ABSTRACT
Cdc42 is a highly conserved master regulator of cell polarity. Here, we investigated the mechanism by which yeast cells never re-establish polarity at cortical sites (cytokinesis remnants [CRMs]) that have previously supported Cdc42-mediated growth as a paradigm to mechanistically understand how Cdc42-inhibitory polarity cues are established. We revealed a two-step mechanism of loading the Cdc42 antagonist Nba1 into CRMs to mark these compartments as refractory for a second round of Cdc42 activation. Our data indicate that Nba1 together with a cortically tethered adaptor protein confers memory of previous polarization events to translate this spatial legacy into a biochemical signal that ensures the local singularity of Cdc42 activation. "Memory loss" mutants that repeatedly use the same polarity site over multiple generations display nuclear segregation defects and a shorter lifespan. Our work thus established CRMs as negative polarity cues that prevent Cdc42 reactivation to sustain the fitness of replicating cells.
Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Asymmetric Cell Division , Cell Cycle Proteins/metabolism , Cell Polarity , Guanine Nucleotide Exchange Factors/metabolism , Membrane Proteins/metabolismABSTRACT
Many cells undergo symmetry-breaking polarization toward a randomly oriented "front" in the absence of spatial cues. In budding yeast, such polarization involves a positive feedback loop that enables amplification of stochastically arising clusters of polarity factors. Previous mathematical modeling suggested that, if more than one cluster were amplified, the clusters would compete for limiting resources and the largest would "win," explaining why yeast cells always make one and only one bud. Here, using imaging with improved spatiotemporal resolution, we show the transient coexistence of multiple clusters during polarity establishment, as predicted by the model. Unexpectedly, we also find that initial polarity factor clustering is oscillatory, revealing the presence of a negative feedback loop that disperses the factors. Mathematical modeling predicts that negative feedback would confer robustness to the polarity circuit and make the kinetics of competition between polarity factor clusters relatively insensitive to polarity factor concentration. These predictions are confirmed experimentally.
Subject(s)
Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Cell Polarity , Feedback, Physiological , Guanine Nucleotide Exchange Factors/metabolism , Models, Biological , Saccharomyces cerevisiae Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Cellular life exhibits order and complexity, which typically increase over the course of evolution. Cell polarization is a well-studied example of an ordering process that breaks the internal symmetry of a cell by establishing a preferential axis. Like many cellular processes, polarization is driven by self-organization, meaning that the macroscopic pattern emerges as a consequence of microscopic molecular interactions at the biophysical level. However, the role of self-organization in the evolution of complex protein networks remains obscure. In this Review, we provide an overview of the evolution of polarization as a self-organizing process, focusing on the model species Saccharomyces cerevisiae and its fungal relatives. Moreover, we use this model system to discuss how self-organization might relate to evolutionary change, offering a shift in perspective on evolution at the microscopic scale.
Subject(s)
Saccharomyces cerevisiae Proteins , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/genetics , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Models, Biological , Evolution, MolecularABSTRACT
Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular behaviors, including the response to stress and cell differentiation, and are highly conserved across eukaryotes. MAPK pathways can be activated by the interaction between the small GTPase Cdc42p and the p21-activated kinase (Ste20p in yeast). By studying MAPK pathway regulation in yeast, we recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is regulated in a similar manner and is turned over by the 26S proteasome. This turnover did not occur when Ste20p was bound to Cdc42p, which presumably stabilized the protein to sustain MAPK pathway signaling. Although Ste20p is a major component of the fMAPK pathway, genetic approaches here identified a Ste20p-independent branch of signaling. Ste20p-independent signaling partially required the fMAPK pathway scaffold and Cdc42p-interacting protein, Bem4p, while Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p. Interestingly, Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p, Rga1p, which unexpectedly dampened basal but not active fMAPK pathway activity. These new regulatory features of the Rho GTPase and p21-activated kinase module may extend to related pathways in other systems.
Subject(s)
Mitogen-Activated Protein Kinases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae , p21-Activated Kinases , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/genetics , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Protein StabilityABSTRACT
How cells generate a single axis of polarity for mating, division, and movement is unknown. In this issue, Howell et al. (2009) use a synthetic biology approach to demonstrate that rapid competition for a soluble signaling component (Bem1) is essential to ensure a unique axis of polarity in budding yeast.
Subject(s)
Cell Polarity , Saccharomyces cerevisiae/cytology , Adaptor Proteins, Signal Transducing/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
For budding yeast to ensure formation of only one bud, cells must polarize toward one, and only one, site. Polarity establishment involves the Rho family GTPase Cdc42, which concentrates at polarization sites via a positive feedback loop. To assess whether singularity is linked to the specific Cdc42 feedback loop, we disabled the yeast cell's endogenous amplification mechanism and synthetically rewired the cells to employ a different positive feedback loop. Rewired cells violated singularity, occasionally making two buds. Even cells that made only one bud sometimes initiated two clusters of Cdc42, but then one cluster became dominant. Mathematical modeling indicated that, given sufficient time, competition between clusters would promote singularity. In rewired cells, competition occurred slowly and sometimes failed to develop a single "winning" cluster before budding. Slowing competition in normal cells also allowed occasional formation of two buds, suggesting that singularity is enforced by rapid competition between Cdc42 clusters.
Subject(s)
Saccharomyces cerevisiae/cytology , Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Feedback, Physiological , Models, Biological , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Polarity decisions are central to many processes, including mitosis and chemotropism. In Saccharomyces cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converges on the small G protein Cdc42. However, pheromone-gradient sensing must override the Rsr1-dependent internal polarity cues used for budding. Using this model system, we asked what happens when intrinsic and extrinsic spatial cues are not aligned. Is there competition, or collaboration? By live-cell microscopy and microfluidics techniques, we uncovered three previously overlooked features of this signaling system. First, the cytokinesis-associated polarization patch serves as a polarity landmark independently of all known cues. Second, the Rax1-Rax2 complex functions as a pheromone-promoted polarity cue in the distal pole of the cells. Third, internal cues remain active during pheromone-gradient tracking and can interfere with this process, biasing the location of MPs. Yeast defective in internal-cue utilization align significantly better than wild type with artificially generated pheromone gradients.
Subject(s)
Cell Polarity , Chemotaxis , Mating Factor/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle , Cytokinesis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in the yeast Saccharomyces cerevisiae, which is controlled by the Rho GTPase Cdc42p. The prevailing view of bud emergence does not account for regulation by extrinsic cues. Here, we show that the filamentous growth mitogen activated protein kinase (fMAPK) pathway regulates bud emergence under nutrient-limiting conditions. The fMAPK pathway regulated the expression of polarity targets including the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and active during the period of the cell cycle leading up to bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Cell Differentiation , Cell Polarity/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/genetics , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which can experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors, we quantified the accuracy of initial polarization during mating encounters. We found that cells bias the orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. Uneven receptor density means that the gradient of ligand-bound receptors does not accurately reflect the external pheromone gradient. Nevertheless, yeast cells appear to avoid being misled by responding to the fraction of occupied receptors rather than simply the concentration of ligand-bound receptors. Such ratiometric sensing also serves to amplify the gradient of active G protein. However, this process is quite error-prone, and initial errors are corrected during a subsequent indecisive phase in which polarity clusters exhibit erratic mobile behavior.
Subject(s)
Gene Expression Regulation, Fungal , Genes, Mating Type, Fungal , Pheromones/metabolism , Saccharomyces cerevisiae/genetics , Signal Transduction/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Receptors, Mating Factor/genetics , Receptors, Mating Factor/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/genetics , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Many cellular processes require cell polarization to be maintained as the cell changes shape, grows or moves. Without feedback mechanisms relaying information about cell shape to the polarity molecular machinery, the coordination between cell polarization and morphogenesis, movement or growth would not be possible. Here we theoretically and computationally study the role of a genetically-encoded mechanical feedback (in the Cell Wall Integrity pathway) as a potential coordination mechanism between cell morphogenesis and polarity during budding yeast mating projection growth. We developed a coarse-grained continuum description of the coupled dynamics of cell polarization and morphogenesis as well as 3D stochastic simulations of the molecular polarization machinery in the evolving cell shape. Both theoretical approaches show that in the absence of mechanical feedback (or in the presence of weak feedback), cell polarity cannot be maintained at the projection tip during growth, with the polarization cap wandering off the projection tip, arresting morphogenesis. In contrast, for mechanical feedback strengths above a threshold, cells can robustly maintain cell polarization at the tip and simultaneously sustain mating projection growth. These results indicate that the mechanical feedback encoded in the Cell Wall Integrity pathway can provide important positional information to the molecular machinery in the cell, thereby enabling the coordination of cell polarization and morphogenesis.
Subject(s)
Cell Polarity/physiology , Feedback, Physiological/physiology , Models, Biological , Morphogenesis/physiology , Biomechanical Phenomena/physiology , Cell Movement/physiology , Cell Wall/physiology , Computational Biology , Computer Simulation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits, despite large variation in the expression of protein signaling components. Therefore, we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model, we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together, our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway.
Subject(s)
GTP-Binding Proteins/metabolism , GTPase-Activating Proteins/physiology , Saccharomyces cerevisiae Proteins/physiology , Cell Polarity , Pheromones/metabolism , Signal Transduction , Transcription, Genetic , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Cell polarization can be defined as the generation and maintenance of directional cellular organization. The spatial distribution and protein or lipid composition of the cell are not symmetric but organized in specialized domains which allow cells to grow and acquire a certain shape that is closely linked to their physiological function. The establishment and maintenance of polarized growth requires the coordination of diverse processes including cytoskeletal dynamics, membrane trafficking, and signaling cascade regulation. Some of the major players involved in the selection and maintenance of sites for polarized growth are Rho GTPases, which recognize the polarization site and transmit the signal to regulatory proteins of the cytoskeleton. Additionally, cytoskeletal organization, polarized secretion, and endocytosis are controlled by signaling pathways including those mediated by mitogen-activated protein kinases (MAPKs). Rho GTPases and the MAPK signaling pathways are strongly conserved from yeast to mammals, suggesting that the basic mechanisms of polarized growth have been maintained throughout evolution. For this reason, the study of how polarized growth is established and regulated in simple organisms such as the fission yeast Schizosaccharomyces pombe has contributed to broaden our knowledge about these processes in multicellular organisms. We review here the function of the Cdc42 GTPase and the stress activated MAPK (SAPK) signaling pathways during fission yeast polarized growth, and discuss the relevance of the crosstalk between both pathways.
Subject(s)
Fungal Proteins , MAP Kinase Signaling System , Schizosaccharomyces/physiology , Stress, Physiological , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Cell Polarity , Phosphorylation , Protein Binding , Protein Transport , Schizosaccharomyces/cytologyABSTRACT
Polarity establishment, the spontaneous generation of asymmetric molecular distributions, is a crucial component of many cellular functions. Saccharomyces cerevisiae (yeast) undergoes directed growth during budding and mating, and is an ideal model organism for studying polarization. In yeast and many other cell types, the Rho GTPase Cdc42 is the key molecular player in polarity establishment. During yeast polarization, multiple patches of Cdc42 initially form, then resolve into a single front. Because polarization relies on strong positive feedback, it is likely that the amplification of molecular-level fluctuations underlies the generation of multiple nascent patches. In the absence of spatial cues, these fluctuations may be key to driving polarization. Here we used particle-based simulations to investigate the role of stochastic effects in a Turing-type model of yeast polarity establishment. In the model, reactions take place either between two molecules on the membrane, or between a cytosolic and a membrane-bound molecule. Thus, we developed a computational platform that explicitly simulates molecules at and near the cell membrane, and implicitly handles molecules away from the membrane. To evaluate stochastic effects, we compared particle simulations to deterministic reaction-diffusion equation simulations. Defining macroscopic rate constants that are consistent with the microscopic parameters for this system is challenging, because diffusion occurs in two dimensions and particles exchange between the membrane and cytoplasm. We address this problem by empirically estimating macroscopic rate constants from appropriately designed particle-based simulations. Ultimately, we find that stochastic fluctuations speed polarity establishment and permit polarization in parameter regions predicted to be Turing stable. These effects can operate at Cdc42 abundances expected of yeast cells, and promote polarization on timescales consistent with experimental results. To our knowledge, our work represents the first particle-based simulations of a model for yeast polarization that is based on a Turing mechanism.
Subject(s)
Cell Membrane Permeability/physiology , Cell Polarity/physiology , Computational Biology/methods , Cell Division , Cell Membrane/metabolism , Computer Simulation , Cytosol/metabolism , Diffusion , Models, Biological , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiology , Stochastic Processes , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
A fundamental problem in cell biology is to understand how spatial information is recognized and integrated into morphogenetic responses. Budding yeast undergoes differentiation to filamentous growth, which involves changes in cell polarity through mechanisms that remain obscure. Here we define a regulatory input where spatial landmarks (bud-site-selection proteins) regulate the MAPK pathway that controls filamentous growth (fMAPK pathway). The bud-site GTPase Rsr1p regulated the fMAPK pathway through Cdc24p, the guanine nucleotide exchange factor for the polarity establishment GTPase Cdc42p. Positional landmarks that direct Rsr1p to bud sites conditionally regulated the fMAPK pathway, corresponding to their roles in regulating bud-site selection. Therefore, cell differentiation is achieved in part by the reorganization of polarity at bud sites. In line with this conclusion, dynamic changes in budding pattern during filamentous growth induced corresponding changes in fMAPK activity. Intrinsic compromise of bud-site selection also impacted fMAPK activity. Therefore, a surveillance mechanism monitors spatial position in response to extrinsic and intrinsic stress and modulates the response through a differentiation MAPK pathway.
Subject(s)
MAP Kinase Signaling System , Saccharomyces cerevisiae/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42-GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on Rsr1 and its GTPase-activating protein (GAP) Bud2. Interestingly, Rga1, a Cdc42 GAP, exhibits transient localization to a site near the bud neck and to the division site during cytokinesis and G1, and this temporal change of Rga1 distribution is necessary for determination of a proper growth site. Mathematical modeling suggests that a proper axis of Cdc42 polarization in haploid cells might be established through a biphasic mechanism involving sequential positive feedback and transient negative feedback.
Subject(s)
Cell Polarity/physiology , GTPase-Activating Proteins/metabolism , Saccharomycetales/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , rab GTP-Binding Proteins/metabolism , Cell Division/physiology , Fungal Proteins/metabolism , G1 Phase/physiology , HaploidyABSTRACT
GTPase-activating proteins (GAPs) play critical roles in the spatial and temporal control of small GTPases. The budding yeast Bem3 is a GAP for Cdc42, a Rho GTPase crucial for actin and septin organization. Bem3 localizes to the sites of polarized growth. However, the amino acid sequence determinants mediating recruitment of Bem3 to its physiological sites of action and those important for Bem3 function are not clear. Here, we show that Bem3's localization is guided by two distinct targeting regions-the PX-PH-domain-containing TD1 and the coiled-coil-containing TD2. TD2 localization is largely mediated by its interaction with the polarisome component Epo1 via heterotypic coiled-coil interaction. This finding reveals a novel role for the polarisome in linking Bem3 to its functional target, Cdc42. We also show that the coiled-coil domain of Bem3 interacts homotypically and this interaction is important for the regulation of Cdc42 by Bem3. Moreover, we show that overexpression of a longer version of the TD2 domain disrupts septin-ring assembly in a RhoGAP-independent manner, suggesting that TD2 may be capable of interacting with proteins implicated in septin-ring assembly. Furthermore, we show that the longer version of TD2 interacts with Kss1, a MAPK involved in filamentous growth. Kss1 is reported to localize mainly in the nucleus. We find that Kss1 also localizes to the sites of polarized growth and Bem3 interacts with Kss1 at the septin-ring assembly site. Our study provides new insights in Bem3's localization and function.
Subject(s)
Carrier Proteins/genetics , GTPase-Activating Proteins/genetics , Mitogen-Activated Protein Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/genetics , Carrier Proteins/metabolism , Cell Polarity/genetics , GTPase-Activating Proteins/metabolism , Gene Expression Regulation, Fungal , Mitogen-Activated Protein Kinases/metabolism , Monomeric GTP-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Septins/genetics , Septins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs) at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae) cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs), which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization.
Subject(s)
Cell Polarity , Electricity , Saccharomycetales/cytology , Cation Transport Proteins/metabolism , Electrochemistry , Lipids/pharmacology , Membrane Potentials , Models, Biological , Optogenetics , Phosphatidylserines/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolismABSTRACT
Spontaneous polarization without spatial cues, or symmetry breaking, is a fundamental problem of spatial organization in biological systems. This question has been extensively studied using yeast models, which revealed the central role of the small GTPase switch Cdc42. Active Cdc42-GTP forms a coherent patch at the cell cortex, thought to result from amplification of a small initial stochastic inhomogeneity through positive feedback mechanisms, which induces cell polarization. Here, I review and discuss the mechanisms of Cdc42 activity self-amplification and dynamic turnover. A robust Cdc42 patch is formed through the combined effects of Cdc42 activity promoting its own activation and active Cdc42-GTP displaying reduced membrane detachment and lateral diffusion compared to inactive Cdc42-GDP. I argue the role of the actin cytoskeleton in symmetry breaking is not primarily to transport Cdc42 to the active site. Finally, negative feedback and competition mechanisms serve to control the number of polarization sites.
Subject(s)
Cell Polarity/physiology , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Actin Cytoskeleton/metabolism , Feedback , Guanosine Triphosphate/chemistry , Models, Biological , Protein Transport , Transport Vesicles/metabolismABSTRACT
Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post-Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP-tag placed on the N-terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post-Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.
Subject(s)
Cell Membrane/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Secretory Vesicles/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/chemistry , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/geneticsABSTRACT
The spatiotemporal control of cell polarity is crucial for the development of multicellular organisms and for reliable polarity switches during cell cycle progression in unicellular systems. A tight control of cell polarity is especially important in haploid budding yeast, where the new polarity site (bud site) is established next to the cell division site after cell separation. How cells coordinate the temporal establishment of two adjacent polarity sites remains elusive. Here, we report that the bud neck associated protein Gps1 (GTPase-mediated polarity switch 1) establishes a novel polarity cue that concomitantly sustains Rho1-dependent polarization and inhibits premature Cdc42 activation at the site of cytokinesis. Failure of Gps1 regulation leads to daughter cell death due to rebudding inside the old bud site. Our findings provide unexpected insights into the temporal control of cytokinesis and describe the importance of a Gps1-dependent mechanism for highly accurate polarity switching between two closely connected locations.