A system of counteracting feedback loops regulates Cdc42p activity during spontaneous cell polarization

Cellular polarization is often a response to distinct extracellular or intracellular cues, such as nutrient gradients or cortical landmarks. However, in the absence of such cues, some cells can still select a polarization axis at random. Positive feedback loops promoting localized activation of the GTPase Cdc42p are central to this process in budding yeast. Here, we explore spontaneous polarization during bud site selection in mutant yeast cells that lack functional landmarks. We find that these cells do not select a single random polarization axis, but continuously change this axis during the G1 phase of the cell cycle. This is reflected in traveling waves of activated Cdc42p which randomly explore the cell periphery. Our integrated computational and in vivo analyses of these waves reveal a negative feedback loop that competes with the aforementioned positive feedback loops to regulate Cdc42p activity and confer dynamic responsiveness on the robust initiation of cell polarization.     

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

Spontaneous Cdc42 Polarization Independent of GDI-Mediated Extraction and Actin-Based Trafficking

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.     

Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p

In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.     

Essential role of the NH2-terminal region of Cdc24 guanine nucleotide exchange factor in its initial polarized localization in Saccharomyces cerevisiae

The cortical recruitment and accumulation of the small GTPase Cdc42 are crucial steps in the establishment of polarity, but this process remains obscure. Cdc24 is an upstream regulator of budding yeast Cdc42 that accelerates the exchange of GDP for GTP in Cdc42 via its Dbl homology (DH) domain. Here, we isolated five novel temperature-sensitive (ts) cdc24 mutants, the green fluorescent protein (GFP)-fused proteins of which lose their polarized localization at the nonpermissive temperature. All amino acid substitutions in the mutants were mapped to the NH2-terminal region of Cdc24, including the calponin homology (CH) domain. These Cdc24-ts mutant proteins did not interact with Bem1 at the COOH-terminal PB1 domain, suggesting a lack of exposure of the PB1 domain in the mutant proteins. The cdc24-ts mutants were also defective in polarization in the absence of Bem1. It was previously reported that a fusion protein containing Cdc24 and the p21-activated kinase (PAK)-like kinase Cla4 could bypass the requirement for Bem1 in polarity cue-independent budding (i.e., symmetry breaking). Cdc24-ts-Cla4 fusion proteins also showed ts localization at the polarity site. We propose that the NH2-terminal region unmasks the DH and PB1 domains, leading to the activation of Cdc42 and interaction with Bem1, respectively, to initiate cell polarization.     

Saccharomyces cerevisiae Cdc42p localizes to cellular membranes and clusters at sites of polarized growth

The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.     

Cdc42p,GTP Hydrolysis and the Cell’s Sense of Direction

The GTPase Cdc42p is essential for polarity establishment in animals and fungi.1 Human Cdc42p can functionally replace yeast Cdc42p,2 indicating a high degree of evolutionary conservation. Current models of Cdc42p action generally follow the signaling paradigm established for Ras, in which receptors responding to an initiating stimulus cause guanine nucleotide exchange factors (GEFs) to trigger GTP-loading of Ras, leading to engagement of downstream effectors and ensuing cell proliferation. Key support for the Ras paradigm came from the finding that oncogenic forms of Ras, unable to hydrolyze GTP and therefore constitutively GTP-bound, mimicked the effect of constitutive signaling by the upstream receptors even in the absence of stimuli. Attempts to assess whether or not this paradigm is valid for Cdc42p-induced polarization of yeast cells have yielded conflicting results.3-6 Here, we discuss the available information on this issue and conclude that unlike Ras signaling, Cdc42p directed polarity establishment additionally requires cycling between GTP- and GDP-bound forms. We suggest that such cycling is critical for a little-studied “function” of Cdc42p: its ability to designate a unique portion of the cell cortex to become the polarization site, and to become concentrated at that site.     

A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast

The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.     

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.     

Interaction between a Ras and a Rho GTPase couples selection of a growth site to the development of cell polarity in yeast

Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.     

Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase–activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site.     

The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate

Signal transduction pathways that co-regulate a given biological process often are organized into networks by molecules that act as coincidence detectors. Phosphoinositides and the Rho-type GTPase Cdc42 regulate overlapping processes in all eukaryotic cells. However, the coincidence detectors that link these pathways into networks remain unknown. Here we show that the p21-activated protein kinase-related kinase Cla4 of yeast integrates signaling by Cdc42 and phosphatidylinositol 4-phosphate (PI4P). We found that the Cla4 pleckstrin homology (PH) domain binds in vitro to several phosphoinositide species. To determine which phosphoinositides regulate Cla4 in vivo, we analyzed phosphatidylinositol kinase mutants (stt4, mss4, and pik1). This indicated that the plasma membrane pool of PI4P, but not phosphatidylinositol 4,5-bisphosphate or the Golgi pool of PI4P, is required for localization of Cla4 to sites of polarized growth. A combination of the Cdc42-binding and PH domains of Cla4 was necessary and sufficient for localization to sites of polarized growth. Point mutations affecting either domain impaired the ability of Cla4 to regulate cell morphogenesis and the mitotic exit network (localization of Lte1). Therefore, Cla4 must retain the ability to bind both Cdc42 and phosphoinositides, the hallmark of a coincidence detector. PI4P may recruit Cla4 to the plasma membrane where Cdc42 activates its kinase activity and refines its localization to cortical sites of polarized growth. In mammalian cells, the myotonic dystrophy-related Cdc42-binding kinase possesses p21-binding and PH domains, suggesting that this kinase may be a coincidence detector of signaling by Cdc42 and phosphoinositides.     

Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.     

Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.     

Mechanistic mathematical model of polarity in yeast

The establishment of cell polarity involves positive-feedback mechanisms that concentrate polarity regulators, including the conserved GTPase Cdc42p, at the "front" of the polarized cell. Previous studies in yeast suggested the presence of two parallel positive-feedback loops, one operating as a diffusion-based system, and the other involving actin-directed trafficking of Cdc42p on vesicles. F-actin (and hence directed vesicle traffic) speeds fluorescence recovery of Cdc42p after photobleaching, suggesting that vesicle traffic of Cdc42p contributes to polarization. We present a mathematical modeling framework that combines previously developed mechanistic reaction-diffusion and vesicle-trafficking models. Surprisingly, the combined model recapitulated the observed effect of vesicle traffic on Cdc42p dynamics even when the vesicles did not carry significant amounts of Cdc42p. Vesicle traffic reduced the concentration of Cdc42p at the front, so that fluorescence recovery mediated by Cdc42p flux from the cytoplasm took less time to replenish the bleached pool. Simulations in which Cdc42p was concentrated into vesicles or depleted from vesicles yielded almost identical predictions, because Cdc42p flux from the cytoplasm was dominant. These findings indicate that vesicle-mediated delivery of Cdc42p is not required to explain the observed Cdc42p dynamics, and raise the question of whether such Cdc42p traffic actually contributes to polarity establishment.     

The Rsr1/Bud1 GTPase Interacts with Itself and the Cdc42 GTPase during Bud-Site Selection and Polarity Establishment in Budding Yeast

Cell polarization occurs along a single axis that is generally determined in response to spatial cues. In budding yeast, the Rsr1 GTPase and its regulators direct the establishment of cell polarity at the proper cortical location in response to cell type–specific cues. Here we use a combination of in vivo and in vitro approaches to understand how Rsr1 polarization is established. We find that Rsr1 associates with itself in a spatially and temporally controlled manner. The homotypic interaction and localization of Rsr1 to the mother-bud neck and to the subsequent division site are dependent on its GDP-GTP exchange factor Bud5. Analyses of rsr1 mutants suggest that Bud5 recruits Rsr1 to these sites and promotes the homodimer formation. Rsr1 also exhibits heterotypic interaction with the Cdc42 GTPase in vivo. We show that the polybasic region of Rsr1 is necessary for the efficient homotypic and heterotypic interactions, selection of a proper growth site, and polarity establishment. Our findings thus suggest that dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry.     

The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites.

Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton. The GTPase Cdc42 regulates actin rearrangements and signal transduction pathways in all eukaryotic cells [1], and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF). Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane [2] [3] at sites of polarized growth [4]. We show that Cdc24p labeled with green fluorescent protein (GFP-Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells. Unexpectedly, GFP-Cdc24p also localized to the nucleus and GFP-Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother-bud necks during cytokinesis. The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p. The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother-bud neck, and mating projection. Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the G(beta) subunit Ste4p. These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.     

Cdc50p,a conserved endosomal membrane protein,controls polarized growth in Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.