Browsing by Subject "cell polarity"
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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 Decoding the Function of Ankyrin-B in Organelle Transport(2016) Qu, FangfeiOrganelle transport in eukaryotic cells is regulated by a precisely coordinated activity of phosphoinositide lipids, small GTPases, and molecular motors. Despite the extensive study of functional activities of individual regulators, how these activities promote precise deliveries of particular membrane proteins to specific cellular locations remained unclear. Ankyrin-B, which is previously well recognized as a plasma membrane adaptor that assembles diverse specialized plasma membrane domains, exhibited an unexpected role in intracellular transport. This thesis establishes ankyrin-B as a master integrator of the polarized long range organelle transport via direct interactions with Rab GTPase Activating Protein 1 Like (RabGAP1L), phosphatidylinositol 3-phosphate (PI3P) and dynactin 4. In Chapter 2, I identified an ankyrin-B death domain binding partner, RabGAP1L, that specifically interacts with ankyrin-B on intracellular organelles and requires ankyrin-B for its proper localization. In Chapter 3, I demonstrated that ankyrin-B is a PI3P-effector in mouse embryonic fibroblasts (MEFs) and promotes the polarized transport of associated organelles in migrating cells in a RabGAP1L-dependent manner. I continued to investigate what membranes/membrane-associated proteins utilize the ankyrin-B/RabGAP1L pathway in Chapter 4 and identified α5β1-integrin as a cargo whose polarized transport and recycling are ankyrin-B-dependent. I further presented that ankyrin-B, through recruiting RabGAP1L to PI3P-positive/Rab22A-associated endosomes containing α5β1-integrin, promotes polarized recycling of α5β1-integrin in migrating mouse embryonic fibroblasts. In collaboration with James Bear (UNC Chapel Hill), we further demonstrated that this ankyrin-B/RabGAP1L-mediated pathway is required for haptotaxis along fibronectin gradients. In Chapter 5, I elucidated the in vivo interaction between ankyrin-B and RabGAP1L. I demonstrated that ankyrin-B specifically interacts with RabGAP1L at long axon tracts in the brain and at costameres in the skeletal muscle. I also show the phenotypic analysis of ankyrin-B floxDD mice as an initial attempt to determine the physiological function of the ankyrin-B death domain in vivo. Together, this thesis dissects an ankyrin-B-mediated molecular mechanism for polarized endosomal trafficking and α5β1-integrin recycling during directional cell migration, and provides new insights into how phosphoinositide lipids, Rab GTPases, and molecular motor activities are coordinated to control the directional transport of specialized membrane cargos.
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 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.
Item Open Access Mechanisms of Chemotropism in Fungi: Saccharomyces cerevisiae as a Model(2021) Clark-Cotton, Manuella RossetteBudding yeast decode pheromone gradients to locate mating partners, providing a model of chemotropism in fungi. 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. Using live-cell fluorescence microscopy, computational modeling, and quantitative autocorrelation analyses, I address the role of the early mobile polarity sites, finding 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, suggesting 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 Open Access Regulation of cell polarity by the cell cycle in Saccharomyces cerevisiae(2019-05-22) Araujo, Ana V.Cell polarity in Saccharomyces cerevisiae is essential for bud formation, which is regulated by the cell cycle. How this regulation occurs is poorly understood. The master regulator of polarity is a Rho-GTPase called Cdc42, which accumulates at a region on the plasma membrane and recruits its downstream effectors and the cell’s cytoskeleton, leading to bud emergence. Previous work suggested that at a time in G1 called Start, the G1 CDK kinase promotes Cdc42 polarization. Recent findings have shown the opposite: Cdc42 is able to polarize prior to Start in daughter cells. Nevertheless, bud growth does not begin until after Start, which lead to the question: what exactly is this kinase regulating? One possibility is that G1 CDK regulates effectors of Cdc42. A partial survey of effectors showed that some were only able to polarize after the kinase activity increased. The aim of this study was to continue surveying effectors of Cdc42, focusing on Gic1 and Gic2. Confocal microscopy was used to obtain movies of yeast cells, which were analyzed using a customized MATLAB program. Gic1 polarization did not occur prior to Start, but Gic2 could polarize pre-Start in daughter cells. Future investigation into the structural difference between Gic1 and Gic2 in combination with that of other effectors may suggest potential ways that G1 CDK regulates effector localization.