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
dystrophy kinase, is required for maintenance of cell polarity and coor-
dinates cell morphogenesis with the cell cycle. Proc. Natl. Acad. Sci.
USA 95, 7526–7531.
3. Zinn, K. (2004). Dendritic tiling: New insights from genetics. Neuron 44,
4. Hergovich, A., Stegert, M.R., Schmitz, D., and Hemmings, B.A. (2006).
NDR kinases regulate essential cell processes from yeast to humans.
Nat. Rev. Mol. Cell Biol. 7, 253–264.
5. Feierbach, B., and Chang, F. (2001). Roles of the fission yeast formin
for3p in cell polarity, actin cable formation and symmetric cell division.
Curr. Biol. 11, 1656–1665.
6. Tatebe, H., Shimada, K., Uzawa, S., Morigasaki, S., and Shiozaki, K.
(2005). Wsh3/Tea4 is a novel cell-end factor essential for bipolar distri-
bution of Tea1 and protects cell polarity under environmental stress in
S. pombe. Curr. Biol. 15, 1006–1015.
7. Feierbach, B., Verde, F., and Chang, F. (2004). Regulation of a formin
complex by the microtubule plus end protein tea1p. J. Cell Biol. 165,
8. Martin, S.G., McDonald, W.H., Yates, J.R., and Chang, F. (2005). Tea4p
linksmicrotubuleplus endswith the formin for3p in the establishmentof
cell polarity. Dev. Cell 8, 479–491.
9. Martin, S.G., Rincon, S.A., Basu, R., Perez, P., and Chang, F. (2007).
Regulation of the formin for3p by cdc42p and bud6p. Mol. Biol. Cell
10. Rincon, S., Coll, P.M., and Perez, P. (2007). Spatial regulation of Cdc42
during cytokinesis. Cell Cycle 6, 1687–1691.
11. Coll, P.M., Rincon, S.A., Izquierdo, R.A., and Perez, P. (2007). Hob3p,
the fission yeast ortholog of human BIN3, localizes Cdc42p to the divi-
sion site and regulates cytokinesis. EMBO J. 26, 1865–1877.
12. Etienne-Manneville, S. (2004). Cdc42–the centre of polarity. J. Cell Sci.
regulates localization of the Rga4 GAP to ensure bipolar activation of
Cdc42 in fission yeast. Curr. Biol. 18, 322–330.
14. Hoffman, G.R., and Cerione, R.A. (2000). Flipping the switch: The struc-
tural basis for signaling through the CRIB motif. Cell 102, 403–406.
15. Snell, V., and Nurse, P. (1994).Genetic analysis of cell morphogenesis in
fission yeast–a role for casein kinase II in the establishment of polarized
growth. EMBO J. 13, 2066–2074.
16. Kanai, M., Kume, K., Miyahara, K., Sakai, K., Nakamura, K., Leonhard,
K., Wiley, D.J., Verde, F., Toda, T., and Hirata, D. (2005). Fission yeast
MO25 protein is localized at SPB and septum and is essential for cell
morphogenesis. EMBO J. 24, 3012–3025.
17. Leonhard, K., and Nurse, P. (2005). Ste20/GCK kinase Nak1/Orb3 polar-
izes the actin cytoskeleton in fission yeast during the cell cycle. J. Cell
Sci. 118, 1033–1044.
18. Hirata, D., Kishimoto, N., Suda, M., Sogabe, Y., Nakagawa, S., Yoshida,
Y., Sakai, K., Mizunuma, M., Miyakawa, T., Ishiguro, J., et al. (2002).
Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is
essentialforcellmorphogenesis andits mutationinduces Wee1-depen-
dent G(2) delay. EMBO J. 21, 4863–4874.
19. Merla, A., and Johnson, D.I. (2000). The Cdc42p GTPase is targeted to
the site of cell division in the fission yeast Schizosaccharomyces
pombe. Eur. J. Cell Biol. 79, 469–477.
20. Coll, P.M., Trillo, Y., Ametzazurra, A., and Perez, P. (2003). Gef1p, a new
guanine nucleotide exchange factor for Cdc42p, regulates polarity in
Schizosaccharomyces pombe. Mol. Biol. Cell 14, 313–323.
21. Murray, J.M., and Johnson, D.I. (2001). The Cdc42p GTPase and its
regulators Nrf1p and Scd1p are involved in endocytic trafficking in
the fission yeast Schizosaccharomyces pombe. J. Biol. Chem. 276,
22. Das, M., Wiley, D.J., Medina, S., Vincent, H.A., Larrea, M., Oriolo, A., and
Verde, F. (2007). Regulation of cell diameter, For3p localization, and
cell symmetry by fission yeast Rho-GAP Rga4p. Mol. Biol. Cell 18,
23. Matsuyama, A., Arai, R., Yashiroda, Y.,Shirai,A., Kamata,A., Sekido, S.,
Kobayashi, Y., Hashimoto, A., Hamamoto, M., Hiraoka, Y., et al. (2006).
ORFeome cloning and global analysis of protein localization in the
fission yeast Schizosaccharomyces pombe. Nat. Biotechnol. 24,
24. Zallen, J.A., Peckol, E.L., Tobin, D.M., and Bargmann, C.I. (2000).
Neuronal cell shape and neurite initiation are regulated by the Ndr
kinase SAX-1, a member of the Orb6/COT-1/warts serine/threonine
kinase family. Mol. Biol. Cell 11, 3177–3190.
25. Stork, O., Zhdanov, A., Kudersky, A., Yoshikawa, T., Obata, K., and
Pape, H.-C. (2004). Neuronal functions of the novel serine/threonine
kinase Ndr2. J. Biol. Chem. 279, 45773–45781.
26. Mazanka, E., Alexander, J., Yeh, B.J., Charoenpong, P., Lowery, D.M.,
Yaffe, M., and Weiss, E.L. (2008). The NDR/LATS family kinase Cbk1
directly controls transcriptional asymmetry. PLoS Biol. 6, e203.
27. Hao, Y., Chun, A., Cheung, K., Rashidi, B., and Yang, X. (2008). Tumor
suppressor LATS1 is a negative regulator of oncogene YAP. J. Biol.
Chem. 283, 5496–5509.
28. Kurischko, C., Kuravi, V.K., Wannissorn, N., Nazarov, P.A., Husain, M.,
Zhang, C., Shokat, K.M., McCaffery, J.M., and Luca, F.C. (2008). The
yeast LATS/Ndr kinase Cbk1 regulates growth via Golgi-dependent
glycosylation and secretion. Mol. Biol. Cell 19, 5559–5578.
29. Govek, E.-E., Newey, S.E., and Van Aelst, L. (2005). The role of the Rho
GTPases in neuronal development. Genes Dev. 19, 1–49.
Spatial Control of Cdc42 GTPase by Orb6 Kinase