Members of the septin family of proteins are found in nearly every eukaryote, typically as hetero-oligomeric complexes, the proper organization of which is required for their function. Although the mechanistic details of their roles remain largely unknown, septins participate in a variety of cellular processes, generally involving plasma membrane remodeling. Mutation or misregulation that upsets the stoichiometry of septin heterooligomers is a common feature of septin-associated human diseases, which include cancer and hereditary neuropathies. In budding yeast, where septins were first identified, hetero-octamers - whose subunit composition is strikingly similar to those within human septin complexes - polymerize into filaments arrayed at sites of cell division and morphogenesis. Septin-containing cellular structures in dividing and differentiating cells are dynamic, undergoing abrupt changes in organization in a temporally and spatially regulated manner. It is not known how assemblies with the proper arrangement of septin subunits are built in the cell, or how they are reorganized during cycles of proliferation and development. The yeast septins represent an elegant and powerful system with which to identify cellular mechanisms regulating the organization of these multi-subunit macromolecular assemblies. Experiments supported by this award will examine how individual septin proteins are incorporated into distinct assemblies tailored to cellular demands, and how covalent modifications and septin-associated factors influence higher-order septin assembly. These studies exploit recently-developed tools for labeling of yeast proteins. Certain of these allow individual septins to be marked with a variety of functional tags and tracked through cell division and differentiation. Others allow visualization in situ with nanometer-scale resolution, and will be used to precisely detemiine the organization of septins within higher-order assemblies in vivo, and validated by a parallel method using ultra-high-resolution light microscopy. An genetic approach will be used to identify cellular factors controlling septin dynamics in vivo, with particular focus on Hsp104, a promising candidate for a novel treatment for diseases caused by septin misassembly.
Although their molecular functions are not understood, it appears that the proper organization of different septin proteins into multisubunit assemblies is critical. Indeed, a number of diseases (ranging from neuronal disorders to cancer) involve mutations in or misregulation of septin genes in ways that upset the balance of septin assembly. This work will identify the ways that yeast cells build and maintain their septin complexes.
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