-
held constant their niches. See Supplemental Sidebar 1 for lease from the membrane, which is mediated by
266 Swaney · Huang · Devreotes

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chemoattractant-induced reductions in phos- Directional

phatidylinositol 4,5-bisphosphate (PIP2 ) levels, Motility sensing Polarity
generates actin barbed ends and contributes
to actin polymerization (114). As with amoe- a
boid cells, the actin-mediated events of fibrob-
lasts and carcinoma cells are coordinated with
myosin-based contraction at the lagging edge,
which is regulated by Rho and calcium signal-
ing. Together, these result in migration that oc-
curs much more slowly than that of amoeboid
cells. Because there is no adaptation in fibrob-
lasts, these cells respond only to absolute con-
centrations of chemoattractant. b
Despite slight differences in the migra-

tory behaviors and specific signaling compo-
nents of Dictyostelium, neutrophils, and the Shallow gradient
other cell types, the overall pathways that

regulate chemotaxis are similar. Furthermore,
many of the guidance mechanisms in these cell
types are evolutionarily conserved among most
migratory eukaryotic cells, including neurons
(reviewed in References 100 and 119). For the
remainder of this review, we focus primarily on c
chemoattractant-mediated signaling events in
Dictyostelium, in which genetic analysis has fa-
cilitated a thorough assessment of chemotactic Steep gradient
behavior (Supplemental Sidebar 2).
Figure 1
Chemotaxis is composed of motility, polarity, and directional sensing. In the
presence of a chemoattractant (or chemorepellent) gradient, cells move toward
MOTILITY, DIRECTIONAL
(or away from) higher concentrations. (a) Left: Free amoeboid cells
SENSING, AND POLARITY rhythmically extend pseudopodia and move in random directions. Middle:
Chemotaxis can be conceptually divided into Spatial sensing, a means of directional sensing, can be demonstrated by the
gradient-mediated relocalization of proteins in cells immobilized by actin
the processes of motility, directional sensing,
inhibitors. Right: Chemotactic cells are often polarized, with a stable leading
and polarity (Figure 1). In the absence of a edge from which pseudopodia are extended. (b) In a shallow gradient, polarized
stimulus, cells provided with a suitable surface cells display biased patterns of pseudopodia extension at the leading edge that
will crawl about, a process referred to as motil- cause cells to turn gradually toward higher concentrations of chemoattractant.
ity. Amoeboid cells, such as Dictyostelium and (c) Sufficiently steep gradients can trigger new projections anywhere along the
cell periphery.
neutrophils, extend pseudopodia rhythmically,
propelling the cell in random directions. When
the cells are exposed to a gradient of chemoat- molecules within immobilized cells can move
tractant or chemorepellent, their motility toward external stimuli and can dynamically
is biased toward or away from, respectively, track changes in gradient direction. Finally,
higher concentrations. The molecular mech- chemotactic cells often display a relatively sta-
anisms that read the gradient and provide this ble axis of polarity, which restricts pseudopodia
chemotactic bias are referred to as directional extension to the cell anterior. Polarity is also
sensing and correspond to the cells’ internal separable from directional sensing, as cells in
compass described above. However, motility uniform chemoattractant can be polarized.
and directional sensing are separable, since Although polarized cells move with more
www.annualreviews.org • Signaling Events in Chemotaxis 267

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persistence than unpolarized cells, they do not anywhere around the perimeter often triggers
move in a specific direction. Chemotaxis typi- the formation of a new front. In a highly po-
cally incorporates motility, directional sensing, larized cell, the side and rear are less sensitive
Directional sensing:
the molecular and polarity and should not be confused with than the front, resulting in the turning behavior
mechanisms that read any one of these processes alone. described above. However, even in a highly po-
the direction of larized cell, a sufficiently steep gradient applied
chemoattractant to the side or back can break the polarity and
gradients and provide Motility: Pseudopod Extension create a new front (Figure 1).
a bias to guide the in Migrating Cells
motility of Recent observations of fluorescent cy-
chemotactic cells Several recent reports analyze the motile behav- toskeletal proteins on the basal surfaces of
Polarity: a ior of Dictyostelium cells through observations of migrating neutrophils or Dictyostelium cells
morphological state pseudopod extension in the absence or presence show wave-like propagation through the
with stable, of shallow cAMP gradients (1, 8). These studies cytoskeleton (10, 118, 123; see also Supple-
functionally distinct conclude that gradients modify the basal behav- mental Movie 1 and references therein).
leading and lagging
ior that unstimulated cells already display. In the Actin-binding proteins or Scar/WAVE com-
edges that are
characterized by the absence of chemoattractant, polarized cells ex- plex components are recruited sequentially
preferential tend pseudopodia of uniform size and duration from the cytosol to adjacent points on the basal
localization of specific alternately from either side of the axis of mo- surface, giving rise to a propagated wave. Basic
molecules tion in a behavior reminiscent of “ice skating.” wave propagation has been modeled and re-
Occasionally, the alternation is skipped and quires the mechanisms of signal relay, positive
several subsequent pseudopodia are extended feedback, and reversible inhibition. Waves can
from the same side. Chemotactic gradients originate at random on the basal surface, al-
cause more pseudopodia to be extended to- though in polarized cells they arise more often
ward the “correct” direction. In one model, this toward the anterior and move outward toward
is achieved by more often choosing to retract the edge. The arrival of waves at the perimeter
pseudopodia extended in the “wrong” direc- coincides with the onset of a protrusion and may
tion (1). In another model, the probability of underlie the spontaneous generation of pseu-
extending pseudopodia toward the gradient is dopodia. The cytoskeletal events that mediate
higher; in addition, the angle at which pseu- actin polymerization for the formation of cell
dopodia are extended is altered to favor move- protrusions have been studied extensively and
ment in the correct direction (8). In both mod- are beyond the scope of this review; however,
els, the bias causes cells to turn toward and little is known about how these events link to
remain facing the source of chemoattractant receptor signaling (27, 50). Recent observations
(Figure 1). The ice skating behavior is less ob- suggest that chemoattractants influence wave
vious in neutrophils, in which individual pseu- propagation, which may provide insight into
dopodia are not as readily separable, and an al- the mechanisms by which signaling pathways
ternative mechanism may exist for lamellipod regulate the cytoskeleton and motility.
extension in fibroblasts.
Analyses of cells in shallow gradients suggest
that generation of pseudopodia is autonomous Directional Sensing: Temporal and
and that the gradient can only bias this behav- Spatial Sensing of Chemoattractants
ior; however, a strong chemotactic stimulus can Physiological responses triggered by
also directly elicit de novo production of a pseu- chemoattractants. When differentiated Dic-
dopod (106). Whether a chemotactic stimulus tyostelium cells are exposed to the chemoattrac-
causes turning or triggers a new projection de- tant cAMP, a series of morphological changes
pends on the relative polarity of the cell ver- and physiological responses is triggered
sus the strength of the stimulus. In a weakly (Figure 2a,b; see also Related Resources).
polarized cell, a chemotactic stimulus applied The network of signaling pathways mediating

ANRV411-BB39-14 ARI 12 April 2010 18:44
these responses is discussed in detail below. In and the calcium influx, are nonadapting,
Supplemental Table 1, we list the genes im- whereas most of the other responses, which
plicated in chemotaxis or in the transduction of are transient, are adapting (12, 54, 61, 86). The
cAR: cAMP receptor
chemotactic signals. Within seconds of cAMP molecular level at which adaptation occurs is
FRET: fluorescence
stimulation, the heterotrimeric G-protein G2 an important open question that is addressed
resonance energy
linked to the cAMP receptor cAR1 is activated, below. transfer
as evidenced by a rapid decrease in the fluores-
PTEN: phosphatase
cence resonance energy transfer (FRET) signal Temporal versus spatial sensing. For direc- and Tensin homolog
between the Gα2- and Gβ-subunits (54, 129). tional sensing, cells need to detect nonuniform on chromosome ten
In addition, cAR1 becomes phosphorylated, distributions of chemoattractants. To this end, Spatial versus
the mitogen-activated protein kinase (MAPK) cells must be able to sense changes in recep- temporal sensing:
Erk2 is activated, and a calcium influx is tor occupancy over time, referred to as tempo- the ability of cells to
triggered (11, 86). These responses persist ral sensing, and/or space, referred to as spatial detect differences in
receptor occupancy
during continuous stimulation. However, many sensing. Moving cells can detect spatial gradi- across the cell length
other responses, listed below, are transiently ents using a temporal sensing mechanism be- versus over time
activated upon chemoattractant stimulation. cause their receptor occupancy changes as a
These transient responses typically display an function of time. In fact, chemotactic bacte-
initial rapid activation and a secondary, delayed ria rely on temporal sensing to bias their ran-
peak. For example, multiple Ras proteins with dom walks and to detect spatial gradients (120).
similar kinetics are activated (57, 61, 99). The However, here, the term spatial sensing is re-
adenylyl cyclase ACA and both soluble and served for the detection of differences in recep-
membrane-bound guanylyl cyclases (sGC and tor occupancy across the cell length.
GCA, respectively) are activated, resulting Observations of immobilized cells show that
in cAMP and cGMP production (93, 98). eukaryotic cells are capable of both temporal
Phosphoinositide 3-kinases (PI3Ks) are acti- and spatial sensing (Supplemental Movies 2
vated, resulting in PIP3 production (34, 42). and 3 and references therein). In cells immo-
Many proteins are phosphorylated, including bilized with Latrunculin, an inhibitor of actin
the Gα2-subunit, a series of protein kinase B polymerization, responses that have been ex-
(PKB) substrates, and Myosin II heavy and light amined, such as Ras activation or PIP3 produc-
chains (36, 62, 104, 131). Actin is polymerized tion, adapt normally to constant uniform stim-
and actin-binding proteins are recruited to the uli (Supplemental Movie 2). If the stimuli are
cortex (25, 59, 101). The phosphoinositide further increased, these responses can be reac-
3-phosphatase PTEN (phosphatase and Tensin tivated but again adapt over time. Therefore,
homolog on chromosome ten) dissociates from cells respond to temporal changes in recep-
the membrane, and Myosin II is lost from the tor occupancy rather than the absolute level.
cortex (45; C. Janetopoulos & P. Devreotes, In contrast, immobilized cells placed in sta-
unpublished observations). In growing undif- ble gradients show persistent Ras and PIP3 re-
ferentiated cells, folic acid and pterines trigger sponses toward the high side of the gradient,
many of these same responses (37). as detected by recruitment of Ras-binding do-
The tendency of cellular responses to mains (RBDs) and specific Pleckstrin homol-
subside during constant receptor occupancy is ogy (PH) domains, respectively (Supplemental
known as adaptation. The responses triggered Movie 3). Because these cells are not moving,
by cAMP can be broadly divided into two the steady-state level of receptor occupancy at
groups on the basis of whether or not they adapt each position on the membrane is constant over
to continuous stimulation (Figure 2b). The time. Therefore, cells are able to compare re-
persistent responses, including receptor phos- ceptor occupancy across their lengths and selec-
phorylation, G-protein and Erk2 activation, tively maintain responses only at the high side.

ANRV411-BB39-14 ARI 12 April 2010 18:44
The temporal phenomenon of adaptation responses at the high side of the gradient
leads to spatial sensing, as illustrated by con- arrive at a steady, nonzero level, whereas those
sidering how the steady-state response evolves. at the low side eventually vanish. Thus, adap-
When cells are first exposed to a gradient, tation still occurs, but instead of eliminating
receptor occupancy increases everywhere and the signal, it allows cells to respond to the
cells show initial global responses similar to difference in receptor occupancy rather than
those induced by uniform stimuli. Over time, the absolute level, resulting in an internal
a Add Cell behavior

chemoattractant
b Time
Nonadapting responses
Add
chemoattractant G-protein loss of FRET
cAR1 phosphorylation
Ca2+ influx
Erk2 activity
Response
Time
Adapting responses
Add
chemoattractant Decreasing responses:
Membrane PTEN
Cortical myosin
Response
Increasing responses:
Ras activity
PIP3
PKB substrate phosphorylations
Time
cAMP
cGMP
Cortical MyoB, Dynacortin, LimE
Actin polymerization
c Local Excitation Global Inhibition model
Time Spatial gradient
Front Back
Excitation Excitation
Response at front at back
Excitation at
Relative amount
Relative amount
front or back
n n
tio tio
i bi ibi
Inh Inh
Note persistent
response at front
Time (min) Time (min)

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amplification of the gradient. So far, mutants cell, and there is no response. That is, the cells
that fail to adapt have not been identified. adapt. In a chemoattractant gradient at steady
However, defects in adaptation can be mim- state, local excitation is higher toward the gra-
Local Excitation
icked by removing negative regulators such as dient, whereas inhibition is nearly uniform and Global Inhibition
PTEN or the Ras GTPase activating protein intermediate in strength. Therefore, excitation (LEGI) model: a
(GAP) NF1 (45, 134). These cells have pro- exceeds inhibition at the high side, whereas model, involving a
longed and poorly localized PIP3 responses, the opposite occurs at the low side. In this balance between local
excitatory and global
resulting in impaired chemotaxis. case, adaptation results in a persistent response
inhibitory processes,
toward the gradient. The model therefore that can explain the
recapitulates the observed temporal and spatial temporal and spatial
Adaptation and the Local Excitation Global behaviors of immobilized cells as outlined responses of
Inhibition model. If responses such as Ras above. Two predictions of the LEGI model immobilized cells to
chemoattractant
activation and PIP3 accumulation adapt to con- have been verified experimentally (55). For
stimulation
stant receptor occupancy, how can they persist example, when gradients change direction, the
Dispersion length:
locally in an immobilized cell in a stable gradi- responses reorient to the new high side. In
the effective range of a
ent? The apparent paradox posed by this ques- addition, localized responses can be induced signaling molecule,
tion can be explained by the Local Excitation simultaneously at two points on the membrane determined by the
Global Inhibition (LEGI) model (Figure 2c) by applying two steep gradients. diffusion coefficient
(78, 91; reviewed in Reference 44). According Four points about the LEGI model need to and half-life of the
molecule
to the LEGI model, two processes are elicited be clarified. First, the nonadapting property of
by chemotactic stimuli: a fast excitation that excitation and inhibition does not imply that the
reflects local receptor occupancy and a delayed molecules involved are stable. Because the exci-
inhibition that is broader and more closely tatory molecules are constantly diffusing, they
reflects the mean receptor occupancy. Neither must be continually inactivated for excitation
process adapts; both persist as long as stimuli to remain localized and adjust to directional
are maintained. The balance between excitation changes in the gradient (94). Second, local and
and inhibition determines the magnitude of the global are relative terms. The LEGI model pre-
observed responses, such as Ras activation and dictions hold true as long as the inhibitor has
PIP3 production. Because excitation is faster a longer range of action, or dispersion length,
than inhibition, there is a positive response im- than the excitor. The dispersion length of a
√
mediately after stimuli are applied. However, molecule is defined as D/k, where D is its dif-
with a uniform stimulus at steady state, inhi- fusion coefficient and k is its rate constant for
bition eventually equals excitation all over the inactivation. Thus, the inhibitor should have
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 2
Temporal and spatial responses triggered by chemoattractants and the Local Excitation Global Inhibition (LEGI) model. (a) When
exposed to a sudden increase in cAMP, cells retract projections or cringe; then, they periodically extend and retract projections at
random sites on the periphery until, after several minutes, they regain their polarized morphology. (b) The biochemical responses
triggered by cAMP can be divided into two groups on the basis of whether or not they adapt to constant stimuli. Some responses, such
as G-protein activation, are nonadapting and persist as long as the stimuli are maintained. Of the adapting responses, most, such as
PIP3 production, transiently increase, whereas others, such as membrane-localized PTEN, transiently decrease. The timescales shown
in panels a and b are the same so that the cell behavior in panel a can be directly compared to the response curves in panel b. (c) To
explain the temporal and spatial adapting responses of immobilized cells, the LEGI model proposes that chemotactic stimuli elicit an
excitor that reflects local receptor occupancy, as well as an inhibitor that is broader and more closely reflects the mean receptor
occupancy. Excitation rises faster than inhibition, resulting in an initial response. Left: In a uniform stimulus at steady state, excitation
equals inhibition throughout the cell, which explains the experimentally observed disappearance of the initial response. Right: In a
gradient at steady state, excitation exceeds inhibition at the high side and vice versa at the low side; therefore, the response persists only
at the high side of the gradient, as seen experimentally and indicated by arrows. Abbreviations: cAMP, 3′ ,5′ -cyclic adenosine
monophosphate; cGMP, 3′ 5′ -cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; PIP3 ,
phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; PTEN, phosphatase and Tensin homolog on chromosome ten.

ANRV411-BB39-14 ARI 12 April 2010 18:44
either a faster diffusion rate or a longer half- also been implicated as a potential negative
life (or both) than the excitor. Third, the LEGI regulator of the pathway, as disruption of the
model can only fully explain responses in un- α9-subunit causes prolonged activation of
polarized cells such as those immobilized by ACA and increases the size of multicellular
Latrunculin treatment. In polarized cells, the aggregates (13). However, there is still no
differential sensitivities at the opposing ends of direct evidence that Gα9 regulates Ras activity.
the cell must be taken into account (see below). Models based on positive feedback mecha-
Fourth, although the inhibitor is often assumed nisms have also been proposed to explain the
to be a different molecule than the excitor, this asymmetric responses of cells (48, 82, 94, 124).
does not have to be the case. Hypothetically, if These models describe the observed responses
cAMP stimulation resulted in rapid activation of polarized cells, which cannot be readily ex-
and then slow inactivation of the receptor, the plained by the LEGI model. However, the pos-
receptor itself could act as the excitor when first itive feedback models cannot easily account for
stimulated and become the inhibitor as adapta- the rapid reorientation of responses to changes
tion ensued. In this case, the inhibitor would in the direction of gradients or the dual re-
have a longer half-life and therefore dispersion sponses induced by two sharply localized stim-
length than the excitor, thus satisfying the re- uli observed in Latrunculin-treated cells. It is
quirements of the LEGI model. possible that some combination of the LEGI
While the LEGI model successfully explains and the positive feedback models is necessary
the responses of cells to uniform stimuli and to account for the entire set of physiological
gradients, it is not known where adaptation, the responses of chemotactic cells.
balance between excitation and inhibition, oc-
curs along the signaling pathway. FRET experi-
ments of the heterotrimeric G-protein indicate Polarity: Selective Localization
that dissociation of the α- and β-subunits, of Molecules and Reactions to the
a readout for G-protein activation, does not Front or Back of Cells
adapt to persistent stimulation, whereas the The polarization component of chemotaxis
activation of the Ras proteins does adapt manifests itself in two ways. First, cells take
(Figure 2b; Supplemental Movie 4 and on an elongated morphology that becomes in-
references therein). Furthermore, prolonged creasingly pronounced as they differentiate.
Ras activation leads to prolonged downstream Mutants that cannot maintain this morphol-
responses (134). Together, these results imply ogy have reduced directional persistence, re-
that the major site of adaptation occurs after sulting in poor chemotaxis (see below). Second,
the G-protein dissociates and before the signal in polarized cells, certain molecules are spa-
reaches the Ras proteins. The molecular links tially restricted to the leading or lagging edge.
from the heterotrimeric G-protein to the Ras These localizations are maintained even in the
guanine nucleotide exchange factors (GEFs) absence of a gradient. The clustering of sig-
are unknown, and exactly where and how naling molecules generates regions at opposing
adaptation occurs are still open questions. ends of the cell that have distinct functions and
One possible link is an extracellular super- different sensitivities to chemoattractant, which
oxide dismutase (SodC) that was identified alters the way the cell responds to a gradient
in a restriction enzyme-mediated insertional (Figure 1). Less differentiated cells, which have
(REMI) mutagenesis screen (Supplemental low or moderate polarity, can easily form new
Sidebar 2) as having elevated PIP3 levels on fronts, whereas fully differentiated cells, which
the membrane (117). Disruption of SodC are extremely polarized, tend to persist along a
leads to continuous recruitment of RBD to the constant path with increased velocity.
membrane, indicating persistent activation of Although it is thought that positive feed-
Ras proteins. The G-protein α9-subunit has back must be involved, the molecules that are

ANRV411-BB39-14 ARI 12 April 2010 18:44
responsible for initiating polarity are still un- therein). In polarized cells, PI3Ks are found
known. In neutrophils, a positive feedback loop at the leading edge, whereas PTEN is found
involving PIP3 and actin has been described (48, at the lagging edge, creating a localized accu-
TorC2: Tor (Target
124). However, the factors mediating polariza- mulation of PIP3 and PH domain-containing of Rapamycin)
tion must be redundant with the PIP3 pathway, effectors at the front (Figure 3). In addition, Complex 2
because neutrophils or Dictyostelium cells lack- actin and many actin-binding proteins have
ing PI3K activity can initiate and maintain rela- been identified in the cortex at the front of the
tively normal polarity (33, 40). cGMP signaling cell. Interestingly, S-adenosylhomocysteine hy-
may contribute to polarity as discussed below drolase (SAHH) and the Shwachman-Bodian-
(9, 88). It is also apparent from studies of mu- Diamond syndrome protein (SBDS) localize to
tants and chemical inhibitors that the cytoskele- the entire pseudopod at the anterior of the cell.
ton is required for polarity. Evidence for the Several other proteins are localized uniformly
requirement of microtubules comes from over- on the membrane or in the cytosol but are enzy-
expression of Lissencephaly protein 1 (Lis1) matically active specifically at the leading edge,
or fragments of Dynein, as well as Lis1 hypo- including Ras, Target of Rapamycin Complex
morphic mutants (97). In all three cases, mi- 2 (TorC2) subunits, and the PKB-related pro-
crotubules are unable to attach to the cortical tein PKBR1. In contrast, both cAR1 and the
shell, resulting in a flattened morphology with heterotrimeric G-proteins, as well as their ac-
increased lateral protrusions and reduced polar- tivities, are uniformly localized throughout the
ity. Furthermore, cells lacking Tsunami (TsuA) membrane in polarized cells (58, 128).
have normal motility and directional sensing Although most asymmetrically localized
capabilities but cannot properly orient their proteins are found at the leading edge, several
microtubules, which causes defects in polarity have also been identified at the lagging edge
and chemotaxis (107). In addition, drugs, such (Figure 3; Supplemental Table 2). These pro-
as Benomyl, that depolymerize microtubules teins are found at the lateral and rear edges of
eliminate polarity (107). The actin cytoskele- the cell at the outset of differentiation but be-
ton is also required for polarity. Treatment with come more tightly localized specifically to the
Latrunculin not only immobilizes cells, but also back as differentiation progresses (K. Swaney &
disrupts their stable axis of polarity and abol- P. Devreotes, unpublished observations). These
ishes spatially restricted protein localizations proteins include PTEN, as described above, as
(55, 73). Furthermore, pten- and tsuA- cells have well as Myosin II and the actin-binding protein
high levels of F-actin, resulting in the forma- Cortexillin I, which localize in the cortex and
tion of lateral pseudopodia, reduced polarity, mediate rear contractility. Furthermore, p21-
and impaired chemotaxis (45, 107). From these activated protein kinase A (PakA) is at the lag-
and other mutants, including disruptions in ging edge and is proposed to prevent Myosin
the MAPKK MEK1, Tortoise (TorA), and the II dissociation by inhibiting Myosin II Heavy
Na-H exchanger Nhe1, which also cause rather Chain (MHC) kinases (MHCKs). ACA is the
specific defects in polarity, it is apparent that an only known integral membrane protein in the
increase in the number of lateral pseudopodia is group of lagging edge proteins. The distribu-
correlated with impaired chemotaxis (Supple- tion of ACA has been proposed to create a lo-
mental Table 1 and references therein). calized synthesis and release of cAMP at the
Many molecules involved in chemotaxis, in- lagging edge, which attracts other nearby cells
cluding both lipids and proteins, are localized and is important for generating the head-to-tail
on the membrane or in the cortex specifi- migration, or streaming, characteristic of polar-
cally at either the leading or lagging edge of ized cells.
polarized cells, and examples of spatially re- Proteins that localize asymmetrically in
stricted proteins are continuously being iden- polarized cells display characteristic behav-
tified (Supplemental Table 2 and references iors in cells stimulated by chemoattractants

ANRV411-BB39-14 ARI 12 April 2010 18:44
a b c
Polarized cells or
cAMP gradient Uniform cAMP stimulation Cytokinesis
Leading
edge
proteins
0s 12 s 18 s 24 s
Lagging
edge
proteins
0s 12 s 18 s 36 s 132 s
Figure 3
Localization of signaling components to the leading or lagging edge. The distributions of leading edge proteins are represented by a
PIP3 -specific PH domain tagged with GFP, and those of lagging edge proteins are represented by PTEN-GFP. (a) In polarized or
chemotaxing cells, many proteins are recruited to the leading or lagging edge. Arrows reflect the direction of migration. (b) When
unpolarized cells are stimulated globally with cAMP, “leading edge” proteins, such as PI3Ks and several actin-associated proteins,
translocate uniformly to the plasma membrane or cortex and then return to the cytosol. Conversely, “lagging edge” proteins, such as
PTEN or Myosin II, transiently fall off the membrane or cortex (arrows) and then return to the periphery. Time in seconds after the
addition of chemoattractant is indicated for each frame. (c) During cytokinesis, “leading edge” proteins localize to the poles, whereas
“lagging edge” proteins are targeted to the cleavage furrow (arrows). Images in panel c are reproduced from Reference 53.
Abbreviations: cAMP, 3′ ,5′ -cyclic adenosine monophosphate; GFP, green fluorescent protein; PIP3 , phosphatidylinositol
3,4,5-trisphosphate; PTEN, phosphatase and Tensin homolog on chromosome ten.
(Figure 3; Supplemental Table 2 and Sup- as the cells become more polarized. Leading
plemental Movies 5 and 6). In less polarized edge proteins are found at the front of polar-
cells, “leading edge” proteins such as PI3Ks ized cells but are additionally and transiently
accumulate in the cytosol and on membrane recruited to the membrane globally with uni-
protrusions, whereas “lagging edge” proteins form stimuli. Lagging edge proteins, already
such as PTEN are localized uniformly on the sharply localized to the back of cells, display
plasma membrane with the exception of mem- limited redistribution in response to uniform
brane protrusions. Upon the addition of cAMP, stimuli in polarized cells. Protein localizations
both sets of proteins respond by transiently at the leading or lagging edge are maintained in
relocalizing with respect to the plasma mem- polarized cells even in the absence of a gradi-
brane or cytoskeletal cortex. The leading edge ent, but, as noted above, treatment with Latrun-
proteins translocate uniformly to the periph- culin eliminates these spatially restricted distri-
ery within 10 s and then return to the cytosol butions (55, 73). In Latrunculin-treated cells,
roughly 30 s after stimulation (Supplemental many of these proteins translocate in response
Movie 5 and references therein). With the same to uniform stimuli as they do during early dif-
kinetics, most of the lagging edge proteins tran- ferentiation and localize toward or away from
siently fall off the cell periphery and into the the source of chemoattractant gradients (47, 55,
cytosol before returning to the membrane or 90, 99).
cortex (Supplemental Movie 6 and references Current evidence, although limited, implies
therein). These redistribution patterns change that the preferential targeting of proteins to

ANRV411-BB39-14 ARI 12 April 2010 18:44
the leading or lagging edge of migrating cells of the cell. For example, the PKB substrate
is important for polarity and chemotaxis. For PakA resides at the lagging edge despite the
example, removing the PIP2 -binding domain restriction of PKB kinase activity to the lead-
of PTEN causes mislocalization of the protein ing edge (21). It is possible that phosphory-
to the cytosol, resulting in a broader distribu- lation by PKBs is not required for or related
tion of PIP3 , loss of polarity, and chemotac- to PakA function at the lagging edge, but this
tic impairment that resemble the phenotype of apparent paradox requires further study. Simi-
pten- cells (see below) (47). Similarly, expression larly, the activation of the lagging edge integral
of PI3Ks tagged with CAAX or myristoylation membrane protein ACA requires both Cytoso-
motifs, which target the proteins uniformly to lic Regulator of Adenylyl Cyclase (Crac), which
the plasma membrane, in wild-type cells mimics is recruited by its PH domain to PIP3 at the
the pten- cell phenotype (34, 41). Furthermore, leading edge, and Pianissimo (PiaA), a TorC2
the mislocalization of Myosin II and its regu- component (18, 90). The mechanism of ACA
latory proteins causes cells to lose polarity and activation by two proteins that are active at the
impairs chemotaxis. For example, excessive re- leading edge is still unclear.
cruitment of MHCKA to the membrane results The asymmetric distribution of proteins has
in the overall cortical loss of Myosin II and the implications beyond polarity and chemotaxis,
overproduction of pseudopodia from the lat- because the same proteins display character-
eral edges of the cell (9, 87). In the future, it istic localization patterns when cells undergo
will be important to disrupt the localizations of morphological changes in general (Figure 3;
other spatially restricted proteins and to char- Supplemental Table 2). For example, in
acterize the effects of these changes on polarity growing cells, both “leading edge” and “lag-
and chemotaxis. However, the mechanisms that ging edge” proteins have localizations that
target many of these asymmetrically localized resemble those of early differentiation dis-
proteins, including PI3Ks and PTEN, to the cussed above. During phagocytosis, many of
leading or lagging edge are still unclear, which the leading edge proteins, such as Crac and
currently hinders the investigation of protein the LIM domain-containing protein, LimE,
mislocalizations. localize to the phagocytic cup and phagosome,
Several intriguing observations suggest that whereas lagging edge proteins, such as PTEN,
different components of the same signaling are excluded from these structures (22, 29).
pathway sometimes have different localizations. During cytokinesis, PI3Ks and other leading
For example, consider the Ras-TorC2-PKB edge proteins are localized to the poles,
pathway. The RasGEF Aimless (AleA) activates whereas lagging edge proteins, such as PTEN
RasC, which activates TorC2, which in turn and Myosin II, are restricted to the cleavage
mediates the phosphorylation and activation furrow (53; see also Supplemental Table 2).
of PKBs, which then phosphorylate a number It will be interesting to determine how the
of substrates (49, 60, 62, 75). The AleA, Ras, signals that target these proteins change during
TorC2, and PKB proteins are localized globally these different morphological states.
on the membrane or in the cytosol, but their ac-
tivities appear to be localized specifically at the
leading edge (62, 99). The dispersion lengths A NETWORK OF SIGNALING
of the active forms of these molecules must be PATHWAYS CONTROLS
sufficiently short to maintain localized down- CHEMOTAXIS
stream responses, implying that these proteins Studies of numerous Dictyostelium mutants
are deactivated before diffusing away from the with single or multiple gene deletions, or
cell anterior. More puzzling, there are cases expressing various mutant proteins, have fa-
in which an upstream activator and its down- cilitated a molecular analysis of chemotaxis
stream effector are localized at opposite ends (Supplemental Sidebar 2). With current

ANRV411-BB39-14 ARI 12 April 2010 18:44
cAMP
Receptor, Cell-to-cell
G-protein signaling
G-protein-
independent cARs NRs
responses
Gα2 Gα4/5 Gα9 SodC

Signaling for YakA
development Gβγ
Erk2 Ca2+
cGMP/ RasB/Rap1/
Myosin II MHCK RasC RasG PIP2 PLA2
GbpD GEFQ AleA GEFR NF1 Gα1
sGC
GCA PdeD
PLC
Rap1 RasB RasC RasG
cGMP
PIP2
PKB PIP3
GbpC Phg2 TorC2 PI3Ks PTEN
Myosin II PIP3
MHCKA
PKBA
PKBR1 cAMP
Back contraction Crac PhdA,
and adhesion PKB others
substrates
Positive links GacQ, TalinB, PI5K, ACA
Inhibitory links PakA, GEFS, GEFN cAMP
RegA
Small-molecule
reactions
Less well- Chemotaxis Actin polymerization
characterized
links Front projection
and adhesion
Feedback loop
Figure 4
A network of signal responses controls chemotaxis. The interactions among the signaling components that generate chemotactic
responses in Dicytostelium cells are shown. Symbols used to indicate positive or inhibitory links, small molecule reactions, and less-well
characterized connections are shown in the key located in the lower left corner of the figure. The network is divided into several
modules, which are contained in shaded boxes of different colors. Experimental data supporting the links between different components
are discussed in detail in the main text. Abbreviations: AleA, RasGEF Aimless; cAMP, 3′ ,5′ -cyclic adenosine monophosphate; cARs,
cAMP receptors; cGMP, 3′ 5′ -cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; GEF, guanonucleotide
exchange factors; PIP3 , phosphatidylinositol 3,4,5-trisphosphate; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PLA2 ,
phospholipase A2; PTEN, phosphatase and Tensin homolog on chromosome ten; TorC2, Target of Rapamycin Complex 2.
techniques, it appears that most of the iso- account for many experimental observations.
lated mutants display general chemotactic This network is characterized by redundancy
impairments rather than defects specific to and cross-talk among the pathways. For ease of
motility, directional sensing, or polarity. In discussion, the network is divided into a series
Figure 4, a subset of the genes listed in of overlapping modules where, to a rough ap-
Supplemental Table 1 is organized into an in- proximation, the output of one module serves
ternally consistent network of pathways that can as an input for the next.

ANRV411-BB39-14 ARI 12 April 2010 18:44
Receptor and G-Protein Module Evidence suggests that the cARs are directly
linked to the heterotrimeric G-protein, G2,
All responses to cAMP are initiated by a set
consisting of α2-, β-, and γ-subunits (reviewed
of similar cAMP receptors, cAR1-4, that are
in Reference 17). The β- and γ-subunits have
expressed at different stages of differentiation
been shown experimentally to directly interact,
(reviewed in Reference 85; see also Supple-
and cAMP triggers a rapid loss of FRET
mental Table 1). When the cAMP receptors
between the α2-subunit and the βγ-complex
are deleted and cells are assessed in early de-
(43, 54, 129). In gα2- or gβ- cells, cAMP does
velopment, car1- cells respond weakly to high
not activate actin polymerization, ACA, sGC or
doses of cAMP, whereas car1-/car3- cells do
GCA, or PI3Ks, nor does it mediate chemotaxis
not respond at all (51). When cAR1, cAR3,
(42, 74, 127, 135). Cells expressing a dominant
or cAR2 is expressed ectopically in car1-/car3-
negative γ-subunit or gγ - cells display a
cells, each receptor can mediate the same set
similar phenotype (133; M. Ueda, personal
of physiological responses but requires a pro-
communication). Observations suggest that the

portionally higher stimulus concentration cor-
βγ-complex mediates downstream signaling,
responding to its respective affinity for cAMP
whereas the α2-subunit is required to link
(67). Differences in affinity have been traced to
the heterotrimeric G-protein to the cARs

sequence differences in the extracellular loop
(127). The Dictyostelium genome contains 13
linking transmembrane domains four and five
additional Gα-subunits, and evidence suggests
(69).
that heterotrimeric G-proteins, consisting of
The properties of cAR1 have been exten-
either α4- or α5-subunits and sharing the βγ-
sively characterized. As with most GPCRs,
complex, link to a set of undefined nutrient re-
there are low- and high-affinity cAMP binding
ceptors (32, 37). Consistently, in growth-stage
sites, the latter of which are sensitive to GTP
gα2- cells, but not gβ- cells, folic acid activates
in isolated membranes (111). The dissociation
cGMP production and mediates chemotaxis
rate of cAMP from the receptor is on the or-
(74, 127). The Dictyostelium genome has at
der of a few seconds (108, 111). cAR1 under-
least seven Regulator of G-protein Signaling
goes robust agonist-induced phosphorylation,
(RGS) domain-containing proteins, which
which rises to a steady-state level proportional
are typically negative regulators of G-protein
to the level of receptor occupancy, with a half-
signaling, although none has been connected to
time of about 45 s (39). Upon removal of the
specific Gα-subunits so far (32). Disruption or
stimulus, dephosphorylation ensues with a half-
overexpression of one of these, RGS domain-
time of about 2 min (115). A phosphorylated
containing protein kinase 1 (RCK1), results in
receptor displays an average fivefold-lowered
a corresponding enhancement or reduction of
affinity for cAMP compared with its native
chemotactic speed (Supplemental Table 1).
counterpart (14). The shift in affinity does not
Expression of genes in early, but not late,
occur if specific phosphorylated serines are re-
development and progression through the en-
moved or replaced by alanines. However, the
tire developmental program require oscillatory
remainder of downstream responses still adapt
cAMP signaling through the receptor (Sup-
to constant stimuli, suggesting that receptor
plemental Sidebar 2 and references therein).
phosphorylation is not the mechanism control-
Cells that do not oscillate, such as those with
ling adaptation (70). A series of point mutations
disruptions in ACA, PiaA, or Crac, can still dif-
in cAR1 have been described that alter its affin-
ferentiate when pulsed with exogenous cAMP.
ity or lock it in constitutively active or various
However, cells lacking cARs or G2 fail to enter
intermediate functional states. All of these point
the developmental program even when supple-
mutations result in corresponding chemotactic
mented with exogenous cAMP (102, 127). The
defects that are consistent with the biochemical
initiation and progression of the developmental
properties of the receptors (68, 132).
program also require the protein kinase YakA,

ANRV411-BB39-14 ARI 12 April 2010 18:44
which appears to act immediately downstream rasG- cells, suggesting that PIP3 levels are not
of the heterotrimeric G-protein in the media- elevated (4). However, PIP3 -binding PH do-
tion of signaling events (110; see also Supple- mains are still recruited to the membrane (C.
mental Table 1). Disruption of YakA results in Janetopoulos, personal communication). These
cells that cannot transduce signals in response apparent discrepancies could be explained by
to cAMP or folic acid stimulation, the latter of variations in experimental conditions or com-
which does not require differentiation. Consti- pensation by RasD. A link between RasG and
tutive activation of cAMP-dependent protein PI3Ks is also supported by the disruption of
kinase A (PKA) can partially bypass the require- the RasGAP NF1, which coincidently prolongs
ment for cAMP oscillations in the developmen- the activation of both proteins (134). The re-
tal program (121). sulting excessive PIP3 levels cause a chemo-
The cAMP receptors are capable of trig- tactic phenotype resembling that of pten- cells
gering certain physiological responses in the (see below). Disruption of RasG or inhibition
absence of functional G-proteins (reviewed of PI3K activity partially rescues the nf1- cell
in Reference 11). In gα2- or gβ- cells, phenotype. The prolonged PIP3 production in
chemoattractant-induced phosphorylation of nf1- cells is consistent with adaptation occur-
cAR1 occurs normally and calcium influx and ring upstream of the Ras proteins. Both TorC2
Erk2 activation are reduced by only 50% (84, and PI3Ks lead to multiple downstream re-
86). Abrogation of calcium influx by disrupting sponses, including chemotaxis and the activa-
the putative inositol 1,4,5-trisphosphate (IP3 ) tion of ACA. This can be partially reconciled
receptor (IplA) has little effect on chemotaxis with early studies, which suggested that RasC
under normal conditions, possibly owing to re- regulates ACA activation, whereas RasG reg-
dundancy (Supplemental Table 1). Further ulates chemotaxis, although the actual roles of
study is necessary to determine the physiologi- the Ras proteins are probably more complex
cal importance of this response. than originally thought (4).
RasC and RasG Modules PIP3 and PIP2 Modules

The receptor- and G-protein-mediated tran- The activation of Ras proteins leads to increases
sient activations of Ras proteins appear to be in PIP3 , mediated by a burst in PI3K activity
significant early steps in the network (reviewed and a kinetically similar loss of PTEN from the
in Reference 122). Although genetic analy- membrane (28, 42, 45, 46, 83). These regula-
sis of these proteins is complicated by appar- tions are independent of PIP3 levels (47). Ev-
ent redundancy and/or compensation among idence suggests that PI3K activation requires
the pathways they trigger, some of the unique both recruitment to the membrane and inter-
roles have been established. For example, AleA action with Ras (34, 42). N-terminal regions of
and GEFR appear to be the predominant ex- PI3Ks, lacking the RBD and kinase domain, are
change factors for RasC and RasG, respectively sufficient for recruitment of GFP to the mem-
(60). In addition, studies of rasC-, rasG-, and brane in a chemoattractant-dependent manner.
rasC-/G- cells suggest that RasC and RasG PI3Ks lacking the N-terminal regions cannot
activate TorC2 and PI3Ks, respectively, with rescue the pi3k1-/pi3k2- cell phenotype, but ad-
possible overlaps in specificity (62, 99). In rasC- dition of a CAAX motif to a truncated protein
or rasC-/G- cells, phosphorylation of PKBR1, can produce a membrane-associated enzyme
a readout of TorC2 activation, is reduced by that is active in the absence of chemoattrac-
70% (62). The residual activation may be at- tant (34, 41, 42). For PTEN, an N-terminal,
tributable to the upregulation of RasD in rasG- 15-residue PIP2 -binding motif is required for
and rasC-/G- cells (66). It has been reported that membrane association, activity in cells, and
PKBA is not activated by chemoattractant in reversal of the pten- phenotype (47). The

ANRV411-BB39-14 ARI 12 April 2010 18:44
essential function of this motif is conserved directed migration, and cell-cell signaling (45).
in human PTEN (hPTEN), which inciden- Taken together, these results suggest that par-
tally can also rescue Dictyostelium pten- cells tially redundant pathways act in parallel with
(77, 116). In HeLa cells, rapid depletion of PIP3 signaling to mediate chemotaxis. One such
PIP2 causes hPTEN to immediately dissoci- pathway appears to involve Phospholipase A2
ate from the membrane, which further sug- (PLA2 ), because the simultaneous loss or in-
gests that PIP2 anchors PTEN to the mem- hibition of PI3K and PLA2 activities causes
brane (96). It is speculated that temporal and a stronger chemotactic defect than does the
spatial regulation of PIP2 levels by chemoat- loss of either activity alone (15, 112). Another
tractant occur primarily through Phospholipase parallel pathway described in detail below in-
C (PLC) in Dictyostelium (71). In theory, activa- volves TorC2- and PKB-mediated phosphory-
tion of PLC leads to a decrease in PIP2 levels, lation events.
dissociation of PTEN from the membrane, and
an enhanced increase in PIP3 . In chemotaxing

plc- cells, PTEN is not dissociated from the PKB and PKB Substrate Modules
membrane at the front and PIP3 increases at the Activated Ras proteins and PIP3 accumula-
leading edge are reduced. The chemorepellent tions are involved in the regulation of the
8-CPT-cAMP, a cAMP analog, acts through the chemoattractant-induced activities of two ma-
G-protein α1-subunit to inhibit PLC activity, jor kinases, PKBA and PKBR1, and the phos-
which is presumed to increase PIP2 and recruit phorylation of PKB substrates (Supplemental
PTEN toward the high side of the gradient, Table 1). Like mammalian PKBs, each kinase
flipping the axis of polarization (65). is phosphorylated within a hydrophobic mo-
Local accumulations of PIP3 lead to the re- tif (HM) by TorC2 and within an activation
cruitment of multiple PH domain–containing loop (AL), presumably by phosphoinositide-
proteins to the membrane (Supplemental dependent protein kinases (PDKs). In cells
Table 2). The PH domain–containing proteins lacking putative TorC2 subunits PiaA or Ras-
that translocate to the membrane in response interacting protein 3 (Rip3), chemoattractants
to chemoattractant stimulation in Dictyostelium fail to elicit phosphorylation of the HM of ei-
include Crac, PKBA, and PH domain protein ther PKB (62). The absence of HM phospho-
A (PhdA), and cells lacking these proteins have rylation prevents phosphorylation of the AL of
been reported to display weak defects in chemo- PKBR1 and substantially decreases phospho-
taxis (23, 90; see also Supplemental Table 2). rylation of the AL of PKBA. Unlike typical
Characterization of the other translocating PH PKBs, including PKBA, PKBR1 lacks a PIP3 -
domain-containing proteins is in progress, and sensitive PH domain and is tethered consti-
many more that have yet to be studied are tutively to the membrane by myristoylation,
currently being evaluated for responsiveness to and therefore its activation is independent of
PIP3 signaling. PIP3 .
Chemoattractant-induced PIP3 production The two PKBs act somewhat redundantly
is a highly conserved signature of chemotactic to phosphorylate a series of substrates, includ-
signaling in many cell types, yet inhibiting this ing TalinB, the RacGAP GacQ, the RasGEFs
response alone does not always block chemo- GEFS and GEFN, PakA, and a putative
taxis (16, 33, 40, 63, 105). Dictyostelium cells or phosphoinositide 5-kinase (PI5K) (21, 62).
neutrophils lacking PI3Ks still carry out essen- The bulk of chemoattractant-mediated PKB-
tially normal chemotaxis in steep gradients or specific phosphorylation events are insensitive
under specific adhesive conditions. In contrast, to inhibition or disruption of PI3Ks, indicating
pten- cells have high basal PIP3 levels and ex- that PKBR1 is the predominate kinase. The
aggerated PIP3 increases with chemoattractant phosphorylation of substrates is substantially
stimulation, causing defects in cell morphology, reduced in pkbR1- cells and nearly abolished

ANRV411-BB39-14 ARI 12 April 2010 18:44
in piaA- cells. Consistently, pkbR1- and piaA- C. Parent, F. Comer, S. Das & P. Devreotes,
cells have impaired chemotaxis (18, 62). Fur- manuscript in preparation).
thermore, phosphorylation of specific PKB cAMP is synthesized by ACA and degraded
substrates is exaggerated and prolonged in both intracellularly by the phosphodiesterase
pten- cells, which have extraneous projections RegA and extracellularly by membrane-bound
that interfere with chemotaxis. These defects and secreted phosphodiesterases (2). A circuit
are suppressed by simultaneous disruption of involving Erk2, RegA, ACA, and PKA has
PKBA, indicating that the effects of PIP3 are been proposed to contribute to the spontaneous
mediated by this protein (M. Tang, M. Iijima, oscillations of cAMP levels that occur dur-
Y. Kamimura & P. Devreotes, manuscript in ing development (80; see also Supplemental
preparation). Thus, in this series of mutants, Sidebar 2). In addition to activating ACA, a re-
the level of PKB substrate phosphorylation cor- ceptor also activates Erk2, which inhibits RegA,
relates well with the observed behavior of the thus preventing the degradation of cAMP. The
cells. Consistently, inhibition of PKBs in mam- resulting elevated levels of cAMP activate PKA,
malian cells has been reported to interfere with which in turn deactivates ACA. In this model,
chemotaxis (35, 130). The functions of specific the oscillations arise from a combination of this
PKB substrates in Dictyostelium are currently inhibition and a positive feedback loop in which
being investigated; it is expected that each has cAMP stimulates the receptor.
a distinct role in mediating chemotaxis.
cGMP/Myosin II Module
cAMP Module Activation of the receptor and heterotrimeric
The oscillatory production and secretion of G-protein triggers a transient burst of cGMP
cAMP by ACA mediate cell-cell signal relay, that is closely associated with the chemotac-
and, although not necessary for chemotaxis in tic response in Dictyostelium (reviewed in Ref-
differentiated cells, the regulation of this re- erence 113). The cGMP increase is mediated
sponse provides important insights into the by membrane-bound and soluble guanylyl cy-
chemotactic signaling networks (93; see also clases (GCA and sGC, respectively) (98). Curi-
Supplemental Sidebar 2). Activation of ACA ously, GCA has 12 transmembrane domains, as
requires PIP3 accumulation and PKB activa- do adenylyl cyclases; both GCs have catalytic
tion. An early study showed that, in vitro, ad- domains that are similar to those of the ACs.
dition of supernatants from piaA- or crac- cells Unlike mammalian GCs, neither Dictyostelium
restored nonhydrolyzable GTP (GTPγS) stim- GC contains a heme group. Simultaneous dis-
ulation of ACA to extracts from crac- or piaA- ruption of GCA and sGC prevents cGMP ac-
cells, respectively, but only wild-type super- cumulation and leads to a defect in chemotaxis.
natants restored activation to extracts of crac-/ Mutations in the complementation group re-
piaA- cells (18). For ACA activation, Crac ferred to as streamer F, which maps to the gene
requires an intact PH domain, which medi- for a cGMP-specific phosphodiesterase, PdeD,
ates the recruitment of Crac to PIP3 at the were some of the earliest described chemotactic
leading edge of the cell (90). Consistently, in- defects (26). Streamer F mutants have exces-
hibitors of PI3Ks block activation of ACA in sive and prolonged stimulus-mediated cGMP
vitro, and pten- cells display excessive ACA ac- accumulation, have excessive Myosin II associ-
tivation (24, 45). The involvement of TorC2 ation with the cortex, and are hyperpolarized
strongly suggests that activation of ACA also (reviewed in Reference 88). These findings led
requires PKB activity and probably phosphory- to the concept that cGMP positively regulates
lation of one or more PKB substrates. Prelim- cell polarity.
inary evidence shows that ACA is not activated The Roco protein kinase family member
in pkbR1-/pkba- cells (H. Cai, Y. Kamimura, cGMP-binding protein C (GbpC) appears to

ANRV411-BB39-14 ARI 12 April 2010 18:44
bind to and mediate the effects of cGMP (9). (87). Rap1 and its putative effector, tyrosine
GbpC contains Leucine-rich repeat (LRR), kinase-like protein Phg2, are also speculated
Ras, and MAPKKK domains, like other Roco to activate MHCK at the leading edge of the
family members, and also a unique C-terminal cell (56, 57, 72). Rap1 is activated by the GEF
extension with cyclic nucleotide binding and domain of cGMP-binding protein D (GbpD),
GEF domains (reviewed in Reference 81). In and although GbpD contains cyclic nucleotide-
vitro, cGMP activates the GbpC GEF domain, binding domains, it is not stimulated by cGMP
which in turn activates the Ras domain and leads or cAMP (6, 72). Disruption of GbpD or ex-
to the subsequent activation of the MAPKKK pression of dominant-negative Rap1 leads to
domain (109). GbpC activity mediates the re- excessive polarization and reduced lateral pseu-
cruitment of Myosin II to the cortex at the rear dopodia extension, whereas overexpression of
of the cell, which is important for generating the GbpD, expression of constitutive-active Rap1,
tension and contraction that facilitate chemo- or disruption of RapGAP1 leads to enhanced
taxis (6, 9). Disruption of the gbpC gene causes adhesion, many protrusions, and inhibition of
defects in polarity and chemotaxis that resem- chemotaxis (56, 57, 72). This phenotype is par-
ble the loss of GCs or Myosin II (6, 9, 98, 126). tially suppressed when GbpD is expressed in
Recent evidence suggests that PIP3 may also be phg2- cells, suggesting that Phg2 mediates some
involved in restricting Myosin II to the lagging of the effects of GbpD and Rap1 (72). As ex-
edge, because Myosin II localization is abnor- pected from the restriction of MHCK to the
mal in pten- cells during cytokinesis and other leading edge, Phg2, RapGAP1, and markers
growth stages (53, 95, 125). that should be specific for Rap1-GTP are all
localized to the front of cells (56, 57). Recent
evidence suggests that Rap1 activity is regulated
RasB/Rap1/MHCK Module by RasG, which is consistent with the slower
The activation of receptor and G-protein chemoattractant-mediated activation of Rap1
triggers the activation of RasB and Rap1, compared with the other Ras proteins (5, 57).
which regulate Myosin Heavy Chain Kinases However, other reports state that prolonged ac-
(MHCKs) and Myosin II. The phosphoryla- tivation of RasG does not appear to increase
tion of Myosin II Heavy Chain (MHCA) by Rap1 activation (134).
MHCKs promotes the disassembly and release
of Myosin II from the cortex and opposes
cGMP-mediated Myosin II assembly (reviewed Linking the Signaling Network
in Reference 7). RasB and Rap1 are thought to to the Cytoskeleton
mediate the chemoattractant-induced recruit- Chemotaxis depends on both myosin-mediated
ment and activation of MHCKA at the front of contraction at the rear of the cell and actin
cells, resulting in the restriction of Myosin II to polymerization at the front. It is thought that
the rear (31, 79). Furthermore, it has been pro- signaling through the Rac proteins mediates
posed that PakA, which is localized at the lag- actin polymerization in Dictyostelium as in neu-
ging edge, inhibits MHCK activity at the rear trophils and other cell types. Disruption of
(19). some of the individual Dictyostelium Rac pro-
Several studies support the involvement of teins or putative Rac exchange factors and ef-
RasB or Rap1 in the activation of MHCKs (56, fectors results in impaired chemotaxis (20, 38,
57, 72, 87). Excessive activation of RasB, caused 52, 76, 89, 92, 103). However, a definitive
by the overexpression of the GEF domain from role for the Racs has been elusive owing to
GEFQ, recruits MHCKA to the membrane and redundancy between the many Rac proteins.
phenocopies mhcA- cells (87, 126). Conversely, Indeed, although there has been intense inves-
gefQ- cells underphosphorylate and overassem- tigation of the cytoskeletal events involved in
ble Myosin II, which also impairs chemotaxis actin polymerization, little is known about the

ANRV411-BB39-14 ARI 12 April 2010 18:44
mechanisms by which signaling events regu- substrates may be an important link between re-
late the actin cytoskeleton, but there are some ceptor signaling and actin polymerization. Sec-
clues. First, excessive or prolonged levels of ond, signals mediated by contractive forces at
PIP3 , as found in pten- and nf1- cells, elevate the rear may regulate actin polymerization as
the actin polymerization response and inter- part of a negative feedback loop. For exam-
fere with chemotaxis (16, 45, 134). The ef- ple, mhcA- cells have an increased number of
fects of elevated PIP3 in these mutants may lateral pseudopodia and chemotactic defects,
be a result of excessive PKB substrate phos- similar to pten- cells (45, 126). By reading ex-
phorylation, because disruption of PKBA in tracellular gradients and integrating the reg-
pten- cells restores polarity, suppresses ex- ulation of actin polymerization and myosin-
traneous pseudopodia, and enhances chemo- based contraction, the signaling network can
taxis (M. Tang, M. Iijima, Y. Kamimura & interpret the intracellular compass and trans-
P. Devreotes, manuscript in preparation). late inputs from chemical stimuli into directed
Therefore, phosphorylation of certain PKB migration.
SUMMARY POINTS
1. A conserved process referred to as chemotaxis guides the migration of cells, such as

neurons, leukocytes, stem cells, and simple amoeba, along chemical gradients.
2. Chemotaxis can be conceptually divided into the processes of motility, directional sensing,
and polarity.
3. Eukaryotic cells can compare and react to the small concentration differences across
their dimensions. In some cells, adaptation to constant chemotactic stimulation allows
subtraction of ambient chemoattractant concentrations and greatly increases the accuracy
of gradient sensing.
4. Genetic analysis of Dictyostelium is revealing that chemotaxis depends on a network of
overlapping interactions among receptors, G-proteins, Ras proteins, PI3Ks, protein ki-
nases, and phosphatases.
FUTURE ISSUES
1. What are the components involved in the generation and propagation of waves of cy-
toskeletal proteins that are seen on the basal cortex of migrating cells? What is the
causal relationship between these waves and cell motility, and how are they affected by
chemotactic stimuli and gradients?
2. What is the molecular mechanism of adaptation? Evidence suggests that it occurs between
G-proteins and Ras proteins. How are the Ras proteins activated and inactivated by G-
protein signaling?
3. What is the nature of the positive feedback mechanisms that drive polarity, and how
are proteins restricted to the front or back of polarized cells? How is it that different
components of the same signaling pathway sometimes show widely different localization
patterns?
4. What is the relative importance of the parallel pathways in the chemotactic signaling
network, and what is the purpose of the apparent redundancy?

ANRV411-BB39-14 ARI 12 April 2010 18:44
5. How is actin polymerization activated and regulated by the chemotactic signaling net-
work?
6. To what extent is the chemotactic signaling network described in Dictyostelium applicable
to other systems, such as leukocytes, fibroblasts, and cancer cells?
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
Kristen F. Swaney and Chuan-Hsiang Huang contributed equally to this review. The authors wish
to thank Pablo Iglesias and Yulia Artemenko for critical reading of the manuscript and Peter van
Haastert for scientific suggestions. KFS is supported by the American Heart Association. CHH
is a Harold L. Plotnick Fellow of the Damon Runyon Cancer Research Foundation. This work
was supported by NIH grants GM28007 and GM34933 to PND.
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Annual Review of
Contents Biophysics
Volume 39, 2010
Adventures in Physical Chemistry

Harden McConnell ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 1
Global Dynamics of Proteins: Bridging Between Structure

and Function
Ivet Bahar, Timothy R. Lezon, Lee-Wei Yang, and Eran Eyal ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣23
Simplified Models of Biological Networks

Kim Sneppen, Sandeep Krishna, and Szabolcs Semsey ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣43
Compact Intermediates in RNA Folding
Sarah A. Woodson ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣61
Nanopore Analysis of Nucleic Acids Bound to Exonucleases
and Polymerases
David Deamer ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣79
Actin Dynamics: From Nanoscale to Microscale
Anders E. Carlsson ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣91
Eukaryotic Mechanosensitive Channels
Jóhanna Árnadóttir and Martin Chalfie ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 111
Protein Crystallization Using Microfluidic Technologies Based on
Valves, Droplets, and SlipChip
Liang Li and Rustem F. Ismagilov ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 139
Theoretical Perspectives on Protein Folding
D. Thirumalai, Edward P. O’Brien, Greg Morrison, and Changbong Hyeon ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 159
Bacterial Microcompartment Organelles: Protein Shell Structure
and Evolution
Todd O. Yeates, Christopher S. Crowley, and Shiho Tanaka ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 185
Phase Separation in Biological Membranes: Integration of Theory
and Experiment
Elliot L. Elson, Eliot Fried, John E. Dolbow, and Guy M. Genin ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 207
v
AR411-FM ARI 2 April 2010 20:22
Ribosome Structure and Dynamics During Translocation

and Termination
Jack A. Dunkle and Jamie H.D. Cate ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 227
Expanding Roles for Diverse Physical Phenomena During the Origin
of Life
Itay Budin and Jack W. Szostak ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 245
Eukaryotic Chemotaxis: A Network of Signaling Pathways Controls
Motility, Directional Sensing, and Polarity
Kristen F. Swaney, Chuan-Hsiang Huang, and Peter N. Devreotes ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 265
Protein Quantitation Using Isotope-Assisted Mass Spectrometry
Kelli G. Kline and Michael R. Sussman ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 291

Structure and Activation of the Visual Pigment Rhodopsin
Steven O. Smith ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 309
Optical Control of Neuronal Activity

Stephanie Szobota and Ehud Y. Isacoff ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 329
Biophysics of Knotting
Dario Meluzzi, Douglas E. Smith, and Gaurav Arya ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 349
Lessons Learned from UvrD Helicase: Mechanism for
Directional Movement
Wei Yang ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 367
Protein NMR Using Paramagnetic Ions
Gottfried Otting ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 387
The Distribution and Function of Phosphatidylserine
in Cellular Membranes
Peter A. Leventis and Sergio Grinstein ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 407
Single-Molecule Studies of the Replisome
Antoine M. van Oijen and Joseph J. Loparo ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 429
Control of Actin Filament Treadmilling in Cell Motility
Beáta Bugyi and Marie-France Carlier ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 449
Chromatin Dynamics
Michael R. Hübner and David L. Spector ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 471
Single Ribosome Dynamics and the Mechanism of Translation
Colin Echeverrı́a Aitken, Alexey Petrov, and Joseph D. Puglisi ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 491
Rewiring Cells: Synthetic Biology as a Tool to Interrogate the
Organizational Principles of Living Systems
Caleb J. Bashor, Andrew A. Horwitz, Sergio G. Peisajovich, and Wendell A. Lim ♣ ♣ ♣ ♣ ♣ 515
vi Contents
AR411-FM ARI 2 April 2010 20:22
Structural and Functional Insights into the Myosin Motor Mechanism

H. Lee Sweeney and Anne Houdusse ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 539
Lipids and Cholesterol as Regulators of Traffic in the
Endomembrane System
Jennifer Lippincott-Schwartz and Robert D. Phair ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 559
Index
Cumulative Index of Contributing Authors, Volumes 35–39 ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 579

Errata
An online log of corrections to Annual Review of Biophysics articles may be found at

http://biophys.annualreviews.org/errata.shtml
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ANRV411-BB39-14 ARI 12 April 2010 18:44

Kristen F. Swaney, Chuan-Hsiang Huang,

Annu. Rev. Biophys. 2010. 39:265–89 Key Words

examples of the importance of chemotaxis in

of neutrophils toward a variety of chemokines

G-protein coupled receptors (GPCRs), result-

266 Swaney · Huang · Devreotes

chemoattractant-induced reductions in phos- Directional

Despite slight differences in the migra-

other cell types, the overall pathways that

www.annualreviews.org • Signaling Events in Chemotaxis 267

268 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 269

a Add Cell behavior

Time Spatial gradient

270 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 271

272 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 273

274 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 275

Gα2 Gα4/5 Gα9 SodC

GbpC Phg2 TorC2 PI3Ks PTEN

276 Swaney · Huang · Devreotes

communication). Observations suggest that the

the heterotrimeric G-protein to the cARs

www.annualreviews.org • Signaling Events in Chemotaxis 277

RasC and RasG Modules PIP3 and PIP2 Modules

278 Swaney · Huang · Devreotes

an enhanced increase in PIP3 . In chemotaxing

www.annualreviews.org • Signaling Events in Chemotaxis 279

280 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 281

Therefore, phosphorylation of certain PKB migration.

1. A conserved process referred to as chemotaxis guides the migration of cells, such as

282 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 283

actin-mediated rocketing of phagosomes. Mol. Biol. Cell 17:4866–75

284 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 285

of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis. Mol. Biol. Cell 18:4772–79

286 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 287

288 Swaney · Huang · Devreotes

www.annualreviews.org • Signaling Events in Chemotaxis 289

Adventures in Physical Chemistry

Global Dynamics of Proteins: Bridging Between Structure

Simplified Models of Biological Networks

Ribosome Structure and Dynamics During Translocation

Kelli G. Kline and Michael R. Sussman ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 291

Optical Control of Neuronal Activity

Structural and Functional Insights into the Myosin Motor Mechanism

Cumulative Index of Contributing Authors, Volumes 35–39 ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 579

An online log of corrections to Annual Review of Biophysics articles may be found at

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