Turnover and bypass of p21-activated kinase during Cdc42-dependent MAPK signaling in yeast.

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.

Complexity and self-organization in the evolution of cell polarization.

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.

Regulation of Cdc42 protein turnover modulates the filamentous growth MAPK pathway.

Rho GTPases are central regulators of cell polarity and signaling. How Rho GTPases are directed to function in certain settings remains unclear. Here, we show the protein levels of the yeast Rho GTPase Cdc42p are regulated, which impacts a subset of its biological functions. Specifically, the active conformation of Cdc42p was ubiquitinated by the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones and turned over in the proteasome. A GTP-locked (Q61L) turnover-defective (TD) version, Cdc42pQ61L+TD, hyperactivated the MAPK pathway that regulates filamentous growth (fMAPK). Cdc42pQ61L+TD did not influence the activity of the mating pathway, which shares components with the fMAPK pathway. The fMAPK pathway adaptor, Bem4p, stabilized Cdc42p levels, which resulted in elevated fMAPK pathway signaling. Our results identify Cdc42p turnover regulation as being critical for the regulation of a MAPK pathway. The control of Rho GTPase levels by stabilization and turnover may be a general feature of signaling pathway regulation, which can result in the execution of a specific developmental program.

How cells determine the number of polarity sites.

The diversity of cell morphologies arises, in part, through regulation of cell polarity by Rho-family GTPases. A poorly understood but fundamental question concerns the regulatory mechanisms by which different cells generate different numbers of polarity sites. Mass-conserved activator-substrate (MCAS) models that describe polarity circuits develop multiple initial polarity sites, but then those sites engage in competition, leaving a single winner. Theoretical analyses predicted that competition would slow dramatically as GTPase concentrations at different polarity sites increase toward a 'saturation point', allowing polarity sites to coexist. Here, we test this prediction using budding yeast cells, and confirm that increasing the amount of key polarity proteins results in multiple polarity sites and simultaneous budding. Further, we elucidate a novel design principle whereby cells can switch from competition to equalization among polarity sites. These findings provide insight into how cells with diverse morphologies may determine the number of polarity sites.

A time-resolved interaction analysis of Bem1 reconstructs the flow of Cdc42 during polar growth.

Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing front of the yeast cell, the scaffold protein Bem1, is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in these transformations, we systematically tracked its protein interactions during one cell cycle to define the ensemble of Bem1 interaction states for each cell cycle stage. Mutants of Bem1 that interact with only a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle-specific shuttle that distributes active Cdc42 from its source to its effectors. It further suggests that Bem1 might convert the PAKs Cla4 and Ste20 into their active conformations.

Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling.

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.

Regulation of intrinsic polarity establishment by a differentiation-type MAPK pathway in S. cerevisiae.

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.

Ratiometric GPCR signaling enables directional sensing in yeast.

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.

Actin and Nuclear Envelope Components Influence Ectopic Recombination in the Absence of Swr1.

The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast Saccharomyces cerevisiae show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Importantly, these phenotypes can be specifically suppressed by mutations in: (1) chromatin-anchorage internal nuclear membrane components (mps3∆75-150 and src1∆); (2) actin and actin regulators (act1-157, act1-159, crn1∆, and cdc42-6); or (3) the SWR subunit Swc5 and the SWR substrate Htz1 However, they are not suppressed by global disruption of actin filaments or by the absence of Csm4 (a component of the external nuclear membrane that forms a bridging complex with Mps3, thus connecting the actin cytoskeleton with chromatin). Moreover, swr1∆-induced Rad52 foci and intramolecular recombination are not associated with tethering recombinogenic DNA lesions to the nuclear periphery. In conclusion, the absence of Swr1 impairs efficient recombinational repair of specific DNA lesions by mechanisms that are influenced by SWR subunits, including actin, and nuclear envelope components. We suggest that these recombinational phenotypes might be associated with a pathological effect on homologous recombination of actin-containing complexes.

Cell-cycle control of cell polarity in yeast.

In many cells, morphogenetic events are coordinated with the cell cycle by cyclin-dependent kinases (CDKs). For example, many mammalian cells display extended morphologies during interphase but round up into more spherical shapes during mitosis (high CDK activity) and constrict a furrow during cytokinesis (low CDK activity). In the budding yeast Saccharomyces cerevisiae, bud formation reproducibly initiates near the G1/S transition and requires activation of CDKs at a point called "start" in G1. Previous work suggested that CDKs acted by controlling the ability of cells to polarize Cdc42, a conserved Rho-family GTPase that regulates cell polarity and the actin cytoskeleton in many systems. However, we report that yeast daughter cells can polarize Cdc42 before CDK activation at start. This polarization operates via a positive feedback loop mediated by the Cdc42 effector Ste20. We further identify a major and novel locus of CDK action downstream of Cdc42 polarization, affecting the ability of several other Cdc42 effectors to localize to the polarity site.

A Positive Feedback between Growth and Polarity Provides Directional Persistency and Flexibility to the Process of Tip Growth.

Polar cell growth is a conserved morphogenetic process needed for survival, mating, and infection [1, 2]. It typically implicates the assembly and spatial stabilization of a cortical polar domain of the active form of a small GTPase of the Rho family, such as Cdc42, which promotes cytoskeleton assembly and secretion needed for local surface expansion [3-6]. In multiple physiological instances, polarity domains may switch from being spatially unstable, exhibiting a wandering behavior around the cell surface, to being stable at a fixed cellular location [7-11]. Here, we show that the rate of surface growth may be a key determinant in controlling the spatial stability of active Cdc42 domains. Reducing the growth rate of single rod-shaped fission yeast cells using chemical, genetic, and mechanical means systematically causes polar domains to detach from cell tips and oscillate around the cell surface within minutes. Conversely, an abrupt increase in growth rate improves domain stabilization. A candidate screen identifies vesicular transport along actin cables as an important module mediating this process. Similar behavior observed in distant filamentous fungi suggests that this positive feedback between growth and polarity could represent a basal property of eukaryotic polarization, promoting persistent polar growth as well as growth redirection with respect to the mechanical environment of cells.

Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells.

Cdc42 was originally discovered as a key regulator of bud site assembly and polarity in S. cerevisiae. Recent genetic studies have shown that the function of Cdc42 in regulating cell polarity appears highly conserved from budding yeast to humans. The role of Cdc42 in hematopoietic cell transformation and leukemia progression has been studied in an acute myeloid leukemia model using the MLL-AF9 oncogene-induced transformation and a Cdc42 conditional gene-targeted mouse model. Here we describe the leukemia cell polarity and division symmetry assays in the context of leukemia cell fate determination.

Self-organization principles of intracellular pattern formation.

Dynamic patterning of specific proteins is essential for the spatio-temporal regulation of many important intracellular processes in prokaryotes, eukaryotes and multicellular organisms. The emergence of patterns generated by interactions of diffusing proteins is a paradigmatic example for self-organization. In this article, we review quantitative models for intracellular Min protein patterns in Escherichia coli, Cdc42 polarization in Saccharomyces cerevisiae and the bipolar PAR protein patterns found in Caenorhabditis elegans By analysing the molecular processes driving these systems we derive a theoretical perspective on general principles underlying self-organized pattern formation. We argue that intracellular pattern formation is not captured by concepts such as 'activators', 'inhibitors' or 'substrate depletion'. Instead, intracellular pattern formation is based on the redistribution of proteins by cytosolic diffusion, and the cycling of proteins between distinct conformational states. Therefore, mass-conserving reaction-diffusion equations provide the most appropriate framework to study intracellular pattern formation. We conclude that directed transport, e.g. cytosolic diffusion along an actively maintained cytosolic gradient, is the key process underlying pattern formation. Thus the basic principle of self-organization is the establishment and maintenance of directed transport by intracellular protein dynamics.This article is part of the theme issue 'Self-organization in cell biology'.

Membrane curvature directs the localization of Cdc42p to novel foci required for cell-cell fusion.

Cell fusion is ubiquitous in eukaryotic fertilization and development. The highly conserved Rho-GTPase Cdc42p promotes yeast fusion through interaction with Fus2p, a pheromone-induced amphiphysin-like protein. We show that in prezygotes, Cdc42p forms a novel Fus2p-dependent focus at the center of the zone of cell fusion (ZCF) and remains associated with remnant cell walls after initial fusion. At the ZCF and during fusion, Cdc42p and Fus2p colocalized. In contrast, in shmoos, both proteins were near the cortex but spatially separate. Cdc42p focus formation depends on ZCF membrane curvature: mutant analysis showed that Cdc42p localization is negatively affected by shmoo-like positive ZCF curvature, consistent with the flattening of the ZCF during fusion. BAR-domain proteins such as the fusion proteins Fus2p and Rvs161p are known to recognize membrane curvature. We find that mutations that disrupt binding of the Fus2p/Rvs161p heterodimer to membranes affect Cdc42p ZCF localization. We propose that Fus2p localizes Cdc42p to the flat ZCF to promote cell wall degradation.

Roles of an N-terminal coiled-coil-containing domain in the localization and function of Bem3, a Rho GTPase-activating protein in budding yeast.

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.

Gic1 is a novel heterochromatin boundary protein in vivo.

In Saccharomyces cerevisiae, HMR/HML, telomeres and ribosomal DNA are heterochromatin-like regions in which gene transcription is prevented by the silent information regulator (Sir) complex. The Sir complex (Sir2, Sir3 and Sir4) can spread through chromatin from the silencer. Boundaries prevent Sir complex spreading, and we previously identified 55 boundary genes among all ~6,000 yeast genes. These boundary proteins can be distinguished into two types: those that activate transcription to prevent spreading of silencing, and those that prevent gene silencing by forming a boundary. We selected 44 transcription-independent boundary proteins from the 55 boundary genes by performing a one-hybrid assay and focused on GIC1 (GTPase interaction component 1). Gic1 is an effector of Cdc42, which belongs to the Rho family of small GTPases, and has not been reported to function in heterochromatin boundaries in vivo. We detected a novel boundary-forming activity of Gic1 at HMR-left and telomeric regions by conducting a chromatin immunoprecipitation assay with an anti-Sir3 antibody. We also found that Gic1 bound weakly to histones in two-hybrid analysis. Moreover, we performed domain analysis to identify domain(s) of Gic1 that are important for its boundary activity, and identified two minimum domains, which are located outside its Cdc42-binding domain.

A Cellular System for Spatial Signal Decoding in Chemical Gradients.

Directional cell growth requires that cells read and interpret shallow chemical gradients, but how the gradient directional information is identified remains elusive. We use single-cell analysis and mathematical modeling to define the cellular gradient decoding network in yeast. Our results demonstrate that the spatial information of the gradient signal is read locally within the polarity site complex using double-positive feedback between the GTPase Cdc42 and trafficking of the receptor Ste2. Spatial decoding critically depends on low Cdc42 activity, which is maintained by the MAPK Fus3 through sequestration of the Cdc42 activator Cdc24. Deregulated Cdc42 or Ste2 trafficking prevents gradient decoding and leads to mis-oriented growth. Our work discovers how a conserved set of components assembles a network integrating signal intensity and directionality to decode the spatial information contained in chemical gradients.

Quantitative analysis of membrane trafficking in regulation of Cdc42 polarity.

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.

Secretory vesicles deliver Cdc42p to sites of polarized growth in S. cerevisiae.

The activation and localization of the Rho-family GTPase Cdc42p at one pole of a cell is necessary for maintaining an axis of polarized growth in many animal and fungal cells. How the asymmetric distribution of this key regulator of polarized morphogenesis is maintained is not fully understood, though divergent models have emerged from a congruence of multiple studies, including one that posits a role for polarized secretion. Here we show with S. cerevisiae that Cdc42p associates with secretory vesicles in vivo.

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.