Septins are widely conserved among eukaryotes and implicated in a variety of cellular processes, although their precise molecular functions remain unknown. In every organism so far examined, multiple septin proteins are found in hetero-oligomeric complexes, the proper organization of which is required for their function. Mutation or misregulation that upsets the stoichiometry of septin hetero-oligomers is a common feature of septin-associated human diseases, which include cancer and hereditary neuropathies. Septin-containing cellular structures in dividing and differentiating cells are dynamic, undergoing abrupt changes in organization in a temporally and spatially regulated manner. The recent high-resolution structures of a human septin hexamer and a budding yeast octamer reveal the striking similarity of the constituent subunits and the interfaces by which they assemble. 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. In this regard, the yeast septins represent a simple model system with which to identify cellular mechanisms regulating the structural organization of these multi- subunit macromolecular assemblies. Experiments supported by this award will examine how individual septin proteins are used in different assemblies, and how covalent modifications and septin-associated factors influence the kinetics of higher-order septin assembly. These studies adapt SNAP-tag technology for labeling of yeast proteins, allowing individual septins to be marked with a variety of functional tags and tracked through cell division and differentiation. Yeast septins are thought to form filaments at the mother-bud neck, but there has been no direct test of whether and how septins are organized in these structures. Experiments directly testing a model based on the structure of septin octamers aim to resolve this long-standing question. Finally, the same SNAP-Tag approach used to study septin dynamics will be applied to another dynamic assembly (myosin II-based filaments), to determine the generality of the new mechanistic insights revealed by the proposed research. Together, this work will lay the foundation for a long- term direction of study as an independent investigator.
Although their molecular functions are not understood, it appears that the proper organization of different septin proteinss 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.
