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pubs.acs.org/acschemicalbiology
Importazole, a Small Molecule Inhibitor of the Transport Receptor
Importin-β
Jonathan F. Soderholm,†,||,z Stephen L. Bird,†,z Petr Kalab,†,‡ Yasaswini Sampathkumar,† Keisuke Hasegawa,‡
Michael Uehara-Bingen,§,^ Karsten Weis,†,* and Rebecca Heald†,*
†
The Department of Molecular and Cell Biology, University of California, Berkeley, MC 3200 LSA, Berkeley, California 94720-3200,
United States
‡
National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, Maryland 20892-4256, United States
§
The Small Molecule Discovery Center, University of California, San Francisco, MC2552 Byers Hall S504, 1700 fourth Street,
San Francisco, California 94158-2330, United States
bS Supporting Information
ABSTRACT: During interphase, the transport receptor importin-β carries cargoes
into the nucleus, where RanGTP releases them. A similar mechanism operates in
mitosis to generate a gradient of active spindle assembly factors around mitotic
chromosomes. Importin-β and RanGTP have been implicated in additional cellular
processes, but the precise roles of the Ran/importin-β pathway throughout the cell
cycle remain poorly understood. We implemented a FRET-based, high-throughput
small molecule screen for compounds that interfere with the interaction between
RanGTP and importin-β and identified importazole, a 2,4-diaminoquinazoline.
Importazole specifically blocks importin-β-mediated nuclear import both in Xenopus
egg extracts and cultured cells, without disrupting transportin-mediated nuclear
import or CRM1-mediated nuclear export. When added during mitosis, importazole
impairs the release of an importin-β cargo FRET probe and causes both predicted and
novel defects in spindle assembly. Together, these results indicate that importazole
specifically inhibits the function of importin-β, likely by altering its interaction with RanGTP. Importazole is a valuable tool to
evaluate the function of the importin-β/RanGTP pathway at specific stages during the cell cycle.
I
mportin-β transport receptors, which comprise at least 22
members in vertebrates,1 bind to cargo molecules and mediate
their import or export through nuclear pores.2 Directionality of
transport depends on the nature of the receptor as well as the
asymmetric distribution of nucleotide states of the small GTPase
Ran, which is GTP-bound in the nucleus due to the chromatin
interaction of its guanine exchange factor (GEF) RCC1 and
GDP-bound in the cytoplasm where its GTPase activating
protein, RanGAP, is localized. The founding member of this
family, importin-β, together with its partner importin-R, recognize nuclear localization signal (NLS)-containing cargo molecules and transport them into the nucleus where RanGTP binds
directly to importin-β, causing a conformational change that
releases importin-R and NLS cargoes. In addition to its vital
interphase functions, importin-β and Ran are also important
regulators during mitosis, contributing to chromatin-mediated
spindle assembly.35 During mitosis, importin-β has an inhibitory function toward NLS-containing spindle assembly factors,
binding them in the cytoplasm and impairing their microtubulestabilizing or -organizing activities. However, RanGTP remains
enriched around condensed mitotic chromosomes in mitosis
and generates a gradient of released cargoes that triggers
spindle assembly.6,7 The importin-β/RanGTP pathway has
also been implicated in a variety of other cellular processes
including postmitotic nuclear envelope assembly, nuclear pore
r 2011 American Chemical Society
complex assembly, protein ubiquitylation, and primary cilium
formation.812
Small-molecule inhibitors provide a promising approach to
study the multifunctional importin-β/Ran pathway in living cells
by acting like conditional mutations that allow disruption of a
protein with temporal precision, at any phase of the cell cycle.
Compounds targeting microtubules or microtubule-based motors have been successfully used to dissect their mitotic functions
and also gain mechanistic insight into the complex events of
mitosis. For example, the drug monastrol inhibits kinesin-5
(Eg5)13 and causes a loss of spindle bipolarity, consistent with
this motor’s microtubule cross-linking and sliding function, as well
as the results of immunodepletion and antibody microinjection
experiments.14,15 However, monastrol has also provided novel
insights through drug-washout experiments and, in combination
with other inhibitors, to assess how spindle bipolarity and microtubule attachment to chromosomes are established16,17 and how
the cell division cleavage plane is positioned.18 Because of its
fundamental role in many cellular functions including mitosis,
nuclear transport is also an attractive target for small molecule
inhibition. However, despite the importance of this process,
Received: January 25, 2011
Accepted: April 6, 2011
Published: April 06, 2011
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surprisingly few inhibitors have been identified. With respect to
nuclear export, leptomycin is a potent inhibitor but binds covalently to its target, preventing washout experiments.19 Peptide
inhibitors20 and small molecule peptidomimetic inhibitors21 of
importin-R/β have been designed and used to study nuclear
import in vivo. However, these inhibitors are not cell-permeable.
Recently, a new cell-permeable small molecule inhibitor of the
RanGTP/importin-β interaction named karyostatin 1A that binds
specifically to importin-β and blocks importin-β-mediated nuclear
import has been identified.22 However, the effects of karyostatin
1A on mitotic events have not yet been demonstrated.
To gain a better understanding of the functions of the
importin-β/Ran pathway in mammalian cells without the limitations associated with microinjection of proteins or antibodies or
the time required for efficacy of RNA interference or peptide
inhibitors,7 we aimed to identify a cell-permeable, specific, and
reversible small molecule inhibitor that would provide high
temporal precision, allowing dissection of the role of importinβ/RanGTP throughout the cell cycle. Here we report the
discovery of importazole, which meets these criteria and suggests
at least one previously uncharacterized role for this pathway in
mitosis.
’ RESULTS AND DISCUSSION
Figure 1. A high-throughput screen identifies importazole as an inhibitor of FRET between CFP-Ran and YFP-importin-β. (a) Schematic
of the fusion proteins that bind and undergo FRET in the presence
of Ran-GTP but not Ran-GDP. (b) Fluorescence emission of the FRET
pair detected between 460 and 550 nm following excitation at 435 nm,
showing strong emission of CFP (475 nm) in the presence of GDP (red
curve) that decreases in the presence of GTP (blue curve) concomitant
with an increase at the emission wavelength of YFP (525 nm), indicative
of FRET. (c) Summary of the screen. Of 137,284 small molecules
screened in duplicate using the FRET-based assay, 141 putative hits were
subjected to three secondary screens designed to eliminate false
positives. Of the 10 compounds remaining after the secondary screens,
only a single compound reproducibly diminished the FRET signal
generated by CFP-Ran and YFP-importin-β in the original assay (d).
(e) The structure of importazole, a 2,4-diaminoquinazoline.
Identification of Importazole in a High-Throughput
Screen. We applied a reverse chemical genetic high-throughput
screen (HTS) to identify compounds that affect the interaction
between RanGTP and importin-β using a fluorescence resonance energy transfer (FRET)-based assay with CFP-tagged Ran
and YFP-tagged importin-β. These proteins bind one another
only when CFP-Ran is GTP-bound, which can be detected by
changes in FRET (Figure 1, panels a and b). When CFP-Ran is
incubated with RCC1, GTP, and YFP-importin-β, and the
mixture is excited with 435 nm fluorescence in a fluorometer, a
strong FRET signal is generated, as indicated by a decrease in the
fluorescence intensity at 475 nm (the emission wavelength of
CFP) and an increase in the fluorescence intensity at 525 nm
(the emission wavelength of YFP). No FRET signal is generated
if GDP is substituted for GTP, and nucleotide-specific interaction could also be observed biochemically, as S-tagged YFPimportin-β pulls CFP-Ran out of solution only in the presence of
GTP (Figure S1 in Supporting Information). These results
demonstrate that the FRET signal generated by CFP-RanGTP
and YFP-importin-β is due to a physical interaction dependent
upon the nucleotide state of Ran and that our approach could be
used to identify compounds that interfere with the interaction
between CFP-RanGTP and YFP-importin-β, resulting in a
reduced FRET signal.
The assay was tested for suitability for HTS using a 384-well
format and a fluorescence plate reader. We calculated FRET
ratios (IFRET/ICFP) for each well and determined two commonly
used statistical parameters, the coefficient of variation (CV),
which was 0.95% and 1.24% for reactions containing the GDP
and GTP, respectively, and the Z0 value, which was 0.81, indicating that our assay was robust and appropriate for HTS.23 To
facilitate rapid data analysis, we developed software to generate
color-coded plate maps to identify compounds that reduced
the FRET ratio by both an increase in CFP emission and a
decrease in YFP emission, thereby eliminating compounds that
altered the FRET ratio by contributing their own fluorescence at
wavelengths in the range of our probes (Figure S2 in Supporting
Information).
In total, we screened 137,284 compounds in duplicate
(Figure 1, panel c), and selected 141 “hits” for further analysis.
Compounds that showed activity upon retesting in the original
assay were analyzed in a unimolecular CFP and YFP FRET-based
assay using a YIC sensor6 to confirm that the observed changes
were not due to nonspecific quenching or augmentation of
either CFP or YFP emission. In a third assay, each compound
was tested for its tendency to form aggregates that nonspecifically
inhibit β-lactamase.24 The 10 compounds that survived the
secondary assays were obtained in larger quantities and tested
again in the original CFP-Ran/YFP-importin-β FRET assay
using a spectrofluorometer. Only one of these compounds, a
2,4-diaminoquinazoline that we named “importazole”, reproducibly disrupted the FRET signal generated by CFP-Ran and YFPimportin-β and was analyzed further (Figure 1, panels d and e).
Importazole Binds Importin-β in Vitro. Although importazole
blocked the FRET interaction between CFP-RanGTP and YFPimportin-β in vitro, it did not obviously affect the binding of the two
proteins in pull-down assays (Figure S1 in Supporting Information).
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Figure 3. Importazole inhibits importin-β NLS-mediated nuclear import but not transportin M9-mediated import. (a) NLS-GFP (importinβ import substrate) was added with Xenopus egg extract to permeabilized
HeLa cells and assayed by fluorescence microscopy for nuclear import
in the presence of DMSO or 100 μM importazole. (b) NLS-GFP
accumulation at the nuclear rim in the presence of importazole. (c) M9YFP (transportin import substrate) in the same assay. In all figures, scale
bar = 10 μm.
significantly affect the melting curves of related importin-β family
members transportin and CRM1 or that of RanGTP, suggesting
that importazole binds preferentially to importin-β (Figure 2, panel
c, Figure S5 in Supporting Information). Going forward, a crystal
structure of the RanGTP/importin-β complex in the presence of
importazole will likely be necessary to fully elucidate its biochemical
mechanism of action.
Importazole Disrupts Importin-β/RanGTP-Mediated Nuclear Import. If importazole binds importin-β and affects the
RanGTP/importin-β interaction, it should inhibit the nuclear
import of any protein bearing a classical NLS. We first tested this
prediction using permeabilized HeLa cells, in which nuclear
import of a GFP-NLS reporter can be reconstituted in vitro.29
Digitonin-permeabilized cells were incubated with a GFP-NLS
reporter plus Xenopus laevis egg extracts as a source of soluble
transport factors including Ran, importin-R, and importin-β.
Whereas rapid nuclear accumulation of GFP-NLS occurred in
the presence of the solvent DMSO, importazole blocked import
and the reporter became enriched at the nuclear envelope, where
RanGTP functions to induce cargo release from importin-β 30,31
(Figure 3, panels a and b). In contrast, importazole did not
block nuclear import mediated by transportin, an importin-β
family member that utilizes the M9 import signal together with
RanGTP to import hnRNP proteins (Figure 3, panel c).32
To investigate whether importazole is cell-permeable and
active in living human cells, we generated a cell line that stably
expresses a GFP-tagged version of the transcription factor
NFAT, which shuttles between the nucleus and the cytoplasm
in a calcium-regulated manner33,34 and is imported by importinR/β and exported by CRM1.35,36 At steady state NFAT is
predominantly cytoplasmic. An increase in cytoplasmic calcium
induced by the ionophore ionomycin leads to the accumulation
of NFAT in the nucleus (Figure 4, panel a). NFAT import can be
reverted upon ionophore withdrawal (Figure 5, panel a), providing an inducible system ideal for testing the effects of importazole
on importin-β-mediated nuclear import and CRM1-mediated
nuclear export, both of which are dependent upon RanGTP.
Cells were pretreated with 40 μM importazole for 1 h followed
by 30 min of ionomycin treatment in the continued presence of
importazole. Whereas control cells treated with DMSO or the
control compound 3016 displayed a robust nuclear accumulation
of the NFAT-GFP reporter after ionomycin addition, there was
Figure 2. Importazole binds specifically to importin-β. (a) Negative
first derivatives of melting curves of 2 μM importin-β in the presence of
50 μM importazole or DMSO where the minima indicate the melting
temperature. Melting curves show the results from six experiments
conducted in quadruplicate using the Applied Biosystems 7500 qPCR
machine. (b) Negative first derivatives of melting curves of 2 μM
RanQ69L in the presence of 50 μM importazole or DMSO as control
where the minima indicate the melting temperature. (c) Mean changes
in melting temperature of 2 μM importin-β, RanQ69L, transportin, and
CRM1 in the presence of 50 μM importazole. Error bars indicate
standard error; asterisks denote statistical significance (p < 0.01).
To begin elucidating the mechanism of importazole action, we
tested whether importazole could alter the ability of importin-β to
protect RanGTP from RanGAP-stimulated hydrolysis in vitro.25,26
Binding curves calculated from these data do not indicate that
importazole disrupts the RanGTP/importin-β interaction and, if
anything, suggest that importazole may slightly stabilize the complex
(Figure S3 in Supporting Information). The inability of importazole
to disrupt the RanGTP/importin-β interaction is not entirely
surprising considering the multiple large interaction surfaces between the two proteins.27 One possible explanation for the importazole-induced FRET change is that importazole binding causes
a conformational change that disrupts the CFP-RanGTP/YFPimportin-β FRET interaction without preventing binding. To test
whether importazole binds to importin-β in vitro, we used a
fluorescent thermal shift assay with the dye Sypro Orange, since
small molecule binding is expected to affect the thermal stability of a
protein.28 Importazole reduced the melting temperature of importin-β by 1.72 ( 0.27 C (Figure 2, panels a and b) but was
unaffected by a related compound of comparable hydrophobicity
that did not interfere with CFP-RanGTP/YFP-importin-β FRET
(compound 3016, Figure S4 in Supporting Information, panels c
and d, and data not shown). In contrast, importazole did not
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Figure 4. Importazole reversibly blocks importin-β-mediated nuclear import in living cells. (a) Schematic showing that the GFP-tagged, NLScontaining transcription factor NFAT enters the nucleus upon treatment with the ionophore ionomycin in a RanGTP- and importin-β-dependent
manner. (b) HEK 293 cells stably expressing GFP-NFAT were treated with DMSO or 40 μM importazole for 1 h prior to a 30 min treatment with
ionomycin to induce nuclear import. Importazole was washed out and after 1 h prior to ionomycin retreatment. (c) Results were quantified as the
percentage of cells with nuclear NFAT-GFP. N = 3; 100 or more cells counted under each condition. Bars represent standard error.
Figure 5. Importazole does not inhibit Crm1-mediated nuclear export. (a) Schematic illustrating that upon removal of the ionophore ionomycin, GFPNFAT exits the nucleus in a RanGTP- and Crm1-dependent manner. (b) Cells were treated with ionomycin to induce nuclear import of NFAT-GFP,
then washed and treated with DMSO, importazole, leptomycin B, or importazole þ leptomycin B for 1 h. (c) Results were quantified as the percentage of
cells with nuclear NFAT-GFP. N = 3; 100 or more cells counted under each condition. Bars represent standard error.
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Figure 6. Importazole impairs spindle assembly in Xenopus egg extracts but does not affect pure microtubule polymerization. (a) Spindle assembly
reactions containing X-rhodamine labeled tubulin in the presence of DMSO, 100 μM importazole, or a truncated form of importin-β that is unable to
bind to RanGTP. Microtubules are red, and DNA is blue. (b) Quantification of the percentage of normal spindle structures. N = 3; 100 structures
counted under each condition. Bars represent standard error. (c) Aster assembly induced by addition of 5% DMSO to extracts containing X-rhodamine
labeled tubulin in the presence of DMSO or importazole. (d) Quantification of the number of asters per field. Ten fields were counted under each
condition. (e) DMSO induced pure tubulin polymerization assay. Reactions were supplemented with additional DMSO, importazole, or nocodazole,
and microtubules pelleted through a sucrose cushion and samples from the pellet (P) and supernatant (S) were analyzed by SDSPAGE.
virtually no import of NFAT-GFP in importazole-treated cells
(Figure 4, panel b, quantified in panel c and Figure S6 in
Supporting Information). Importazole displayed an IC50 of
approximately 15 μM for inhibition of NFAT-GFP import
(data not shown). This effect was reversible upon importazole
washout, which restored ionomycin-induced import of NFATGFP to near control levels (Figure 4, panels b and c). Thus,
it should be possible to use importazole in drug-washout
experiments to study the Ran/importin-β pathway in cells. The
reversibility of importazole required 1 h of recovery time
between washing out the drug and adding ionomycin and did
not require new protein synthesis (data not shown).
To further assess the specificity of importazole, we tested
its effects on CRM1-mediated export of NFAT-GFP. Export
of NFAT-GFP occurred efficiently in the presence or absence of
importazole but was blocked by leptomycin B, a specific CRM1
inhibitor37 (Figure 5 panel b, quantified in panel c). Importantly,
when cells were treated with both leptomycin B and importazole
upon ionomycin washout, NFAT-GFP was still restricted to the
nucleus (Figure 5, panels b and c), confirming that importazole
treatment does not nonspecifically damage the nuclear envelope
allowing proteins to leak out into the cytoplasm. Consistent
with the concentration of importazole sufficient to impair
nuclear import, we found that importazole kills HeLa cells with
an IC50 of approximately 22.5 μM (Figure S7 in Supporting
Information).
Taken together our nuclear import experiments indicate that
importazole is likely specific for importin-β-mediated protein
import. Although we have not tested importazole’s effect on all
importin-β family members, no effect on transportin-mediated
import or CRM1-mediated export was detected. Furthermore,
these results also suggest that importazole does not impair
RCC1-dependent loading of Ran with GTP or the function of
RanGTP itself since the export function of CRM1 critically
depends on the formation and function of RanGTP.
Importazole Blocks Spindle Assembly in Xenopus Egg
Extracts but Does Not Affect Pure Microtubules. A specific
inhibitor of importin-β/RanGTP should also disrupt mitosis.
We first tested importazole in metaphase-arrested Xenopus egg
extracts, which rely heavily on a RanGTP gradient for spindle
assembly around sperm chromosomes. Addition of 100 μM
importazole, but not the solvent DMSO, strongly inhibited
spindle assembly, preventing normal bipolar microtubule structures from forming around 80% of sperm nuclei (Figure 6, panels
a and b). The effect was similar to that of adding a truncated
importin-β (amino acids 71876), a version that no longer binds
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Figure 7. Importazole disrupts mitotic cargo release monitored by the FRET probe Rango. Donor fluorescence (top panels) and pseudocolored FLIM
images (bottom panels) of mitotic HeLa cells expressing the Rango-3 FRET sensor. Rango-3 displays a greater fluorescence lifetime around the
chromosomes of cells treated with importazole compared to that of cells treated with DMSO, resulting from importazole’s disruption of sensor release
from importin-β. N = 3; 30 cells counted for each condition.
to RanGTP and therefore sequesters its cargoes.38 Although
importazole significantly weakened spindle microtubule density,
it was not a general microtubule inhibitor, since it did not impair
the formation of microtubule asters in the extract induced by the
microtubule stabilizing agent DMSO (Figure 6, panels c and d)
or affect the polymerization of pure microtubules, in contrast
to nocodazole (Figure 6, panel e). Thus, importazole caused
dramatic effects on spindle assembly consistent with the known
role of the importin-β /RanGTP pathway in the Xenopus egg
extract system and is not a general microtubule inhibitor.
Importazole Impairs Mitotic Cargo Release and Reveals
Novel Functions for the Importin-β/RanGTP Pathway in
Human Cells. A major advantage of a cell-permeable importinβ/RanGTP inhibitor is its potential for dissecting novel roles of
this pathway in dividing human cells, which also provides a
system to analyze mitotic gradients of released cargos using
FRET probes.7 If importazole disrupts the interaction of importin-β with RanGTP, then the chromatin-localized FRET
gradient of the cargo probe Rango should be reduced, since it
undergoes FRET when released from importin-β in HeLa cells.7
As predicted, the difference in fluorescence lifetime of the donor
GFP of the Rango-3 probe between the chromosomes and distal
cytoplasm was significantly reduced in the presence of importazole compared to controls, from an average of 0.12 ( 0.4 ns
to 0.07(0.03 ns due to reduced FRET (Figure 7, p-value: 1.3
107). Thus, importazole impairs mitotic importin-β cargo
release in HeLa cells.
To examine the consequences of importazole on mitosis,
HeLa cells treated for 1 h were fixed and stained for tubulin
and chromosomes. Control metaphase figures displayed robust
spindles with a mean area of 105 μm2 and were centrally located
within the cell with chromosomes aligned on the metaphase plate
(Figure 7, panels a and d). Importazole treatment caused dosedependent defects in spindle assembly, chromosome alignment,
and spindle size (Figure 8, Supplemental Movie 1 in Supporting
Information). Interestingly, importazole also led to spindle
positioning defects, with more than 40% of the cells displaying
off-center spindles (Figure 8, panels a and b). Spindle positioning was not previously attributed to the Ran pathway, and
this phenotype may be a consequence of astral microtubule
disruption by importazole (data not shown). Previous studies
have most likely not revealed this role of the Ran pathway in
mitosis because they were performed in cell-free systems such as
Xenopus extracts where spindle positioning could not be assessed.
The discovery of this spindle misalignment phenotype demonstrates the importance of importazole as a tool to study the Ran
pathway in mitosis.
The Ran pathway members Ran and importin-β are highly
conserved, and an inhibitor of the RanGTP/importin-β interaction may have considerable value as a research tool across
multiple species. Additionally, as the Ran pathway has been
shown to be upregulated in some forms of cancer,39 importazole
may have some potential as a therapeutic compound. Development of more potent, related compounds should allow a more
complete disruption of the Ran/importin-β interaction as well as
limit any possible nonspecific effects of compound treatment,
further increasing the value of these inhibitors in both the
academic and medical fields. Overall, we have shown importazole
to be an effective inhibitor of the Ran/importin-β interaction
in vitro and in cells with great potential for future use as a tool to
study the Ran pathway in mitosis.
’ METHODS
Protein Expression and Purification. pET30a-derived constructs encoding importin-β with an N-terminal YFP fusion (pKW1532),
a CFP-Ran fusion (pKW1543), and importin-β (pKW485) were transformed into BL21 cells (Invitrogen). Additionally, pQE32-derived Ran
constructs (pKW356 [WT Ran], pKW 590 [RanQ69L]), a pQE9-derived
Crm1 construct (pKW812), and a pQE60-derived transportin construct
(pKW738) were transformed into SG13 cells. All constructs were induced
with IPTG at RT. Harvested cells were lysed using a French press. Fusion
proteins were purified with Ni NTA resin using a standard protocol
followed by gel filtration. RCC1 was purified as described previously.40
FRET Assay. The following reaction buffer was used for all FRET
assays, including the high-throughput screen: 1X PBS, 5% glycerol,
2 mM MgCl2, 1 mM DTT, 0.01% NP-40. For standard CFP-Ran/YFPimportin-β FRET assays, 50100 nM CFP-Ran mixed with 20 nM
RCC1 and 200 μM GDP or 200 μM GTP was immediately followed by
addition of 50100 nM YFP-importin-β. The reaction was excited using
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Figure 8. Importazole disrupts mitotic spindles in living HeLa cells. (a) Asynchronously growing cultures were treated with DMSO or importazole for 1
h prior to fixation and staining for DNA (blue) and tubulin (red). Note defects including chromosome congression (white arrowheads point to
misaligned chromosomes) and spindle positioning upon importazole treatment. Dashed white lines indicate cell boundaries. (b) Quantification of
spindle defects in cells treated with DMSO, 20 μM importazole, or 40 μM importazole. N = 5. In each case, 100 metaphase cells were counted, and the
fraction of those displaying defects were scored. (c) Time-lapse fluorescence microscopy of a metaphase HeLa cells treated with 50 μM importazole.
Frames were captured every 3 min. See Supplemental Movie 1 in Supporting Information. (d) Asynchronous HeLa cells were treated with 040 μM
importazole for 1 h prior to fixation, and the size of the spindle in mitotic cells was measured. N = 4; 100 metaphase spindles were measured per
condition. Bars represent standard error.
β-lactamase-based assay as described.24 Importazole was found to be
soluble up to approximately 100 μM in water. Additionally, importazole
was characterized by mass spectrometry and NMR to confirm its identity
and purity.
Fluorescent Thermal Shift Assay. Experiments were performed
using Applied Biosystems StepOnePlus Real-Time PCR (RT-PCR)
System as previously described.28,41 Protein stocks were diluted in PBS
and added 70% v/v to a Microamp Fast 96-well Reaction Plate and
maintained on ice. Compounds (importazole and control compound
3016) were then added at 30% v/v in 3% DMSO. Freshly prepared 100X
water based-dilution of Sypro Orange Protein Gel Stain was then
added at 1% v/v to reach a final reaction volume of 20 μL. Samples
were mixed by gentle pipetting. After sealing the plates with Microamp
Optical Adhesive Film, the plate was subjected to a heating cycle
composed of a 10 s prewarming step at 25 C and a gradient between
25 and 95 C with a 0.3 C ramp. Data was analyzed using the
StepOnePlusSoftware v2.1.
Cell Lines and Tissue Culture. A GFP-NFAT expression plasmid
(pKW520) was generated by inserting a BamH I/Hind III-cleaved
a Fluorolog 3 spectrofluorometer with 435 nm fluorescence, and the
emission was read between 460 and 550 nm. For the high-throughput
screen, the concentrations of reaction components were as follows:
CFP-Ran, 62.5 nM; YFP-importin-β, 62.5 nM; GTP or GDP, 200 μM;
RCC1, 20 nM
High-Throughput Screen. The screen was carried out in collaboration with the Small Molecule Discovery Center (SMDC) at the
University of California, San Francisco. Compounds were from ChemBridge, ChemDiv, SPECS, ChemRX, and Microsource. The complete
content of this library can be found through the Small Molecule Discover
Center Web site (http://smdc.ucsf.edu/). A detailed screening protocol
can be found in the Supporting Information section. The software used
to analyze the screening data is available upon request.
Secondary Screening. A total of 141 hit compounds from the
primary screen were tested for nonspecific effects on fluorescence
with FRET probe YIC that contains the importin-β-binding domain
of importin-R flanked by CFP and YFP. When unbound in solution,
this probe undergoes intramolecular FRET.6 In the second assay, we
tested our 141 hits for nonspecific inhibition due to aggregation using a
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In Vitro Microtubule Polymerization and Spindle Assembly. Xenopus laevis egg extracts were prepared as described.42 For in vitro
spindle assembly, Xenopus laevis sperm DNA was added to egg extracts
supplemented with rhodamine-labeled tubulin. Asters were formed by
addition of 5% DMSO. DNA was stained with Hoechst dye. The
formation of microtubule-based structures was assessed using epifluorescence microscopy after a 30 min RT incubation. In vitro microtubule
polymerization and pelleting assays were performed by incubating
25 μM bovine tubulin, 1 mM GTP, and 5% DMSO in BRB80 buffer
(80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8) at 37 C for
30 min. Polymerized microtubules were pelleted through a sucrose
cushion, resuspended, and analyzed by SDSPAGE.
Fluorescence Lifetime Imaging Microscopy (FLIM). The
Rango-3 FRET sensor is an improved version of Rango and was created
by replacing the Cerulean-EYFP donoracceptor pair in Rango 7 with
EGFP as a donor and nonfluorescent acceptor sREACh,43 which was
modified by the introduction of mild dimerization mutations. Timecorrelated single photon counting (TCSPC) data sets were acquired
with a Plan-Apochromat 63x/1.40 NA oil immersion lens on an inverted
Zeiss LSM710 NLO microscope equipped with a Becker & Hickl SPC830 TCSPC controller. Samples were excited by one-photon 485 nm
pulses generated by a frequency doubling 970 nm 80 MHz Ti:sapphire
laser (Coherent MiraSHG). The emission was collected from a custom
side port, filtered through a 525 nm bandpass filter (ET525/50 Chroma)
and detected by a HPM-100-40 module (Becker & Hickl) containing a
hybrid Hamamatsu R10467-40 GaAsP photomultiplier. Two to three
days before the experiment, HeLa cells were transfected with a pSG8
plasmid containing the Rango-3 open reading frame (pK135) to induce
sensor expression. Treatment with importazole or DMSO was started
1 h before imaging and continued for up to 1 h in an environmental
chamber built on the microscope (37 C, 5% CO2). Recording conditions were chosen to limit emission to approx 12 106 counts
per second, and images of 128 128 pixels (1024 time bins/pixel) were
averaged over 60 s. Fluorescence lifetime images were produced and
analyzed using SPCI software (Becker & Hickl).
Immunofluorescence Microscopy. Cells were fixed in 4%
formaldehyde and 0.1% glutaraldehyde in PHEM (60 mM PIPES,
25 mM HEPES, 10 mM EGTA, 2 mM MgSO4) at 37 C for 15 min
followed by permeabilization with 0.1% Triton X-100 for 2 min at RT.
Cells were then washed and blocked (PHEM þ 5% FBS þ 0.2%
saponin) and stained by standard techniques using the E7-A anti-β
tubulin antibody (Developmental Studies Hybridoma Bank) diluted
1:1000 and Hoechst dye.
’ ASSOCIATED CONTENT
bS
Supporting Information. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: kweis@berkeley.edu; bheald@berkeley.edu.
Present Addresses
)
NFATC1 cDNA fragment into pEGFP-C1 (Clontech) digested with
BglII and Hind III. The plasmid was a gift of K. Reif. The construct
was stably transfected into HEK 293 cells and a single clone expressing
moderate levels of NFAT-GFP was selected and maintained in Opti-mem
media (Gibco) plus 4% fetal bovine serum, 1% penicillin/streptomycin,
and 200 μg/mL G418. HeLa cells were grown and maintained according
to standard protocols.
Nuclear Import with Permeabilized HeLa Cells. HeLa cells
were permeabilized and treated with an import reporter and cytosol
from Xenopus laevis oocytes as described previously.29
NFAT-GFP Nuclear Import and Export. For all import and
export experiments, HEK 293 cells stably expressing NFAT-GFP were
grown on glass coverslips to approximately 50% confluency prior to drug
treatment. In all cases, importazole was used at 40 μM, and leptomycin B
was used at 10 ng/mL. For controls, DMSO was used at a concentration
of 0.4%. Ionomycin was added at 1.25 μM. Importazole and leptomycin
B treatments were all for 1 h. In all experiments cells were fixed with 4%
formaldehyde prior to fluorescence microscopy. DNA was visualized
with 1 μg/mL Hoechst dye. For quantification, 100 cells from each
condition were analyzed, and the percentage that showed nuclear
accumulation of NFAT-GFP calculated.
ARTICLES
Yonsei University, The Underwood International College and
College of Life Science and Biotechnology, Seoul, Korea.
^
Yowza Software, P.O. Box 642413, San Francisco, CA.
Author Contributions
z
These authors contributed equally to this work.
’ ACKNOWLEDGMENT
The authors thank Brian Wolff, Janice Williams, Brian Feng,
and James Wells at the Small Molecule Discovery Center at
UCSF for help with the screen, Elisa Dultz for live cell imaging of
imporatzole effects, Karin Reif for NFAT cDNA, David Halpin
for biotin-labeled RCC1, and Lili Wang at the Broad Institute for
developing the thermal shift assay to probe binding of importazole to importin-β . This work was supported by the National
Institutes of Health (R01 GM065232 and R21 NS53592, K.W. and
R.H.), the Cancer Research Coordinating Committee (S.L.B.), and
the Intramural Research Program of the Center for Cancer
Research, NCI, National Institutes of Health (P.K. and K.H.).
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