Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogen Candida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) (), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth in S. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother-daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.     

Phr1p, a glycosylphosphatidylinsitol-anchored β(1,3)-glucanosyltransferase critical for hyphal wall formation, localizes to the apical growth sites and septa in Candida albicans

Cell wall biogenesis is a dynamic process relying on the coordinated activity of several extracellular enzymes. PHR1 is a pH-regulated gene of Candida albicans encoding a glycosylphosphatidylinositol-anchored β(1,3)-glucanosyltransferase of family GH72 which acts as a cell wall remodelling enzyme and is crucial for morphogenesis and virulence. In order to explore the function of Phr1p, we obtained a green fluorescent protein (GFP) fusion to determine its localization. During induction of vegetative growth, Phr1p-GFP was concentrated in the plasma membrane of the growing bud, in the mother-bud neck, and in the septum. Phr1p-GFP was recovered in the detergent-resistant membranes indicating its association with the lipid rafts as the wild type Phr1p. Upon induction of hyphal growth, Phr1p-GFP highly concentrated at the apex of the germ tubes and progressively distributed along the lateral sides of the hyphae. Phr1p-GFP also labelled the hyphal septa, where it colocalized with chitin. Localization to the hyphal septa was perturbed in nocodazole-treated cells, whereas inhibition of actin polymerization hindered the apical localization. Electron Microscopy analysis of the hyphal wall ultrastructure of a PHR1 null mutant showed loss of compactness and irregular organization of the surface layer. These observations indicate that Phr1p plays a crucial role in hyphal wall formation, a highly regulated process on which morphogenesis and virulence rely.     

Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus,Ustilago maydis

Ustilago maydis is a dimorphic Basidiomycete fungus with a yeast-like form and a hyphal form. Here we present a comprehensive analysis of bud formation and the actin and microtubule cytoskeletons of the yeast-like form during the cell cycle. We show that bud morphogenesis entails a series of shape changes, initially a tubular or conical structure, culminating in a cigar-shaped cell connected to the mother cell by a narrow neck. Labelling of cells with concanavalin A demonstrated that growth occurs at bud tip. Indirect immunofluorescence studies revealed that the actin cytoskeleton consists of patches and cables that polarize to the presumptive bud site and the bud tip and an actin ring that forms at the neck region. Because the bud tip corresponds to the site of active cell wall growth, we hypothesize that actin is involved in secretion of cell wall components. The microtubule cytoskeleton has recently been shown to consist of a cytoplasmic network during interphase that disassembles at mitosis when a spindle and astral microtubules are formed. We have carried out studies of U. maydis cells synchronized by the microtubule-depolymerizing drug thiabendazole which allow us to construct a temporal sequence of steps in spindle formation and spindle elongation during the cell cycle. These studies suggest that astral microtubules may be involved in early stages of spindle orientation and migration of the nucleus into the bud and that the spindle pole bodies may be involved in reestablishment of the cytoplasmic microtubule network.     

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth

Morphogenesis in the fungal pathogen Candida albicans is an important virulence-determining factor, as a dimorphic switch between yeast and hyphal growth forms can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase (PLK) in C. albicans and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked early in nuclear division with very short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle elongation at an earlier point in the cell cycle than that described for the homologue Cdc5p in yeast. Strikingly, the cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Filament growth was determinate, as the filaments started to die after 24 h. The filaments resembled serum-induced hyphae with respect to morphology, organization of cytoplasmic microtubules, localization of nuclei, and expression of hyphal-specific components. Filament formation required CaCDC35, but not EFG1 or CPH1. Similar defects in spindle elongation and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea. Because CaCdc5p does not appear to act as a direct repressor of hyphal growth, the data suggest that a target of CaCdc5p function is associated with hyphal-like development. Thus, an internal, cell cycle-related cue can activate hyphal regulatory networks in Candida.     

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Summary Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.Abbrevations CE cytochalasin E - DMSO dimethyl sulphoxide - RP rhodamine phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Sep7 is essential to modify septin ring dynamics and inhibit cell separation during Candida albicans hyphal growth

When Candida albicans yeast cells receive the appropriate stimulus, they switch to hyphal growth, characterized by continuous apical elongation and the inhibition of cell separation. The molecular basis of this inhibition is poorly known, despite its crucial importance for hyphal development. In C. albicans, septins are important for hypha formation and virulence. Here, we used fluorescence recovery after photobleaching analysis to characterize the dynamics of septin rings during yeast and hyphal growth. On hyphal induction, septin rings are converted to a hyphal-specific state, characterized by the presence of a frozen core formed by Sep7/Shs1, Cdc3 and Cdc12, whereas Cdc10 is highly dynamic and oscillates between the ring and the cytoplasm. Conversion of septin rings to the hyphal-specific state inhibits the translocation of Cdc14 phosphatase, which controls cell separation, to the hyphal septum. Modification of septin ring dynamics during hyphal growth is dependent on Sep7 and the hyphal-specific cyclin Hgc1, which partially controls Sep7 phosphorylation status and protein levels. Our results reveal a link between the cell cycle machinery and septin cytoskeleton dynamics, which inhibits cell separation in the filaments and is essential for hyphal morphogenesis.     

Ectopic expression of a constitutively active Cdc42 small GTPase alters the morphology of haploid and dikaryotic hyphae in the filamentous homobasidiomycete Schizophyllum commune

Cloning of the Cdc42 gene from Schizophyllum commune enabled investigation of the role of ScCdc42 in the regulation of vegetative growth and sexual reproduction in this fungus, which has a well-characterized hyphal cell structure, cytoskeleton, and mating system. Ectopic expression of the constitutively active Sccdc42(G12V) or Sccdc42(Q61L) alleles from native or inducible ScCel1 promoters in haploid hyphae had dramatic effects on hyphal morphology, cytoskeletal structure, and Cdc42 localization. For transformants with constitutively active Sccdc42, polar tip growth of apical cells in the leading hyphae was normal but polar tip growth in side branches was altered, implying different regulation of polarity establishment in the two groups of apical cells. Branch emergence at exceptional sites and isotropic growth of cells near the septum indicated that ScCdc42 regulates branch site selection and subsequent hyphal development. Poor dikaryotization along with irregular clamp connections in mates expressing Sccdc42(G12V) or Sccdc42(Q61L) suggested that Cdc42 also contributes to efficient mating in S. commune.     

The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae

Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by G1-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305- 1320). We isolated slt2-1 as a mutation that enhances the division defect of cdc28 mutants with defects at Start. Slt2p(Mpk1p) is a member of the MAP kinase family (Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsumoto, and D. E. Levin. 1993. Mol. Cell. Biol. 13:3067-3075). We show that slt2 mutants exhibit phenotypes similar to those shown by mutants of the yeast actin cytoskeleton, including delocalization of chitin deposition and of actin cortical spots and the accumulation of secretory pathway membranes and vesicles. Furthermore, slt2::HIS3 act1-1 and slt2::HIS3 myo2-66 double mutants are inviable. We suggest that Slt2p functions downstream or in parallel with Cdc28p in promoting bud formation and apical growth.     

Septin function in Candida albicans morphogenesis

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.     

Ultrastructural localization of anionic sites on the surface of yeast, hyphal and germ-tube forming cells of Candida albicans

The cell wall of Candida albicans contains chitin, beta-glucans and phosphorylated mannoproteins, and possesses a fuzzy coat which is thought to play a role in pathogenicity, phagocytosis, and adherence of this dimorphic yeast. Using scanning electron microscopy and the gold method, mannoproteins were detected on the whole surface of blastoconidia including the bud scars, but chitin was absent even after alpha-mannosidase treatment of the cells. The presence of surface beta-(1----6)glucan (but not beta(1----3)glucan) was observed only after extensive alpha-mannosidase and alkaline phosphatase treatments of blastoconidia. Using transmission and scanning electron microscopy, the locations of anionic sites were revealed by polycationic colloidal gold-chitosan complexes on the surface of blastoconidia, germ tubes and hyphae. Anionic sites were dispersed evenly over the surface of blastoconidia bearing bud scars. Depending upon the growth conditions, anionic sites could be detected on emerging buds and young cells. However, bud scars were always free of marking. When germ-tube formation was induced, anionic sites were present at different densities on all cell surfaces, the highest density being observed on cells with bud scars. Anionic sites were detected at a remarkably high density on all hyphal surfaces. An apical concentration of anionic sites was observed on germ tubes and hyphae. The distribution of anionic sites was not modified by endoglucosaminidase treatment of blastoconidia, germ tubes and hyphae. The anionic sites were associated with the fuzzy coat. As the hyphal form is regarded as possessing the greatest invasiveness, it is suggested that anionic sites play an important role in establishing tissue colonization by this human pathogen.     

Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localization

The location of the septin ring in the germ tubes of Candida albicans hyphae and pseudohyphae was studied using an antibody to Saccharomyces cerevisiae Cdc11p. In pseudohyphae induced by growth at 35 degrees C in YEPD or Lee's medium, a septin ring formed at or near (mean 1.8 microm) the neck between the mother cell and the germ tube. This became double later in the cycle, and the first mitosis took place across the plane of this double ring. A septin ring also formed at the germ tube neck of developing hyphae induced by serum or growth on Lee's medium at 37 degrees C. However, at later times, this ring became disorganized and disappeared. A second double ring then appeared 10-15 microm (mean 12.5 microm) along the length of the germ tube. The nucleus subsequently migrated out of the mother cell into the germ tube, and the first mitosis took place across the plane of this second septin ring. The relocation of the septin ring in developing hyphae provides a clear-cut molecular distinction between hyphae and pseudohyphae. Commitment to one type of septin localization and mitosis was shown to occur early in the first mitotic cycle, well before evagination. Germ tubes of hyphae and pseudohyphae also have different widths. A point of commitment to germ tube width was also demonstrated, but occurred later in the cycle, approximately coincident with the time of evagination.     

CDK-dependent phosphorylation of Mob2 is essential for hyphal development in Candida albicans

Nuclear Dbf2-related (NDR) protein kinases are essential components of regulatory pathways involved in cell morphogenesis, cell cycle control, and viability in eukaryotic cells. For their activity and function, these kinases require interaction with Mob proteins. However, little is known about how the Mob proteins are regulated. In Candida albicans, the cyclin-dependent kinase (CDK) Cdc28 and the NDR kinase Cbk1 are required for hyphal growth. Here we demonstrate that Mob2, the Cbk1 activator, undergoes a Cdc28-dependent differential phosphorylation on hyphal induction. Mutations in the four CDK consensus sites in Mob2 to Ala significantly impaired hyphal development. The mutant cells produced short hyphae with enlarged tips that displayed an illicit activation of cell separation. We also show that Cdc28 phosphorylation of Mob2 is essential for the maintenance of polarisome components at hyphal tips but not at bud tips during yeast growth. Thus we have found a novel signaling pathway by which Cdc28 controls Cbk1 through the regulatory phosphorylation of Mob2, which is crucial for normal hyphal development.     

A Candida albicans Temperature-Sensitive cdc12-6 Mutant Identifies Roles for Septins in Selection of Sites of Germ Tube Formation and Hyphal Morphogenesis

Septins were identified for their role in septation in Saccharomyces cerevisiae and were subsequently implicated in other morphogenic processes. To study septins in Candida albicans hyphal morphogenesis, a temperature-sensitive mutation was created that altered the C terminus of the essential Cdc12 septin. The cdc12-6 cells grew well at room temperature, but at 37°C they displayed expected defects in septation, nuclear localization, and bud morphogenesis. Although serum stimulated the cdc12-6 cells at 37°C to form germ tube outgrowths, the mutant could not maintain polarized hyphal growth and instead formed chains of elongated cell compartments. Serum also stimulated the cdc12-6 mutant to induce a hyphal reporter gene (HWP1-GFP) and a characteristic zone of filipin staining at the leading edge of growth. Interestingly, cdc12-6 cells shifted to 37°C in the absence of serum gradually displayed enriched filipin staining at the tip, which may be due to the altered cell cycle regulation. A striking difference from the wild type was that the cdc12-6 cells frequently formed a second germ tube in close proximity to the first. The mutant cells also failed to form the diffuse band of septins at the base of germ tubes and hyphae, indicating that this septin band plays a role in preventing proximal formation of germ tubes in a manner analogous to bud site selection. These studies demonstrate that not only are septins important for cytokinesis, but they also promote polarized morphogenesis and selection of germ tube sites that may help disseminate an infection in host tissues.     

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.     

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.     

An IQGAP-related protein,encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii

In filamentous ascomycetes hyphae are compartmentalized by septation in which the cytoplasm of the compartments are interconnected via septal pores. Thus, septation in filamentous fungi is different from cytokinesis in yeast like fungi. We have identified an Ashbya gossypii orthologue of the Saccharomyces cerevisiae CYK1 gene which belongs to the IQGAP-protein family. In contrast to S. cerevisiae disruption of AgCYK1 yields viable mutant strains that exhibit wildtype-like polarized hyphal growth rates. In the Agcyk1 mutant cortical actin patches localize to growing hyphal tips like wildtype, however, mutant hyphae are totally devoid of actin rings at presumptive septal sites. Septation in wildtype results in the formation of chitin rings. Agcyk1 mutant hyphae are aseptate and do not accumulate chitin in their cell walls. Agcyk1 mutant strains are completely asporogenous indicating that septation is essential for the formation of sporangia in A. gossypii. AgCyk1p-GFP localizes to sites of future septation as a ring prior to chitin depositioning. Furthermore, decrease in Cyk1p-ring diameter was found to be a prerequisite for the accumulation of chitin and septum formation.     

Regulation of Cdc42 GTPase activity in the formation of hyphae in Candida albicans

The human fungal pathogen Candida albicans can switch between yeast, pseudohyphal, and hyphal morphologies. To investigate whether the distinctive characteristics of hyphae are due to increased activity of the Cdc42 GTPase, strains lacking negative regulators of Cdc42 were constructed. Unexpectedly, the deletion of the Cdc42 Rho guanine dissociation inhibitor RDI1 resulted in reduced rather than enhanced polarized growth. However, when cells lacking both Cdc42 GTPase-activating proteins, encoded by RGA2 and BEM3, were grown under pseudohyphal-promoting conditions the bud was highly elongated and lacked a constriction at its base, so that its shape resembled a hyphal germ tube. Moreover, a Spitzenk?rper was present at the bud tip, a band of disorganized septin was present at bud base, true septin rings formed within the bud, and nuclei migrated out of the mother cell before the first mitosis. These are all characteristic features of a hyphal germ tube. Intriguingly, we observed hyphal-specific phosphorylation of Rga2, suggesting a possible mechanism for Cdc42 activation during normal hyphal development. In contrast, expression of Cdc42(G12V), which is constitutively GTP bound because it lacks GTPase activity, resulted in swollen cells with prominent and stable septin bars. These results suggest the development of hyphal-specific characteristics is promoted by Cdc42-GTP in a process that also requires the intrinsic GTPase activity of Cdc42.