Current Biology 19, 1314–1319, August 11, 2009 ª2009 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2009.06.057
The Conserved NDR Kinase Orb6 Controls
Polarized Cell Growth by Spatial
Regulation of the Small GTPase Cdc42
Maitreyi Das,1David J. Wiley,1,3Xi Chen,1Kavita Shah,2
and Fulvia Verde1,*
1Department of Molecular and Cellular Pharmacology,
University of Miami Miller School of Medicine, Miami,
FL 33101-1015, USA
2Department of Chemistry and Purdue University Center for
Cancer Research, Purdue University, West Lafayette,
IN 47907, USA
The conserved NDR kinase regulates cell morphogenesis
from yeast to neurons [1–4]. Although studies have unrav-
eled the mechanism of regulation of NDR kinase activity ,
the mechanism of morphology control by NDR and the effec-
tors that mediate NDR function are unknown. Via a chemical
genetic approach, we show that the fission yeast NDR
homolog, Orb6 kinase, maintains polarized cell growth at
the cell tips by spatially regulating the localization of Cdc42
GTPase, a key morphology regulator. Loss of Orb6 kinase
activity leads to the recruitment of Cdc42 GTPase and the
Cdc42-dependent formin For3, normally found only at the
cell tips, to the cell sides. Furthermore, we show that loss
of Orb6 kinase activity leads to ectopic lateral localization
of the Cdc42 guanine nucleotide exchange factor (GEF)
Gef1, but not of the other Cdc42 GEF, Scd1. Consistent
with these observations, gef1 deletion suppresses the
increased cell diameter phenotype of orb6 mutants. In
contrast, the microtubule cytoskeleton and the localization
of the microtubule-dependent polarity markers Tea1 and
Tea4 are not altered by loss of Orb6 kinase activity. Our find-
ings indicate that the conserved NDR kinase Orb6 regulates
cell polarity by spatially restricting the localization and
activity of Cdc42 GTPase.
Results and Discussion
Loss of Orb6 Kinase Activity Leads to Rapid
Disorganization of the Actin Cytoskeleton and Ectopic Cell
Growth at the Cell Sides
To investigate the role of Orb6 kinase activity in morphology
control, we constructed an analog-sensitive orb6 mutant
(orb6-as2;orb6M170A) that allows the rapid inhibition of
Orb6 kinase activity in vivo by the cell-permeable inhibitor
pyrimidine). Treatment of orb6-as2 mutants with 1-Na-PP1
led to a swift alteration of the actin cytoskeleton organization
(Figure 1D), whereas it did not have any effect on control
orb6+cells (Figure 1B), even after several hours. Actin cables,
which are generally distributed along the cell main axis in
wild-type cells, became randomly distributed and often
crisscrossed, and actin patches, which normally aggregate
closer to the growing cell tips, became randomly distributed
throughout the cell cortex (Figure 1D). As expected, orb6-as2
mutants treated with dimethyl sulfoxide (DMSO) alone (Fig-
ure 1C) or wild-type cells treated with 1-Na-PP1 (Figure 1B)
Consistent with a change in actin organization, the localization
of Myo52-GFP, a barbed-end-directed myosin V, became
more dispersed from the cell tips upon Orb6 kinase inhibition
(Figure 1H). Conversely, the microtubule cytoskeleton did
several hours of treatment with the inhibitor (see Figure S1D
To understand how inhibition of Orb6 kinase activity affects
polarized cell growth, we analyzed the pattern of cell wall
deposition, which defines the sites of polarized secretion,
cell wall remodeling, and cell expansion, following treatment
of orb6-as2 mutant cells with 1-Na-PP1. FITC-concanavalin
A-pretreated (ConA, visualized in green) orb6+cells (Figures
1I and 1J) and orb6-as2 cells (Figures 1K and 1L) were grown
in the presence of DMSO (Figures 1I and 1K) or 1-Na-PP1
(Figures 1J and 1L) for 2 hr. Cells were then stained with calco-
fluor (visualized in red) to define the complete cell wall. As
expected, in orb6+cells treated with DMSO (Figure 1I) or
1-Na-PP1 (Figure 1J) and in orb6-as2 cells treated with DMSO
(Figure 1K), cell tips were stained only with calcofluor, defining
Conversely, orb6-as2 mutant cells treated with 1-Na-PP1
showed zones of green FITC-ConA staining throughout the
whole cell wall, intercalated by areas only stained by calco-
fluor, indicating that cell wall deposition in these mutants
had occurred in several areas of the cell cortex, including the
cell sides (Figure 1L, arrows). Furthermore, orb6-as2 mutants
displayed decreased new cell wall deposition at the cell tips,
as shown by the presence of green FITC-ConA fluorescence
at the cell tips (Figure 1L), as compared to orb6+cells
The effect of this abnormal pattern of cell growth on overall
cell shape became evident 3 hr after Orb6 kinase inhibition.
In orb6-as2 cells, cell diameter increased significantly (4.5 6
0.2 mm; p % 0.0001) (Figure 1P) and cell length at division
shortened significantly (8.9 6 1 mm; p % 0.0001) as compared
to orb6+cells (diameter 3.7 6 0.2 mm; length 14.7 6 0.7 mm)
(Figure 1N). Collectively, these observations indicate that the
normal organization of the actin cytoskeleton is disrupted by
Orb6 kinase inhibition, leading to ectopic cell growth in areas
of the lateral cell cortex and increased cell diameter.
Loss of Orb6 Kinase Activity Leads to Localization
of GTP-Bound Cdc42 GTPase at the Cell Sides
The observation that the directionality of actin cables
undergoes a rapid change upon Orb6 kinase inhibition promp-
ted us to study the localization of For3, the fission yeast formin
implicated in actin cable polymerization , in orb6-as2
the cell cortex shortly after inhibitor addition (Figure 2D).
Conversely, For3-3GFP patches remained localized to the
growing cell tips in orb6+cells (Figure 2B).
3Present address: Department of Microbiology and Immunology,
University of Miami Miller School of Medicine, Miami, FL 33101-1015, USA
Previous studies have shown that For3 localization to the
growing cell tips is dependent in part on the microtubule-
dependent marker proteins Tea1 and Tea4 [6–8]. Loss of
Tea1 or Tea4 leads to unipolar cellgrowth and toFor3 localiza-
tion at only one cell tip but does not change overall cell shape
or cell diameter. We found that Tea1 and Tea4 localization
were not affected by Orb6 kinase inhibition and remained
normal for several hours following 1-Na-PP1 addition to
orb6-as2 cells (Figures S1H and S1L).
dent on the Cdc42 GTPase, a key regulator of cell morphology
in eukaryotic cells . In fission yeast, Cdc42 is found predom-
ature-sensitive cdc42-1625 mutant alleles fail to accumulate
For3 at the cell tips and display a decrease in actin cables
[9, 11]. To test whether Cdc42 localization changes upon Orb6
kinase inhibition, we visualized GFP-Cdc42 in orb6-as2 cells
following addition of 1-Na-PP1 (Figure 2H). Within 15 min of
exposure to the inhibitor, severalareas of the lateral cellcortex
of orb6-as2 cells displayed increased levels of GFP-Cdc42
(Figure 2H, arrows; quantification shown in Figure S2A), while
in orb6+cells (Figures 2E and 2F) and in orb6-as2 cells in
DMSO (Figure 2G), GFP-Cdc42 was found at the growing cell
tips. Consistent with these observations, increased GFP-
Cdc42 localization at several areas along the lateral cell cortex
was also observed in temperature-sensitive orb6-25 mutants
grown at 36?C for 3 hr (Figure S2Cd).
Cdc42 cycles between an active GTP-bound and an inactive
GDP-bound state . To specifically visualize the localization
of active Cdc42 on the cell cortex, we used a GFP-tagged
CRIB (Cdc42/Rac interactive-binding) reporter . The CRIB
domain, which is found in many Cdc42 effector proteins, asso-
ciates with GTP-bound Cdc42 . Similar to GFP-Cdc42,
CRIB-GFP localizes to the cell tips and to the site of cell
division . Consistent with the results obtained with
GFP-Cdc42, the CRIB-GFP reporter was rapidly recruited to
distinct spots on the cell sides in orb6-as2 mutants, within
5 min of inhibitor addition (Figure 2L; quantification shown in
Figure S2B). In contrast, CRIB-GFP localization remained
restricted to the cell tips in control cells treated with DMSO
or 1-Na-PP1 and in orb6-as2 mutant cells treated with DMSO
(Figures 2I, 2J, and 2K, respectively). Consistent with these
results, CRIB-GFP patches were observed along the lateral
cell cortex in temperature-sensitive orb6-25 mutants grown
at 36?C for 3 hr (Figure S2Ch).
Furthermore, to determine whether the abnormal Cdc42-
GTP localization to the cell sides is a consequence rather
than a cause of altered formin For3 dynamics or of perturbed
cell shape, we examined CRIB-GFP localization in for3-
D353-406 mutants  and in several orb mutants . Deletion
within the DID domain renders formin For3p activation inde-
pendent of Cdc42-GTP binding and leads to disorganized
actin cables and altered cell shape . We found that in for3-
D353-406 mutants, CRIB-GFP did not localize to the cell sides,
even when cell shape was altered (Figure S3Ab). Similarly, we
orb mutants, including orb2-34, orb5-19, and orb9-46 (Figures
S3Bd–S3Bf) [1, 2, 15]. Conversely, we found that CRIB-GFP
localized to the cell sides in orb3-167 mutants (Figure S3Bc)
and orb1-3 mutants (data not shown), consistent with the
fact that orb3 is a mutant allele of nak1, which encodes an
upstream regulatory kinase of Orb6 [1, 16, 17], and that orb1
Figure 1. Loss of Orb6 Activity Leads to Loss of
Cell Polarity, Isotropic Growth Pattern, and
Disorganized Actin Cables and Patches
(C and D) cells were treated with DMSO (A and C)
or 50 mM 1-Na-PP1 (B and D) at 32?C, fixed with
formaldehyde, and stained with Alexa Fluor phal-
loidin. 58% of orb6-as2 cells displayed randomly
localized actin dots after 7 min incubation with
1-Na-PP1 (scored as cells containing ten or more
dots not localized at the cell tips), as compared
to 9% of orb6-as2 cells incubated with DMSO or
7% of control cells incubated with 1-Na-PP1 for
up to 1 hr.
(E–H) myo52-GFP (DM3415) (E and F) and orb6-
as2 myo52-GFP (FV778) (G and H) cells were
treated with DMSO (E and G) or 50 mM 1-Na-PP1
(F and H) at 25?C and visualized under a fluores-
cence microscope. Myo52-GFP was observed
at the cell sides in 83% of orb6-as2 cells after
12 min, (n = 50), as compared to the untreated
control (myosin V localization observed at the
cell sides in 20% of cells, n = 150) or to wild-type
cells (observed in 19% of cells, n = 150).
(I–L) orb6+(I and J) and orb6-as2 (K and L) cells
were coated with FITC-concanavalin A (ConA,
green) and then grown in presence of DMSO
(I and K) or 50 mM 1-Na-PP1 (J and L) at 32?C for
2 hr. Cells were then stained with calcofluor (red).
(M–P) orb6+(M and N) and orb6-as2 (O and P)
cells were treated with DMSO (M and O) or
50 mM 1-Na-PP1 (N and P) at 32?C for 3 hr. Cells
were then stained with calcofluor.
Scale bars represent 5 mm.
Spatial Control of Cdc42 GTPase by Orb6 Kinase
the Orb6 pathway [1, 18]. These observations indicate that
ectopic Cdc42 localization is not merely a consequence of
Finally, we tested whether loss of Orb6 kinase activity alters
total Cdc42 protein levels or the levels of GTP-bound Cdc42.
We performed a GST-CRIB pull-down assay  in control
and orb6-25 strains following exposure to the restrictive
temperature for 5 hr. We detected a 1.7-fold increase in
Cdc42-GTP levels in orb6-25 mutants as compared to control
in total Cdc42 protein levels (Figure S4C). Thus, we observed
a modest, albeit statistically significant (p = 0.0012), increase
in Cdc42-GTP levels in orb6-25 cells (Figures S2D and S2E).
Collectively, our results suggest that Orb6 kinase functions
to spatially restrict the localization of active Cdc42 to the cell
tips. Upon loss of Orb6 kinase activity, GTP-bound Cdc42
localizes to ectopic sites of the cell cortex, where it promotes
actin cable polymerization via formin For3 activation and
abnormal cellwalldeposition, presumablybyredirecting intra-
cellular transport to these new sites of cell growth.
orb6-25 Temperature-Sensitive Mutants Are Sensitive
to Increased Cellular Levels of GTP-Bound Cdc42
The transition of Cdc42 between an active GTP-bound and an
inactive GDP-bound state is regulated by GTPase-activating
proteins (GAPs) that accelerate the hydrolysis of bound GTP
and guanine nucleotide exchange factors (GEFs) that promote
the exchange of GDP for GTP . In fission yeast, the known
Cdc42 GEFs are Scd1 and Gef1 [20, 21]. Currently, the only
known fission yeast Cdc42 GAP is Rga4 [13, 22].
To test the effects of altering the function of Cdc42 in orb6
mutants, we increased the levels of GTP-Cdc42 by mildly
overexpressing Gef1 from the attenuated nmt-81 promoter
 (Figures 3A and 3B). We found that increased levels of
Gef1 negatively affected the viability of orb6-25 mutant cells,
but not orb6+cells, at restrictive temperature (Figure 3A).
Furthermore, we found that increased levels of Gef1 led to
complete loss of cell polarity in orb6-25 cells grown at
semipermissive temperature (Figure 3Bd). Conversely, in
orb6+cells, moderate Gef1 overexpression from the attenu-
ated nmt-81 promoter led to increased cell diameter, but
not to loss of cell polarity (Figure 3Bb). These results are
also consistent with the previous finding that overexpression
of the second Cdc42 GEF, Scd1, is toxic to orb6-25 mutant
cells . It should be noted that our results showing a sensi-
tivity of orb6 mutants to Gef1 overexpression appear to be in
disagreement with another observation of the same previous
study . However, more recent independent experiments
from the same laboratory confirm that orb6-25 mutants are
sensitive to increased gef1 expression (P. Coll and P. Perez,
To visualize the effects of increased Gef1 protein dosage on
the localization of GFP-Cdc42, CRIB-GFP, and For3-YFP, we
overexpressed Gef1 from a full-strength nmt1 promoter in
wild-type cells. As reported previously, strong Gef1 overex-
pression from the full-strength nmt1 promoter led to loss of
cell polarity and a rounded cell shape in wild-type cells .
We found that GFP-Cdc42 (Figure 3Cb), CRIB-GFP (data not
shown), and For3-YFP (Figure 3Cd) localized to ectopic sites
on the cell cortex, similar to our observations in orb6-as2
and orb6-25 mutants (Figures 2D, 2H, and 2L; Figures S2Cd
Finally, we increased endogenous GTP-Cdc42 levels in
orb6-as2 cells by deleting Rga4 . rga4 deletion leads to
increased cell diameter, but not to loss of cell polarity .
Consistent with what was observed with Gef1 overexpression,
rga4 deletion in orb6 mutants also resulted in the complete
loss of polarity when rga4D orb6-25 cells were grown at
semipermissive temperature (Figure S5Ad). Together, these
observations indicate that orb6 mutants are hypersensitive
to increased levels of GTP-Cdc42 and display phenotypic
features that are similar to cells overexpressing Gef1.
Orb6 Kinase Spatially Controls Cdc42
GEF Gef1 Localization
To further investigate the mechanism of Cdc42 activation at
the cell sides in orb6 mutants, we analyzed the effects of
Figure 2. Loss of Orb6 Activity Leads to Ectopic
For3 and Cdc42 Localization
(A–D) for3-3GFP (CA5818) (A and B) or orb6-as2
for3-3GFP (FV1106) (C and D) cells treated with
DMSO (A and C) or 50 mM 1-Na-PP1 (B and D)
for 15 min at 32?C.
(E–H) GFP-cdc42 (PPG6303) (E and F) or orb6-
as2 GFP-cdc42 (FV1034) (G and H) cells treated
with DMSO (E and G) or 50 mm 1-Na-PP1 (F and
H) for 15 min at 32?C. Arrows indicate GFP-
Cdc42 localization. It should be noted that cells
expressing GFP-Cdc42 at the endogenous level
have aberrant cell morphology as compared to
tagged GFP-Cdc42 is not fully functional, as
reported previously .
(I–L) orb6+(CA5931) (I and J) and orb6-as2
(FV1109) (K and L). Cells expressing CRIB-3GFP
were treated with DMSO (I and K) or 50 mM
1-Na-PP1 (J and L) at 32?C for 15 min. The CRIB
domain gene sequence was integrated in the
genome and expressed under the control of the
pak1 gene promoter .
Scale bars represent 5 mm.
Current Biology Vol 19 No 15
Orb6 kinase activity inhibition on the localization of known
Cdc42 regulators, the Cdc42 GEF proteins Gef1  and
Scd1  and the Cdc42 GAP protein Rga4 [13, 22]. Under
normal conditions, Gef1 and Scd1 localize to the cell tips
and cell septum [10, 20, 23] and are partially redundant in
function . Rga4 localizes to the cell sides and cell septum
. We found that upon Orb6 kinase inhibition, Gef1-GFP
protein rapidly localized to the cell sides (Figure 4Ad; quanti-
fication shown in Figure S4A). This result was observed in the
cdc25-22 background at permissive temperature, which
allows for easier visualization of Gef1-GFP at the cell cortex
(Figure 4A; Supplemental Experimental Procedures), as
well as in cdc25+cells expressing Gef1-3YFP (Figure S4E).
Gef1-GFP mislocalization in orb6-as2 mutants closely fol-
lowed the pattern observed for CRIB-GFP (Figure S4B).
Conversely, Scd1-GFP remained at the cell tips (Figure 4Ah)
up to 2 hr following 1-Na-PP1 addition. No change was
observed in the localization of the Cdc42 GAP Rga4 (Fig-
ure S5Bd). No substantial changes were observed in the
total Gef1, Scd1, and Rga4 protein levels following Orb6
kinase inhibition (Figure S4C). Thus, our results show that
Orb6 kinase activity restricts Cdc42 GEF Gef1 localization
to the cell tips but does not regulate the cortical local-
ization of the second Cdc42 GEF, Scd1, or the Cdc42 GAP,
To test the hypothesis that Gef1 mislocalization mediates
the lateral cell expansion in orb6 mutants, we deleted the
Cdc42 GEF gef1 in orb6-25 temperature-sensitive mutants
and examined cells after growth at 36?C for 5 hr. Deletion of
gef1 ameliorated the increasedcelldiameter and theabnormal
Increased Cdc42 Activity
(A) orb6+(PN22) and orb6-25 (FV117) cells trans-
formed with empty vector or with a vector
carrying nmt81-gef1 were grown at 25?C and
replica plated onto plates at 36?C.
(Ba–Bd) orb6+(PN22) (Ba and Bb) and orb6-25
(FV117)(Bcand Bd) cells transformed with empty
pRep81X vector (Ba and Bc) or with a pRep81X
vector carrying nmt81-gef1 (Bb and Bd) were
grown in liquid EMM media at 32?C, stained
with calcofluor, and observed under a micro-
(Ca–Cd) pREP3X-gef1-carrying cells expressing
GFP-Cdc42 (PPG6303) (Ca and Cb) or For3-YFP
(BFY81) (Cc and Cd) were grown in the presence
of thiamine (Ca and Cc) or without thiamine (Cb
and Cd) for 16 hr at 32?C.
Scale bars represent 5 mm.
3. orb6MutantsAre Sensitiveto
cell morphology phenotype of orb6-25
cells at restrictive temperature (Fig-
ure 4Bd; quantification in Figure S5D).
abnormal localization of CRIB-GFP was
also restored by gef1 deletion (Fig-
ure 4Cd). Similar results were obtained
by moderately increasing Cdc42 GAP
Rga4 expression in orb6-25 tempera-
and S5D). Rga4 correctly localizes to
the cell sides even when overexpressed
downregulating Cdc42 activation at the cell sides alleviates
the cell diameter defect of orb6 mutants.
The NDR kinase is known to modulate cell morphogenesis in
several eukaryotic cells, including C. elegans, Drosophila, and
rodent neurons [3, 24, 25]. The targets or effectors that mediate
NDR kinase morphology control functions are currently
unknown. We have demonstrated here for the first time that
the conserved NDR kinase Orb6 maintains cell polarity by
spatiallyregulatingCdc42GTPaseatthecellcortex. Our results
the cell tips, thus preventing aberrant Cdc42 localization to the
ization promotes the establishment of polarized cell growth.
In S. pombe, Cdc42 regulators display a characteristic intra-
cellular localization that defines different domains on the cell
cortex. The Cdc42 GEF proteins Gef1 and Scd1 are localized
at the sites of cell growth: the cell tips and septum .
Conversely, the Cdc42 GAP Rga4 is localized at the cell sides,
which do not grow under normal conditions [13, 22]. We found
that Gef1 protein rapidly localizes to the cell sides upon Orb6
kinase inhibition, whereas Scd1 and Rga4 localization remain
unchanged. Although it is currently unclear whether Gef1 is
a bona fide substrate for Orb6 kinase in vivo, it is nevertheless
intriguing that Gef1 can be phosphorylated in vitro by Mob2-
associated Orb6 kinase (Figure S4F). Indeed, Gef1, but not
Scd1, contains potential NDR phosphorylation sites, as
defined for the budding yeast Orb6 homolog Cbk1 and for
human NDR1 and NDR-related LATS1 kinase ([26, 27]; M.D.
Spatial Control of Cdc42 GTPase by Orb6 Kinase
and F.V., unpublished data). Consistent with the idea that Gef1
localization to the cell sides mediates, at least in part, the
lateral cell expansion observed in orb6 mutants, deletion of
gef1, but not scd1 (data not shown), alleviates the morpholog-
ical phenotype of orb6 mutants.
Additionally, it is also possible that other factors participate
in the morphology control function of Orb6 kinase. For
example, Orb6 could also positively regulate the activity of
the Cdc42 GAP Rga4 at the cell sides, thus inhibiting Cdc42
activation. Rga4 physically interacts with Orb6 in the two-
hybrid system  and also contains two putative NDR phos-
phorylation sites (M.D. and F.V., unpublished data). Further-
more, recent observations in S. cerevisiae indicate that the
Orb6 homolog Cbk1 has a function in the control of protein
Studies in higher eukaryotes have demonstrated the signif-
icance of Cdc42 GTPase spatial regulation in promoting polar-
ized growth during cell migration  and cell differentiation
. Interestingly, defects in the C. elegans and Drosophila
Orb6 kinase homologs, SAX1 and Trc, alter neuronal arboriza-
tion by promoting supernumerary neurite formation and
branching [3, 24], a process dependent on the activity of
Cdc42 and Rac GTPases . Thus, it is likely that the role of
the S. pombe NDR kinase Orb6 in spatially regulating Cdc42
localization and thus promoting cell growth asymmetry, as
Figure 4. Loss of Orb6 Kinase Activity Leads to
Lateral Localization of Gef1
(Aa–Ad) cdc25-22 gef1-GFP (PPG3601) (Aa and
Ab) and cdc25-22 orb6-as2 gef1-GFP (FV1157)
(Ac and Ad) cells were treated with DMSO (Aa
and Ac) or 50 mM 1-Na-PP1 (Ab and Ad) at 25?C
for 24 min and observed under a fluorescence
microscope. 50% of cells showed lateral Gef1
localization 8 min following inhibitor addition at
25?C (n = 200). Gef1-GFP localization at the cell
tips is more clearly visualized in the cdc25-22
mutant background  (see Supplemental
as2 scd1-GFP (FV1149) (Ag and Ah) cells were
treated with DMSO (Ae and Ag) or 50 mM 1-Na-
PP1 (Af and Ah) at 32?C for 60 min and observed
under a fluorescence microscope. No (0%) Scd1
localization to the cell sides was observed after
2 hr of exposure to 1-Na-PP1 (n = 200).
(Ba–Bd) orb6+(972) (Ba), gef1D (PG2601) (Bb),
(FV1076) (Bd) cells were grown in liquid media
at 36?C for 5 hr, stained with calcofluor, and
observed under a microscope.
(Ca–Cd) CRIB-GFP localizationin orb6+(CA5931)
(Ca), gef1D (FV1104) (Cb), orb6-25 (FV1115) (Cc),
and gef1D orb6-25 (FV1101) (Cd) cells grown in
liquid media at 36?C for 3 hr. Arrows point to
areas of ectopic CRIB-GFP localization.
Scale bars represent 5 mm.
described in this paper, is conserved in
higher eukaryotes and could be a para-
digm for further functional analysis of
this conserved kinase pathway.
Experimental Procedures, one table, and five
figures and can be found with this article
online at http://www.cell.com/current-biology/supplemental/S0960-9822(09)
We thank K. Shiozaki (University of California, Davis), P. Perez (Consejo
Superior de Investigaciones Cientificas/University of Salamanca), D. John-
son (University of Vermont), F. Chang (Columbia University), E. Manser
(Institute of Medical Biology, Singapore), and K. Gull (University of Oxford)
for various strains, reagents, and antibodies. We also thank S. Lemmon
(University of Miami), G. D’Urso (University of Miami), and P. Perez for crit-
ically reading the manuscript; the Yeast Club at the University of Miami for
useful suggestions; and A. Torbati for technical assistance. This work was
supported by National Science Foundation grant 0745129 and by the
Sylvester Comprehensive Cancer Center at the University of Miami Miller
School of Medicine.
Received: October 16, 2008
Revised: June 10, 2009
Accepted: June 11, 2009
Published online: July 30, 2009
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Spatial Control of Cdc42 GTPase by Orb6 Kinase