Cell Polarity in Yeast.

A conserved molecular machinery centered on the Cdc42 GTPase regulates cell polarity in diverse organisms. Here we review findings from budding and fission yeasts that reveal both a conserved core polarity circuit and several adaptations that each organism exploits to fulfill the needs of its lifestyle. The core circuit involves positive feedback by local activation of Cdc42 to generate a cluster of concentrated GTP-Cdc42 at the membrane. Species-specific pathways regulate the timing of polarization during the cell cycle, as well as the location and number of polarity sites.

Negative feedback enhances robustness in the yeast polarity establishment circuit.

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.

Particle-based simulations reveal two positive feedback loops allow relocation and stabilization of the polarity site during yeast mating.

Many cells adjust the direction of polarized growth or migration in response to external directional cues. The yeast Saccharomyces cerevisiae orient their cell fronts (also called polarity sites) up pheromone gradients in the course of mating. However, the initial polarity site is often not oriented towards the eventual mating partner, and cells relocate the polarity site in an indecisive manner before developing a stable orientation. During this reorientation phase, the polarity site displays erratic assembly-disassembly behavior and moves around the cell cortex. The mechanisms underlying this dynamic behavior remain poorly understood. Particle-based simulations of the core polarity circuit revealed that molecular-level fluctuations are unlikely to overcome the strong positive feedback required for polarization and generate relocating polarity sites. Surprisingly, inclusion of a second pathway that promotes polarity site orientation generated relocating polarity sites with properties similar to those observed experimentally. This pathway forms a second positive feedback loop involving the recruitment of receptors to the cell membrane and couples polarity establishment to gradient sensing. This second positive feedback loop also allows cells to stabilize their polarity site once the site is aligned with the pheromone gradient.

Singularity in polarization: rewiring yeast cells to make two buds.

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.

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.

A novel stochastic simulation approach enables exploration of mechanisms for regulating polarity site movement.

Cells polarize their movement or growth toward external directional cues in many different contexts. For example, budding yeast cells grow toward potential mating partners in response to pheromone gradients. Directed growth is controlled by polarity factors that assemble into clusters at the cell membrane. The clusters assemble, disassemble, and move between different regions of the membrane before eventually forming a stable polarity site directed toward the pheromone source. Pathways that regulate clustering have been identified but the molecular mechanisms that regulate cluster mobility are not well understood. To gain insight into the contribution of chemical noise to cluster behavior we simulated clustering using the reaction-diffusion master equation (RDME) framework to account for molecular-level fluctuations. RDME simulations are a computationally efficient approximation, but their results can diverge from the underlying microscopic dynamics. We implemented novel concentration-dependent rate constants that improved the accuracy of RDME-based simulations, allowing us to efficiently investigate how cluster dynamics might be regulated. Molecular noise was effective in relocating clusters when the clusters contained low numbers of limiting polarity factors, and when Cdc42, the central polarity regulator, exhibited short dwell times at the polarity site. Cluster stabilization occurred when abundances or binding rates were altered to either lengthen dwell times or increase the number of polarity molecules in the cluster. We validated key results using full 3D particle-based simulations. Understanding the mechanisms cells use to regulate the dynamics of polarity clusters should provide insights into how cells dynamically track external directional cues.

Principles that govern competition or co-existence in Rho-GTPase driven polarization.

Rho-GTPases are master regulators of polarity establishment and cell morphology. Positive feedback enables concentration of Rho-GTPases into clusters at the cell cortex, from where they regulate the cytoskeleton. Different cell types reproducibly generate either one (e.g. the front of a migrating cell) or several clusters (e.g. the multiple dendrites of a neuron), but the mechanistic basis for unipolar or multipolar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by two-component reaction-diffusion models based on conserved aspects of Rho-GTPase biochemistry. Some such models display rapid winner-takes-all competition between clusters, yielding a unipolar outcome. Other models allow prolonged co-existence of clusters. We investigate the behavior of a simple class of models and show that while the timescale of competition varies enormously depending on model parameters, a single factor explains a large majority of this variation. The dominant factor concerns the degree to which the maximal active GTPase concentration in a cluster approaches a "saturation point" determined by model parameters. We suggest that both saturation and the effect of saturation on competition reflect fundamental properties of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism, which predict whether the system will generate unipolar or multipolar outcomes.

How do cells know what shape they are?

Studies on a yeast cell cycle checkpoint that can delay mitosis depending on whether cells have built a bud have identified a "sensor" that seems to recognize the organization of filament-forming septin proteins. Innovative work applying correlative light and platinum replica electron microscopy suggests that the informative septin organization involves parallel alignment of septin filaments, and another striking study shows that septin filaments prefer to populate membranes that have positive micron-scale curvature. Together, these findings suggest a model for how cells may monitor aspects of their own shape to influence cell behavior.

Chemical transformation of the multibudding yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/µg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.

Tools for live-cell imaging of cytoskeletal and nuclear behavior in the unconventional yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

Mechanism of commitment to a mating partner in Saccharomyces cerevisiae.

Many cells detect and follow gradients of chemical signals to perform their functions. Yeast cells use gradients of extracellular pheromones to locate mating partners, providing a tractable model for understanding how cells decode the spatial information in gradients. To mate, yeast cells must orient polarity toward the mating partner. Polarity sites are mobile, exploring the cell cortex until they reach the proper position, where they stop moving and "commit" to the partner. A simple model to explain commitment posits that a high concentration of pheromone is detected only upon alignment of partner cells' polarity sites and causes polarity site movement to stop. Here we explore how yeast cells respond to partners that make different amounts of pheromone. Commitment was surprisingly robust to various pheromone levels, ruling out the simple model. We also tested whether adaptive pathways were responsible for the robustness of commitment, but our results show that cells lacking those pathways were still able to accommodate changes in pheromone. To explain this robustness, we suggest that the steep pheromone gradients near each mating partner's polarity site trap the polarity site in place.

Targeted secretion: Myosin V delivers vesicles through formin condensates.

Secretory vesicles are often delivered to very specific targets, like pre-synaptic terminals or cell tips, to focus exocytosis. New work suggests that a biomolecular condensate focuses actin filaments that deliver incoming vesicles through the condensate to the plasma membrane.

Pheromone Guidance of Polarity Site Movement in Yeast.

Cells' ability to track chemical gradients is integral to many biological phenomena, including fertilization, development, accessing nutrients, and combating infection. Mating of the yeast Saccharomyces cerevisiae provides a tractable model to understand how cells interpret the spatial information in chemical gradients. Mating yeast of the two different mating types secrete distinct peptide pheromones, called a-factor and α-factor, to communicate with potential partners. Spatial gradients of pheromones are decoded to guide mobile polarity sites so that polarity sites in mating partners align towards each other, as a prerequisite for cell-cell fusion and zygote formation. In ascomycetes including S. cerevisiae, one pheromone is prenylated (a-factor) while the other is not (α-factor). The difference in physical properties between the pheromones, combined with associated differences in mechanisms of secretion and extracellular pheromone metabolism, suggested that the pheromones might differ in the spatial information that they convey to potential mating partners. However, as mating appears to be isogamous in this species, it is not clear why any such signaling difference would be advantageous. Here we report assays that directly track movement of the polarity site in each partner as a way to understand the spatial information conveyed by each pheromone. Our findings suggest that both pheromones convey very similar information. We speculate that the different pheromones were advantageous in ancestral species with asymmetric mating systems and may represent an evolutionary vestige in yeasts that mate isogamously.

Chemotropism and Cell-Cell Fusion in Fungi.

Fungi exhibit an enormous variety of morphologies, including yeast colonies, hyphal mycelia, and elaborate fruiting bodies. This diversity arises through a combination of polar growth, cell division, and cell fusion. Because fungal cells are nonmotile and surrounded by a protective cell wall that is essential for cell integrity, potential fusion partners must grow toward each other until they touch and then degrade the intervening cell walls without impacting cell integrity. Here, we review recent progress on understanding how fungi overcome these challenges. Extracellular chemoattractants, including small peptide pheromones, mediate communication between potential fusion partners, promoting the local activation of core cell polarity regulators to orient polar growth and cell wall degradation. However, in crowded environments, pheromone gradients can be complex and potentially confusing, raising the question of how cells can effectively find their partners. Recent findings suggest that the cell polarity circuit exhibits searching behavior that can respond to pheromone cues through a remarkably flexible and effective strategy called exploratory polarization.

Cultivating PhD Aspirations during College.

Science, technology, engineering, and mathematics (STEM) career barriers persist for individuals from marginalized communities due to financial and educational inequality, unconscious bias, and other disadvantaging factors. To evaluate differences in plans and interests between historically underrepresented (UR) and well-represented (WR) groups, we surveyed more than 3000 undergraduates enrolled in chemistry courses. Survey responses showed all groups arrived on campus with similar interests in learning more about science research. Over the 4 years of college, WR students maintained their interest levels, but UR students did not, creating a widening gap between the groups. Without intervention, UR students participated in lab research at lower rates than their WR peers. A case study pilot program, Biosciences Collaborative for Research Engagement (BioCoRE), encouraged STEM research exploration by undergraduates from marginalized communities. BioCoRE provided mentoring and programming that increased community cohesion and cultivated students' intrinsic scientific mindsets. Our data showed that there was no statistical significant difference between BioCoRE WR and UR students when surveyed about plans for a medical profession, graduate school, and laboratory scientific research. In addition, BioCoRE participants reported higher levels of confidence in conducting research than non-BioCoRE Scholars. We now have the highest annual number of UR students moving into PhD programs in our institution's history.

Symmetry-breaking polarization driven by a Cdc42p GEF-PAK complex.

BACKGROUND: In 1952, Alan Turing suggested that spatial patterns could arise from homogeneous starting conditions by feedback amplification of stochastic fluctuations. One example of such self-organization, called symmetry breaking, involves spontaneous cell polarization in the absence of spatial cues. The conserved GTPase Cdc42p is essential for both guided and spontaneous polarization, and in budding yeast cells Cdc42p concentrates at a single site (the presumptive bud site) at the cortex. Cdc42p concentrates at a random cortical site during symmetry breaking in a manner that requires the scaffold protein Bem1p. The mechanism whereby Bem1p promotes this polarization was unknown. RESULTS: Here we show that Bem1p promotes symmetry breaking by assembling a complex in which both a Cdc42p-directed guanine nucleotide exchange factor (GEF) and a Cdc42p effector p21-activated kinase (PAK) associate with Bem1p. Analysis of Bem1p mutants indicates that both GEF and PAK must bind to the same molecule of Bem1p, and a protein fusion linking the yeast GEF and PAK bypasses the need for Bem1p. Although mammalian cells lack a Bem1p ortholog, they contain more complex multidomain GEFs that in some cases can directly interact with PAKs, and we show that yeast containing an artificial GEF with similar architecture can break symmetry even without Bem1p. CONCLUSIONS: Yeast symmetry-breaking polarization involves a GEF-PAK complex that binds GTP-Cdc42p via the PAK and promotes local Cdc42p GTP-loading via the GEF. By generating fresh GTP-Cdc42p near pre-existing GTP-Cdc42p, the complex amplifies clusters of GTP-Cdc42p at the cortex. Our findings provide mechanistic insight into an evolutionarily conserved pattern-forming positive-feedback pathway.

Dynamics of septin ring and collar formation in Saccharomyces cerevisiae.

Although the septin ring and collar in budding yeast were described over 20 years ago, there is still controversy regarding the organization of septin filaments within these structures and about the way in which the ring first forms and about how it converts into a collar at the mother-bud neck. Here we present quantitative analyses of the recruitment of fluorescently-tagged septins to the ring and collar through the cell cycle. Septin ring assembly began several minutes after polarity establishment and this interval was longer in daughter than in mother cells, suggesting asymmetric inheritance of septin regulators. Septins formed an initial faint and irregular ring, which became more regular as septins were recruited at a constant rate. This steady rate of septin recruitment continued for several minutes after the ring converted to a collar at bud emergence. We did not detect a stepwise change in septin fluorescence during the ring-to-collar transition. After collar formation, septins continued to accumulate at the bud neck, though at a reduced rate, until the onset of cytokinesis when the amount of neck-localized septins rapidly decreased. Implications for the mechanism of septin ring assembly are discussed.

The morphogenesis checkpoint: how yeast cells watch their figures.

The morphogenesis checkpoint maintains coordination between the process of bud formation and the nuclear events of the cell cycle in yeast. This checkpoint regulates the Wee1 homolog, Swe1p, to induce cell-cycle delay or arrest when aspects of bud formation are defective. A variety of studies have suggested that this checkpoint can monitor actin organization, septin organization, the presence of a bud and even the size of a bud. The evidence for these proposals is reviewed, highlighting recent findings indicating that Swe1p degradation is controlled by the cell shape change that accompanies bud emergence.

Scaffold-mediated symmetry breaking by Cdc42p.

Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking. In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally. The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization. We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments. Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour. Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch.