Regulation of mitotic progression in Saccharomyces cerevisiae by the microtubule-associated proteins Slk19 and Stu1

Mitosis is the process by which eukaryotic cells segregate their chromosomes before division. A critical stage of mitosis is anaphase, when the microtubule-based spindle segregates chromosomes into mother and daughter cells prior to cytokinesis. My dissertation research aimed to provide a better understanding of the regulation of anaphase progression and spindle function during mitosis in Saccharomyces cerevisiae. My research focused on two microtubule-associated proteins, Slk19 (CENP-F homolog) and Stu1 (CLASP homolog). These proteins play fundamental roles in anaphase progression, Stu1 in microtubule spindle stability and Slk19 in spindle midzone organization and Cdc14 phosphatase regulation. I found that Stu1 and Slk19 physically interact and that Slk19 regulates Stu1 localization during anaphase. In addition to its interaction with Slk19, I identified a number of other physical and genetic interactors of Stu1 through mass spectrometry, yeast two-hybrid and synthetic genetic analyses. These interactors provide insight into the role of Stu1 at kinetochores. I also investigated Stu1 function through a protein truncation analysis and purification of full-length Stu1 protein. My truncation analysis revealed that the Stu1 C-terminus is dispensable for viability but is necessary for proper protein localization. The N-terminus, however, is essential for viability. My attempts to purify Stu1 from insect cells were partially successful; the protein is extremely sensitive to proteolytic degradation, and under conditions that limit proteolysis, the protein appears to aggregate or oligomerize in solution.I also investigated the role of Slk19 sumoylation in anaphase progression. I determined that the Cdc14 early anaphase release (FEAR) network protein Slk19 is sumoylated in vivo and that sumoylation is important for restricting Cdc14 phosphatase localization to the nucleus at the end of anaphase. A slk19 sumoylation mutant causes premature Cdc14 movement from the nucleus to the bud neck, which affects mitotic exit, as this slk19 sumoylation mutant can partially rescue the spindle disassembly defect of the mitotic exit network mutant cdc15-2. This slk19 mutant also has aberrant spindle elongation dynamics, which might be due to a change in Cdc14 function during anaphase. In conclusion, my dissertation research has uncovered a number of previously unrecognized interactions among mitotic proteins and has revealed a novel function of sumoylation in the regulation of Cdc14 function during anaphase through the FEAR network protein Slk19