Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating

Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner. Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1. Export of Cdc24 to a site of cell polarization is mediated by two mechanisms. At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28-Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm. Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site. In contrast, in response to mating pheromones, the Far1-Cdc24 complex is exported from the nucleus by Msn5. This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone. Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability. Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24. This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.     

A Cdc24p-Far1p-Gbetagamma protein complex required for yeast orientation during mating

Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a two- hybrid interaction between Cdc24p and Gbeta requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and Gbeta. Binding experiments demonstrate that Cdc24p, Far1p, and Gbeta form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-Gbetagamma complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal.     

Cdc42p GDP/GTP cycling is necessary for efficient cell fusion during yeast mating

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP-GTP cycling is critical for efficient cell fusion.     

Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans

Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.     

Fission yeast Cdc37 is required for multiple cell cycle functions

The identification of a Schizosaccharomyces pombe homologue of the cdc37 gene is described. The gene product is most similar to the budding yeast homologue, but shows similarity to metazoan Cdc37 proteins, with a region of high similarity at the extreme N-terminus. Gene transplacement experiments in diploid cells followed by tetrad dissection show that the gene is essential. Depletion of the gene product after switching off expression of cdc37 from the regulatable nmt81 promoter results in cessation of growth and division. The cells arrest heterogeneously, with a significant proportion showing mitotic defects; paradoxically, a proportion of the cells show a short-cell phenotype consistent with an advanced cell cycle.Communicated by D. Y. Thomas     

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.     

Nucleocytoplasmic shuttling of the Cdc42p exchange factor Cdc24p

Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle-dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p-Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p-Far1p complex which is required for chemotropism.     

The fission yeast Map4 protein is a novel adhesin required for mating

Cell adhesion is required for many cellular processes. In fungi, cell-cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h(+) cells and enhances cell adhesion when overexpressed.     

Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast

Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.     

The nucleotide exchange factor Cdc24p may be regulated by auto-inhibition

Site-specific activation of the Rho-type GTPase Cdc42p by its guanine-nucleotide exchange factor (GEF) Cdc24p is critical for the establishment of cell polarity. Here we show that binding of Cdc24p to the small GTPase Rsr1p/Bud1p is required for its recruitment to the incipient bud site. Rsr1p/Bud1p binds to the CH-domain of Cdc24p, which is essential for its function in vivo. We have identified a cdc24-mutant allele, which is specifically defective for bud-site selection. Our results suggest that Cdc24p is auto-inhibited by an intramolecular interaction with its carboxy-terminal PB1-domain. Rsr1p/Bud1p appears to activate the GEF activity of Cdc24p in vivo, possibly by triggering a conformational change that dissociates the PB1-domain from its intramolecular binding site. Genetic experiments suggest that Bem1p functions as a positive regulator of Cdc24p by binding to the PB1-domain of Cdc24p, thereby preventing its re-binding to the intramolecular inhibitory site. Taken together, our results support a two-step molecular mechanism for the site-specific activation of Cdc24p, which involves Rsr1p/Bud1p and the adaptor protein Bem1p.     

Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gbetagamma to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gbetagamma. Our results imply that Gbetagamma not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo.     

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.     

Cell fusion during yeast mating requires high levels of a-factor mating pheromone

During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.     

Remodeling of organelle-bound actin is required for yeast vacuole fusion

Actin participates in several intracellular trafficking pathways. We now find that actin, bound to the surface of purified yeast vacuoles in the absence of cytosol or cytoskeleton, regulates the last compartment mixing stage of homotypic vacuole fusion. The Cdc42p GTPase is known to be required for vacuole fusion. We now show that proteins of the Cdc42p-regulated actin remodeling cascade (Cdc42p --> Cla4p --> Las17p/Vrp1p --> Arp2/3 complex --> actin) are enriched on isolated vacuoles. Vacuole fusion is dramatically altered by perturbation of the vacuole-bound actin, either by mutation of the ACT1 gene, addition of specific actin ligands such as latrunculin B or jasplakinolide, antibody to the actin regulatory proteins Las17p (yeast Wiskott-Aldrich syndrome protein) or Arp2/3, or deletion of actin regulatory genes. On docked vacuoles, actin is enriched at the "vertex ring" membrane microdomain where fusion occurs and is required for the terminal steps leading to membrane fusion. This role for actin may extend to other trafficking systems.     

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.     

Inhibition of Cdc42 during mitotic exit is required for cytokinesis

The role of Cdc42 and its regulation during cytokinesis is not well understood. Using biochemical and imaging approaches in budding yeast, we demonstrate that Cdc42 activation peaks during the G₁/S transition and during anaphase but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to down-regulate Cdc42 during mitotic exit impairs the normal localization of key cytokinesis regulators—Iqg1 and Inn1—at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase Ste20. Inhibition of Cdc42 and related Rho guanosine triphosphatases may be a general feature of cytokinesis in eukaryotes.     

Nuclear fusion during yeast mating occurs by a three-step pathway

In Saccharomyces cerevisiae, mating culminates in nuclear fusion to produce a diploid zygote. Two models for nuclear fusion have been proposed: a one-step model in which the outer and inner nuclear membranes and the spindle pole bodies (SPBs) fuse simultaneously and a three-step model in which the three events occur separately. To differentiate between these models, we used electron tomography and time-lapse light microscopy of early stage wild-type zygotes. We observe two distinct SPBs in ~80% of zygotes that contain fused nuclei, whereas we only see fused or partially fused SPBs in zygotes in which the site of nuclear envelope (NE) fusion is already dilated. This demonstrates that SPB fusion occurs after NE fusion. Time-lapse microscopy of zygotes containing fluorescent protein tags that localize to either the NE lumen or the nucleoplasm demonstrates that outer membrane fusion precedes inner membrane fusion. We conclude that nuclear fusion occurs by a three-step pathway.     

Cdc6 is a rate-limiting factor for proliferative capacity during HL60 cell differentiation

The DNA replication (or origin) licensing pathway represents a critical step in cell proliferation control downstream of growth signalling pathways. Repression of origin licensing through down-regulation of the MCM licensing factors (Mcm2-7) is emerging as a ubiquitous route for lowering proliferative capacity as metazoan cells exit the cell division cycle into quiescent, terminally differentiated and senescent "out-of-cycle" states. Using the HL60 monocyte/macrophage differentiation model system and a cell-free DNA replication assay, we have undertaken direct biochemical investigations of the coupling of origin licensing to the differentiation process. Our data show that down-regulation of the MCM loading factor Cdc6 acts as a molecular switch that triggers loss of proliferative capacity during early engagement of the somatic differentiation programme. Consequently, addition of recombinant Cdc6 protein to in vitro replication reactions restores DNA replication competence in nuclei prepared from differentiating cells. Differentiating HL60 cells over-expressing either wild-type Cdc6 or a CDK phosphorylation-resistant Cdc6 mutant protein (Cdc6A4) exhibit an extended period of cell proliferation compared to mock-infected cells. Notably, differentiating HL60 cells over-expressing the Cdc6A4 mutant fail to down-regulate Cdc6 protein levels, suggesting that CDK phosphorylation of Cdc6 is linked to its down-regulation during differentiation and the concomitant decrease in cell proliferation. In this experimental model, Cdc6 therefore plays a key role in the sequential molecular events leading to repression of origin licensing and loss of proliferative capacity during execution of the differentiation programme.