This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Septin proteins assemble into a cytoskeletal polymer. Septin cytoskeleton is essential for cytokinesis, the last step in cell division. In budding yeast, filamentous rings of septins have been visualized at the bud neck and constrict it in order to separate the mother and daughter cells. We are using yeast septins that self-assemble in vitro. Mitotic Saccharomyces cerevisiae cells express five septins: Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7. All, but Shs1, are essential for cell division. We have determined the molecular organization of the four essential septins within the minimal complex by labeling techniques using electron microscopy. All of the septins but Cdc10 have a predicted coiled coil which promotes septin assembly into paired filaments. Octameric minimal subunits assemble into filaments when the ionic strength is lowered to 200 mM monovalent salt. Small angle x-ray scattering will be used on both wild type septins and on mutant septins deprived of coiled coils to be able to locate those flexible domains. In addition to that, using a stopped flow mixer, the kinetics of filament formation will be probed. Those kinetics experiments will be performed in the presence of various nucleotides so their effect on the polymerization process can be tested.
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