Mechanisms of Mitotic Spindle Disassembly and Positioning in Saccharomyces cerevisiae

When a cell divides, it must accurately replicate its genetic material and then faithfully segregate this material into the resulting daughter cells. My research addresses the latter half of this problem, focusing on how the cell regulates the function of the mitotic spindle, an elegant microtubule-based machine that attaches to replicated DNA and pulls it apart during mitosis. Here, I present two studies that investigate how the cell disassembles the mitotic spindle at the end of mitosis and how the cell positions the mitotic spindle prior to mitotic completion.I combined genetic analysis with live-cell fluorescence microscopy to identify the subprocesses driving spindle disassembly as well as the proteins that perform these subprocesses. Our results suggest that mechanistically distinct pathways largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8 cooperate to drive spindle disassembly in budding yeast. We also describe the roles of novel disassembly factors such as the spindle protein She1 and the 7-protein Alternative Replication Factor C complex. Together, these pathways disengage the mitotic spindle halves, inhibit spindle microtubule growth, and promote sustained spindle microtubule depolymerization. Strikingly, combined inhibition of pairs of disassembly pathways yielded cells with hyper-stable spindle remnants, which caused dramatic defects in cell cycle progression, thus establishing that regulated and rapid spindle disassembly is crucial for cell proliferation.To better understand the mechanisms of spindle positioning, I examined how the dynein-driven spindle-positioning pathway in budding yeast is silenced. My work suggests that dynein activity is regulated through interaction with the multi-subunit dynactin complex at anaphase and identifies a new cellular factor, She1, which controls this interaction. Dynactin is a well-known dynein activator, and, in budding yeast, the complete complex is required for dynein-dependent spindle movement. I found that localization of the dynactin complex is cell cycle-regulated, such that dynactin is recruited to astral microtubules, via interaction with dynein, primarily during anaphase. Additionally, we discovered that the protein She1 is a cell cycle-regulated inhibitor of dynein activity. Without She1, dynein activity extends beyond anaphase and, as a result, mis-positions the mitotic spindle. Strikingly, loss of She1 also permits recruitment of the dynactin complex to astral microtubules throughout the cell cycle. These results suggest that in wild-type cells, She1 restricts dynein activity to anaphase by preventing the interaction between dynein and the complete dynactin complex.