Browsing by Author "Lew, Daniel J"
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Item Open Access A Role for Gic1 and Gic2 in Promoting Cdc42 Polarization(2018) Daniels, Christine NicoleThe Rho GTPase Cdc42 is a master regulator of cell polarity that orchestrates reorganization of the cytoskeleton. During polarity establishment, active GTP-Cdc42 accumulates at a part of the cell cortex that becomes the front of the cell. Localized GTP-Cdc42 orients the cytoskeleton through a set of “effector” proteins that bind specifically to GTP-Cdc42 and not GDP-Cdc42. A family of Cdc42 effectors, called GICs in yeast and BORGs in mammals, have been implicated in regulation of both the actin cytoskeleton and the septin cytoskeleton. Yeast cells lacking both Gic1 and Gic2 are able to polarize and grow at low temperatures, but many mutant cells fail to polarize the cytoskeleton at high temperature. This led to the conclusion that GICs communicate between Cdc42 and different cytoskeletal elements.
To better characterize the role of GIC proteins in yeast, we utilized time-lapse fluorescent microscopy to examine morphogenetic events in living single cells. Surprisingly, we found that not only the cytoskeleton but also Cdc42 itself failed to polarize in many gic1 gic2 mutant cells at high temperature. This observation indicates that GICs may act upstream of polarization rather than downstream.
Polarization of Cdc42 is triggered by cell-cycle progression, and in particular by G1 Cyclin-dependent kinase (CDK) activity. Using a live-cell reporter for G1 CDK activation, we found that cells lacking GICs were not defective in CDK activation, but showed a specific defect in polarization downstream of the CDK. Previous work had implicated the scaffold protein Bem1 in a positive feedback loop important for polarization. Cells lacking GICs failed to polarize Bem1 as well as Cdc42 at high temperature. Future work will be directed at understanding how GICs contribute to polarity establishment. Because many of the mechanisms and proteins involved in polarization are highly conserved, we anticipate our findings will help inform how this process regulated in higher eukaryotes.
Item Open Access A Role for Inositol Hexakisphosphate in N-terminal Acetylation and Mitochondrial Distribution(2012) Pham, Trang ThuyInositol phosphates (IPs) are versatile metabolites that play important roles in multiple cellular processes. They have been considered signaling messengers that relay extracellular signals via a wave of their production and allosteric regulation of downstream targets. In addition to this classical role, recent studies have revealed that certain IPs can also function as protein structural cofactors. However, except for the two plant hormone receptors TIR1 and COI1, these IP binding proteins have neither sequences nor functions in common. Therefore, to test whether other cellular proteins are also subjected to this type of regulation and whether an IP binding motif exists, more proteins that bind IPs in a similar manner need to be identified. Via a proteome-wide biochemical screen, two yeast proteins were found to contain IP6 as an integral component. One is the N-terminal acetyltransferase A complex (NatA), and the other one is Tif31 (or Clu1). IP6 binding was also observed in NatC, another N-terminal acetyltransferase. The bioinformatics analysis and mutagenesis study showed that tandem tetratricopeptide repeats (TPRs), the only common structural element of NatA and Tif31, were responsible for coordinating IP6. This mechanism of IP6 binding is conserved in the fly homologs of these proteins.
NatA is one of the enzymes that acetylate the α-amino groups at protein N-termini. This widespread protein modification affects a wide range of cellular processes. IP6 was shown to be essential for yeast NatA in vitro thermostability and for some but not all functions of the protein in cells grown under temperature stress. Other multiple phosphate-containing molecules including IP5 species and the bacterial alarmone ppGpp were found to bind NatA and partially compensate for the lack of IP6. IP6 also binds the human NatA homolog. This binding is crucial for hNatA complex formation, in vitro and in vivo activities, and ability to rescue NatA-deficient phenotypes when it is expressed in yeast. Therefore, IP6 acts as a molecular glue that brings hNatA (and hNatE) subunits together. The other protein found in our screen, Tif31, is important for normal mitochondrial morphology and distribution. In cells that cannot produce IP4, IP5 and IP6, Tif31 levels were significantly decreased and these cells exhibited severe mitochondrial aggregation. Tif31 mutants that cannot bind IP6 showed a reduction in cellular levels, a shift to high molecular weight complexes or aggregates, and inability to rescue tif31δ mitochondrial phenotype. This study established the vital role of IP6 and IP5 in maintaining Tif31 stability and Tif31-mediated regulation of mitochondrial distribution.
Collectively, this dissertation discovered two proteins that use IP6 as a structural cofactor. For the first time, a conserved IP6 binding motif has been shown to be present in certain TPR-containing proteins. Via tight binding to these proteins, IP6 stabilizes their structures or subunit interaction. This research provides mechanistic evidence for the interplay between IP biology and N-terminal acetylation as well as between IP biology and mitochondrial morphology.
Item Open Access Aneuploidy Tolerance in a Polyploid Organ(2016) Schoenfelder, Kevin PaulEndopolyploid cells (hereafter - polyploid cells), which contain whole genome duplications in an otherwise diploid organism, play vital roles in development and physiology of diverse organs such as our heart and liver. Polyploidy is also observed with high frequency in many tumors, and division of such cells frequently creates aneuploidy (chromosomal imbalances), a hallmark of cancer. Despite its frequent occurrence and association with aneuploidy, little is known about the specific role that polyploidy plays in diverse contexts. Using a new model tissue, the Drosophila rectal papilla, we sought to uncover connections between polyploidy and aneuploidy during organ development. Our lab previously discovered that the papillar cells of the Drosophila hindgut undergo developmentally programmed polyploid cell divisions, and that these polyploid cell divisions are highly error-prone. Time-lapse studies of polyploid mitosis revealed that the papillar cells undergo a high percentage of tripolar anaphase, which causes extreme aneuploidy. Despite this massive chromosome imbalance, we found the tripolar daughter cells are viable and support normal organ development and function, suggesting acquiring extra genome sets enables a cell to tolerate the genomic alterations incurred by aneuploidy. We further extended these findings by seeking mechanisms by which the papillar cells tolerated this resultant aneuploidy.
Item Open Access Cell Polarity Establishment in the Budding Yeast Saccharomyces Cerevisiae(2009) Howell, AudreyEstablishing an axis of cell polarity is central to cell motility, tissue morphogenesis, and cell proliferation. A highly conserved group of polarity regulators is responsible for organizing a wide variety of polarized morphologies. One of the most widely expressed polarity regulators is the Rho-type GTPase Cdc42. In response to cell cycle cues the budding yeast Saccharomyces cerevisiae polarizes Cdc42p to a discrete site on the cell periphery. GTP-Cdc42p recruits a number of effectors that aid in the organization of a polarized actin cytoskeleton. The polarized actin cytoskeleton acts as tracks to facilitate the delivery of the secretory vesicles that will grow the bud, an essential process for an organism that proliferates by budding. We have employed treatment with the actin depolymerizing drugs Latrunculin A and B as well as high-speed timelapse microscopy of fluorescently labeled polarity proteins to characterize the assembly of the incipient bud site.
Often, ensuring that only a single axis of polarity is established is as important as generating asymmetry in the cell. Even in the absence of positional cues dictating the direction of polarization, many cells are still able to self-organize and establish one, and only one, polarity axis through a process termed symmetry breaking. Symmetry breaking is thought to employ positive feedback to amplify stochastic fluctuations in protein concentration into a larger asymmetry. To test whether singularity could be guaranteed by the amplification mechanism we re-wired yeast to employ a synthetic positive feedback mechanism. The re-wired cells could establish polarity, however they occasionally made two buds simultaneously, suggesting that singularity is guaranteed by the amplification mechanism.
Item Open Access Cell-cycle control of cell polarity in yeast.(The Journal of cell biology, 2019-01) Moran, Kyle D; Kang, Hui; Araujo, Ana V; Zyla, Trevin R; Saito, Koji; Tsygankov, Denis; Lew, Daniel JIn 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.Item Open Access Chemotropism and Cell-Cell Fusion in Fungi.(Microbiology and molecular biology reviews : MMBR, 2022-02-09) Clark-Cotton, Manuella R; Jacobs, Katherine C; Lew, Daniel JFungi 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.Item Open Access Cultivating PhD Aspirations during College.(CBE life sciences education, 2022-06) Jones, Daniela S; Gillette, Devyn D; Cooper, Paige E; Salinas, Raquel Y; Hill, Jennifer L; Black, Sherilynn J; Lew, Daniel J; Canelas, Dorian AScience, 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.Item Open Access Cytoskeletal Dynamics and the Temporal Control of Yeast Morphogenesis(2012) Chen, HsinThe cells of the budding yeast Saccharomyces cerevisiae undergo a robust morphological cycle, involving reorganization of the actin cytoskeleton, septin ring formation, and polarized growth. These events are crucial to the formation of a fully-equipped and properly-shaped bud, which gives rise to the daughter cell. The budding yeast, as a well-established genetic model system, has attracted numerous investigations aimed at uncovering the underlying principles of morphogenesis.
Despite the important roles of the septin ring and collar in morphogenesis and cytokinesis, little is known about how they are assembled. We found that septins are recruited to the ring and collar following a tri-linear assembly/disassembly scheme.
Polarization of actin cables enable directed secretion and growth. The formin Bni1p, an actin nucleator, is thought to polarize actin cables in response to the direct regulation by the master polarity regulator, Cdc42p. However, we found that all the known Bni1p-regulatory pathways are dispensable, including the direct regulation by Cdc42p, and we uncovered a novel pathway linking Bni1p to Cdc42p via the Cdc42p effector, Gic2p.
Yeast morphogenesis is tightly coupled with the cell cycle. Contrary to the prevailing model, we found that G1-CDK activity, albeit required for bud emergence, is not needed to trigger polarization. This finding suggests that cells are in a default polarized state, which is negatively regulated by the G2-CDK.
Item Open Access Experimentally informed bottom-up model of yeast polarity suggests how single cells respond to chemical gradients(2021) Ghose, DebrajHow do single cells—like neutrophils, amoebae, neurons, yeast, etc.—grow or move in a directed fashion in response to spatial chemical gradients? To address this question, we used the mating response in the budding yeast, Saccharomyces cerevisiae, as a biological model. To mate, pairs of yeast cells orient their cell fronts toward each other and fuse. Each cell relies on a pheromone gradient established by its partner to orient correctly. The ability for cells to resolve gradients is striking, because each cell is only ~5 μm wide and is thought to be operating in complex and noisy environments. Interestingly, mating pairs of cells often start out not facing each other. When this happens, the front of each cell—defined by a patch of cortical polarity proteins—undergoes a series of erratic and random movements along the cell cortex till it ‘finds’ the mating partner’s patch. We sought to understand how polarity patches in misaligned cells find each other. To this end, we first characterized patch movement in cells by the distribution of their step-lengths and turning angles and analyzed a bottom-up model of the polarity patch’s dynamics. The final version of our model combines 11 reaction-diffusion equations representing polarity protein dynamics with a stochastic module representing vesicle trafficking on a plane with periodic boundary conditions. We found that the model could not quantitatively reproduce step-length and turning angle distributions, which suggested that some mechanisms driving patch movement may not be present in the model. Incorpo-rating biologically inspired features into the model—such as focused vesicle delivery, sudden fluctuations in vesicle delivery rates, and the presence of polarity inhibitors on vesicles—allowed us to quantitatively match the in vivo polarity patch’s behavior. We then introduced a pathway, which connects pheromone sensing to polarity, to see how the model behaved when exposed to pheromone gradients. Concurrently, we analyzed the behavior of fluores-cently labeled polarity patches in mating pairs of cells. We discovered that the ~1 μm wide patch could (remarkably) sense and bias its movement up pheromone gradients, a result corroborated by our model. Further analysis of the model revealed that while the polarity patch tends to bias the location of a cluster of pheromone-sensing-receptors, the receptors can transform an external pheromone distribution into a peaked non-linear “polarity-activation” profile that “pulls” the patch. Stochastic perturbations cause the patch to “ping-pong” around the activation-profile. In a gradient of pheromone, this ping-ponging be-comes biased, leading to net patch movement up the gradient. We speculate that such a mechanism could be used by single cells with mobile fronts to track chemical gradients.
Item Open Access Exploratory polarization facilitates mating partner selection in Saccharomyces cerevisiae.(Molecular biology of the cell, 2021-05) Clark-Cotton, Manuella R; Henderson, Nicholas T; Pablo, Michael; Ghose, Debraj; Elston, Timothy C; Lew, Daniel JYeast decode pheromone gradients to locate mating partners, providing a model for chemotropism. How yeast polarize toward a single partner in crowded environments is unclear. Initially, cells often polarize in unproductive directions, but then they relocate the polarity site until two partners' polarity sites align, whereupon the cells "commit" to each other by stabilizing polarity to promote fusion. Here we address the role of the early mobile polarity sites. We found that commitment by either partner failed if just one partner was defective in generating, orienting, or stabilizing its mobile polarity sites. Mobile polarity sites were enriched for pheromone receptors and G proteins, and we suggest that such sites engage in an exploratory search of the local pheromone landscape, stabilizing only when they detect elevated pheromone levels. Mobile polarity sites were also enriched for pheromone secretion factors, and simulations suggest that only focal secretion at polarity sites would produce high pheromone concentrations at the partner's polarity site, triggering commitment.Item Unknown From Polarity to Morphogenesis PAK Behaviors and Mechanism for Bud Sensing in Morphogenesis Checkpoint(2016) Kang, HuiBud formation by Saccharomyces cerevisiae is a fundamental process for yeast proliferation. Bud emergence is initiated by the polarization of the cytoskeleton, leading to local secretory vesicle delivery and gulcan synthase activity. The master regulator of polarity establishment is a small Rho-family GTPase – Cdc42. Cdc42 forms a clustered patch at the incipient budding site in late G1 and mediates downstream events which lead to bud emergence. Cdc42 promotes morphogenesis via its various effectors. PAKs (p21-activated kinases) are important Cdc42 effectors which mediate actin cytoskeleton polarization and septin filament assembly. The PAKs Cla4 and Ste20 share common binding domains for GTP-Cdc42 and they are partially redundant in function. However, we found that Cla4 and Ste20 behaved differently during the polarization and this depended on their different membrane interaction domains. Also, Cla4 and Ste20 compete for a limited number of binding sites at the polarity patch during bud emergence. These results suggest that PAKs may be differentially regulated during polarity establishment.
Morphogenesis of yeast must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site prior to bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor”. Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed, and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.
Item Unknown How cells determine the number of polarity sites.(eLife, 2021-04-26) Chiou, Jian-Geng; Moran, Kyle D; Lew, Daniel JThe 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.Item Open Access How Diffusion Impacts Cortical Protein Distribution in Yeasts.(Cells, 2020-04-30) Moran, Kyle D; Lew, Daniel JProteins associated with the yeast plasma membrane often accumulate asymmetrically within the plane of the membrane. Asymmetric accumulation is thought to underlie diverse processes, including polarized growth, stress sensing, and aging. Here, we review our evolving understanding of how cells achieve asymmetric distributions of membrane proteins despite the anticipated dissipative effects of diffusion, and highlight recent findings suggesting that differential diffusion is exploited to create, rather than dissipate, asymmetry. We also highlight open questions about diffusion in yeast plasma membranes that remain unsolved.Item Open Access How Yeast Cells Find Their Mates(2019) Henderson, Nicholas TrubianoExtracellular chemical gradients provide signals that guide a broad spectrum of different cellular processes. By accurately sensing and responding to chemical gradients, immune cells can chase down invading pathogens, sperm cells can locate a distant egg, and growing axons can form connections in the developing nervous system. Haploid cells of the budding yeast Saccharomyces cerevisiae grow up gradients of pheromone in order to locate and fuse with nearby mating partners. Gradient sensing should be challenging for yeast, because they must detect a minute difference in concentration across their small cell bodies. Nevertheless, yeast cells can orient with remarkable accuracy in shallow pheromone gradients. Several mechanisms have been proposed to explain how yeast cells locate their partners, but it remains unclear whether, and to what degree each of the proposed mechanisms contributes.
We imaged fluorescent polarity probes in real time during mating events and found that cells located their partners in a multi-step process. First, cells placed a weak cluster of polarity proteins in approximately the correct location, despite a previously unappreciated challenge posed by asymmetrically distributed pheromone receptors. Cells were able to overcome receptor asymmetry by sensing the ratio, rather than the number, of active pheromone receptors. Second, the polarity cluster proceeded to move erratically around the cortex during an “indecisive phase.” We found evidence that cells switched to a local sensing mechanism during the indecisive phase, wherein cells used the mobile polarity cluster like a nose to search for areas where the local pheromone concentration was high. Third, the polarity cluster stabilized adjacent to a partner cell’s cluster and remained stationary until the partners met in the middle and fused.
Item Open Access Imaging Polarization in Budding Yeast.(Methods Mol Biol, 2016) McClure, Allison W; Wu, Chi-Fang; Johnson, Sam A; Lew, Daniel JWe describe methods for live-cell imaging of yeast cells that we have exploited to image yeast polarity establishment. As a rare event occurring on a fast time-scale, imaging polarization involves a trade-off between spatiotemporal resolution and long-term imaging without excessive phototoxicity. By synchronizing cells in a way that increases resistance to photodamage, we discovered unexpected aspects of polarization including transient intermediates with more than one polarity cluster, oscillatory clustering of polarity factors, and mobile "wandering" polarity sites.Item Open Access Inhibitory GEF phosphorylation provides negative feedback in the yeast polarity circuit(Current Biology, 2014) Kuo, Chun-Chen; Savage, Natasha S; Chen, Hsin; Wu, Chi-Fang; Zyla, Trevin R; Lew, Daniel JCell polarity is critical for the form and function of many cell types. During polarity establishment, cells define a cortical "front" that behaves differently from the rest of the cortex. The front accumulates high levels of the active form of a polarity-determining Rho-family GTPase (Cdc42, Rac, or Rop) that then orients cytoskeletal elements through various effectors to generate the polarized morphology appropriate to the particular cell type [1, 2]. GTPase accumulation is thought to involve positive feedback, such that active GTPase promotes further delivery and/or activation of more GTPase in its vicinity [3]. Recent studies suggest that once a front forms, the concentration of polarity factors at the front can increase and decrease periodically, first clustering the factors at the cortex and then dispersing them back to the cytoplasm [4-7]. Such oscillatory behavior implies the presence of negative feedback in the polarity circuit [8], but the mechanism of negative feedback was not known. Here we show that, in the budding yeast Saccharomyces cerevisiae, the catalytic activity of the Cdc42-directed GEF is inhibited by Cdc42-stimulated effector kinases, thus providing negative feedback. We further show that replacing the GEF with a phosphosite mutant GEF abolishes oscillations and leads to the accumulation of excess GTP-Cdc42 and other polarity factors at the front. These findings reveal a mechanism for negative feedback and suggest that the function of negative feedback via GEF inhibition is to buffer the level of Cdc42 at the polarity site. © 2014 Elsevier Ltd.Item Open Access Mating and Marital Fidelity in Saccharomyces cerevisiae(2023) Robertson, Corrina GHaploid cells of the budding yeast Saccharomyces cerevisiae communicate using secreted pheromones and mate to form diploid zygotes. Mating is monogamous, resulting in the fusion of precisely one cell of each mating type. Monogamous mating in crowded conditions, where cells have access to more than one potential partner, raises the question of how multiple-mating outcomes are prevented. Here we identify mutants capable of mating with multiple partners, revealing the mechanisms that ensure monogamous mating. Before fusion, cells develop polarity sites oriented toward potential partners. Competition between these polarity sites within each cell leads to disassembly of all but one focus, thus favoring a single fusion event. Fusion promotes the formation of heterodimeric complexes between subunits that are uniquely expressed in each mating type. One complex shuts off haploid-specific gene expression, and the other shuts off the ability to respond to pheromone. Zygotes able to form either complex remain monogamous, but zygotes lacking both can re-mate.Guidance of cell growth or movement in response to chemical cues in the environment is critical for many cell behaviors. Budding yeast orientation of polarized growth in response to gradients of mating pheromones provides a tractable model to address how cells accurately assess small spatial differences in chemical concentrations. Pheromones bind to receptors that act through heterotrimeric G proteins to promote activation of the MAPK Fus3. Active Fus3 binds to Gα, which is thought to enhance local phosphorylation of relevant MAPK substrates to promote orientation of polarity towards high-pheromone regions. Polarity is oriented by a pathway in which Gβγ binds the scaffold protein Far1 to activate the conserved polarity regulator Cdc42, which activates the formin Bni1 to orient actin and hence growth. Gβγ, Far1, and Bni1 are all MAPK substrates whose phosphorylation could improve orientation towards high-pheromone regions. Here we show that the Gα-MAPK interaction can enhance the efficiency of polarity site alignment between mating partners, although the magnitude of that effect depends on context. Surprisingly, however, we find no evidence that phosphorylation of Gβγ, Far1, or Bni1 contribute to the benefit conferred by Gα-MAPK interaction. The role of this interaction remains mysterious.
Item Open Access Mating in wild yeast: delayed interest in sex after spore germination.(Molecular biology of the cell, 2018-12) McClure, Allison W; Jacobs, Katherine C; Zyla, Trevin R; Lew, Daniel JStudies of laboratory strains of Saccharomyces cerevisiae have uncovered signaling pathways involved in mating, including information-processing strategies to optimize decisions to mate or to bud. However, lab strains are heterothallic (unable to self-mate), while wild yeast are homothallic. And while mating of lab strains is studied using cycling haploid cells, mating of wild yeast is thought to involve germinating spores. Thus, it was unclear whether lab strategies would be appropriate in the wild. Here, we have investigated the behavior of several yeast strains derived from wild isolates. Following germination, these strains displayed large differences in their propensity to mate or to enter the cell cycle. The variable interest in sex following germination was correlated with differences in pheromone production, which were due to both cis- and trans-acting factors. Our findings suggest that yeast spores germinating in the wild may often enter the cell cycle and form microcolonies before engaging in mating.Item Open Access Mechanisms for Controlling Cell Polarity in Yeast(2022) Moran, Kyle DonovanCell polarity is critical for essential functions in many types of cells. Rho-family GTPases are master regulators of cell polarity. Thus, mechanisms for controlling the activity of Rho GTPases are of both academic interest and practical concern. While much has been discovered about regulation of Rho GTPase activity by partners like GEFs, GAPs, and GDIs, there are still many things which remain unclear about how their behavior is enforced in cells of various kinds.
The budding yeast, Saccharomyces cerevisiae, has long been a model for studying cell polarity. Its Rho GTPase Cdc42 is responsible for defining a single polarity site for the purpose of either mating with a single partner or making a single bud. Other types of yeasts, however, can generate multiple polarity sites utilizing the same core polarity machinery. What are the rules which allow for such differences in behavior while using a similar set of proteins? We highlight key design principles established in mathematical models for Rho GTPase polarity machineries and show that they apply in budding yeast cells: strains featuring specific genetic perturbations which increase the total amount of polarity proteins can go from making one bud, to making multiple buds.
Bud emergence in yeast is enabled by cell cycle activity in G1. It is known that bud emergence also requires cytoskeletal changes which are orchestrated by Cdc42 and its effectors. How are these changes coordinated with cell cycle progression? It seems likely that G1 CDK activity regulates many aspects of Cdc42 polarization. We use live-cell fluorescence microscopy to reveal one such avenue whereby input from the cell cycle is required for many Cdc42 effector proteins to localize to sites with active Cdc42, thus restricting bud formation until the time is right.
Item Open Access Mechanisms of Cdc42 Polarization in Yeast(2016) Woods, Benjamin LeePolarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.
Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.
While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.