Robust cell polarity is a dynamic state established by coupling transport and GTPase signaling.
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Yeast cells can initiate bud formation at the G1/S transition in a cue-independent manner. Here, we investigate the dynamic nature of the polar cap and the regulation of the GTPase Cdc42 in the establishment of cell polarity. Using analysis of fluorescence recovery after photobleaching, we found that Cdc42 exchanged rapidly between the polar caps and cytosol and that this rapid exchange required its GTPase cycle. A previously proposed positive feedback loop involving actomyosin-based transport of the Cdc42 GTPase is required for the generation of robust cell polarity during bud formation in yeast. Inhibition of actin-based transport resulted in unstable Cdc42 polar caps. Unstable polarity was also observed in mutants lacking Bem1, a protein previously implicated in a feedback loop for Cdc42 activation through a signaling pathway. When Bem1 and actin were both inhibited, polarization completely failed. These results suggest that cell polarity is established through coupling of transport and signaling pathways and maintained actively by balance of flux. (+info)
Cyclical regulation of the exocyst and cell polarity determinants for polarized cell growth.
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Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast. (+info)
The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2.
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Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a key second messenger that regulates actin and membrane dynamics, as well as other cellular processes. Many of the effects of PtdIns(4,5)P(2) are mediated by binding to effector proteins that contain a pleckstrin homology (PH) domain. Here, we identify two novel effectors of PtdIns(4,5)P(2) in the budding yeast Saccharomyces cerevisiae: the PH domain containing protein Slm1 and its homolog Slm2. Slm1 and Slm2 serve redundant roles essential for cell growth and actin cytoskeleton polarization. Slm1 and Slm2 bind PtdIns(4,5)P(2) through their PH domains. In addition, Slm1 and Slm2 physically interact with Avo2 and Bit61, two components of the TORC2 signaling complex, which mediates Tor2 signaling to the actin cytoskeleton. Together, these interactions coordinately regulate Slm1 targeting to the plasma membrane. Our results thus identify two novel effectors of PtdIns(4,5)P(2) regulating cell growth and actin organization and suggest that Slm1 and Slm2 integrate inputs from the PtdIns(4,5)P(2) and TORC2 to modulate polarized actin assembly and growth. (+info)
Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20.
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The Saccharomyces cerevisiae PAK (p21-activated kinase) family kinase Ste20 functions in several signal transduction pathways, including pheromone response, filamentous growth, and hyperosmotic resistance. The GTPase Cdc42 localizes and activates Ste20 by binding to an autoinhibitory motif within Ste20 called the CRIB domain. Another factor that functions with Ste20 and Cdc42 is the protein Bem1. Bem1 has two SH3 domains, but target ligands for these domains have not been described. Here we identify an evolutionarily conserved binding site for Bem1 between the CRIB and kinase domains of Ste20. Mutation of tandem proline-rich (PxxP) motifs in this region disrupts Bem1 binding, suggesting that it serves as a ligand for a Bem1 SH3 domain. These PxxP motif mutations affect signaling additively with CRIB domain mutations, indicating that Bem1 and Cdc42 make separable contributions to Ste20 function, which cooperate to promote optimal signaling. This PxxP region also binds another SH3 domain protein, Nbp2, but analysis of bem1Delta versus nbp2Delta strains shows that the signaling defects of PxxP mutants result from impaired binding to Bem1 rather than from impaired binding to Nbp2. Finally, the PxxP mutations also reduce signaling by constitutively active Ste20, suggesting that postactivation functions of PAKs can be promoted by SH3 domain proteins, possibly by colocalizing PAKs with their substrates. The overall results also illustrate how the final signaling function of a protein can be governed by combinatorial addition of multiple, independent protein-protein interaction modules. (+info)
Interplay between septin organization, cell cycle and cell shape in yeast.
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Septins are conserved filament-forming proteins that assemble into cortical cytoskeletal structures in animal and fungal cells. Although rapid progress has been made into the functions of septins, the mechanisms governing their localization and organization remain mysterious. In Saccharomyces cerevisiae, Cdc42p organizes the septin cytoskeleton into a ring in preparation for bud formation, following which septins remain as a collar at the mother-bud neck. We have dissected the phenotype of cdc42(V36T,K94E) cells that display an aberrant cell shape correlated with the development of ectopic septin caps and rings within the bud. The results suggest that a well-assembled septin cortex plays a novel role in directing growth to shape the nascent bud, and that a disorganized septin cortex directs improper growth generating an aberrant neck. Conversely, we found that the elongated bud shape arising as a result of the morphogenesis checkpoint cell cycle delay that accompanies septin perturbation can feed back to exacerbate minor defects in septin organization, by maintaining a bud-tip-localized septin assembly activity that competes with the neck-localized septin cortex. Using this exacerbation as a tool, we uncovered septin organization defects in many mutants not previously known to display such defects, expanding the cast of characters involved in proper assembly of the septin cortex to include CLN1, CLN2, BNI1, BNI4, BUD3, BUD4 and BUD5. (+info)
Ras and the Rho effector Cla4 collaborate to target and anchor Lte1 at the bud cortex.
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Lte1, a protein important for exit from mitosis, localizes to the bud cortex as soon as the bud forms and remains there until cells exit from mitosis. Ras, the Rho GTPase Cdc42 and its effector the protein kinase Cla4 are required for Lte1's association with the bud cortex. Here we investigate how Ras, and the Cdc42 effector Cla4 regulate the localization of Lte1. We find that Ras2 and Lte1 associate in stages of the cell cycle when Lte1 is phosphorylated and associated with the bud cortex and that this association requires CLA4. Additionally, RAS1 and RAS2 are required for CLA4-dependent Lte1 phosphorylation. Our findings suggest that Cla4-dependent phosphorylation promotes the initial association of Lte1 with Ras at the bud cortex and that Ras is required to stabilize phosphorylated forms of Lte1 at the bud cortex. Our results also raise the interesting possibility that the localization of Lte1 affects the protein's ability to promote mitotic exit. (+info)
Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex.
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Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis. (+info)
A system of counteracting feedback loops regulates Cdc42p activity during spontaneous cell polarization.
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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. (+info)