In eukaryotes, different cell morphologies are generated by fine-tuning the spatiotemporal regulation of the polarized growth machinery. Studying hyphal growth in the multimorphic opportunistic yeast pathogen Candida albicans provides a unique opportunity to understand how highly polarized cell structures are generated and maintained, and has the potential to provide insight into mechanisms of pathogenesis. Hyphal cell morphology requires that polarized growth machinery be held at hyphal tips over extreme distances and through multiple cell cycles. Deletion of the bud-site selection GTPase Rsr1 in C. albicans results in defects in cell size and shape not observed in studies of its ortholog in the related yeast Saccharomyces cerevisiae. This suggests that, in addition to its role in bud site selection, Rsr1 has expanded function in C. albicans, which impacts polarized growth and the generation of the hyphal morphology. Here, I show that loss of Rsr1 results in changes to a hyphal-specific tip structure, the Spitzenkörper, a downstream developmental indicator of Cdc42 signaling, and key regulator of polarized growth. Also, my results show that Rsr1's function impacts the spatiotemporal distribution of Bem1 a marker of the active form of Cdc42. Interestingly, the changes in the distribution of Cdc42 activity are also correlated with reduced expression of the hyphal transcriptional program. In addition, I also show the differential effects of the guanine nucleotide binding states of Rsr1, through the manipulation of the Rsr1 GAP, Bud2, and GEF, Bud5. Through the action of Bud2, Rsr1-GDP acts as a global inhibitor that limits competitive, stochastically-activated clusters of Cdc42, and also as a lateral inhibitor of growth at hyphal tips that strongly influences the overall width of the hypha. In contrast, Rsr1-GTP, through Bud5 activity, is needed to efficiently nucleate single clusters of Cdc42 activity during hyphal emergence, and also contributes to the extremely narrow morphology of the hypha. Altogether, the data presented here suggest that Rsr1 cycling supports a conservative mechanism of polarization that optimizes the efficiency with which polarization occurs, which is required for the maintenance of polarized growth.
University of Minnesota Ph.D. dissertation. August 2014. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisors: Dr. Cheryl Gale and Dr. Judith Berman. 1 computer file (PDF) ; ix, 117 pages, appendix A.
A conservative mechanism of polarization drives hyphal growth in the opportunistic yeast pathogen Candida albicans.
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