Mechanisms of Spindle Disassembly and Microtubule-kinetochore Attachment in the Yeast Saccharomyces cerevisiae
- Author(s): Pigula, Adrianne Barber
- Advisor(s): Barnes, Georjana
- Drubin, David
- et al.
The timely regulation of spindle disassembly is essential for cells to coordinate mitotic exit with cytokinesis. In the budding yeast Saccharomyces cerevisiae, the microtubule-associated protein She1 functions in one of at least three parallel pathways that promote spindle disassembly. She1 phosphorylation by the Aurora kinase Ipl1 facilitates a role for She1 in late anaphase, when She1 contributes to the depolymerization of microtubules and shrinkage of spindle halves. By examining genetic interactions between known spindle disassembly genes, I identified components of the Hog (High Osmolarity Glycerol response) pathway, and found that three genes in this pathway, SHO1,PBS2, and HOG1, are necessary for proper localization of She1 to the anaphase spindle. Hog pathway mutants exhibited spindle disassembly defects, as well as mislocalization of the NoCut checkpoint proteins Boi1 and Boi2 from the bud neck. Moreover, Boi2, but not Boi1, plays a role in spindle disassembly that places it in a pathway with Sho1, Pbs2, and Hog1. A non-classical nuclear localization signal, the importin Nmd5, and exportin Crm1/Xpo1 - all necessary for Hog1 trafficking - are also necessary for proper She1 localization to the spindle and bud neck. Together, my data suggest a process by which cells monitor events at the spindle and bud neck, and describe a novel role for Hog pathway signaling in mitosis.
I also studied mechanisms by which spindle microtubules attach to kinetochores. I generated two new alleles of NDC80, a component of the essential Ndc80 complex, which tethers the outer kinetochore to spindle microtubules and participates in chromosome segregation. Deletions of an internal loop within the coiled-coil region of Ndc80 render cells temperature sensitive and unable to make stable microtubule-kinetochore attachments, resulting in metaphase arrest and DNA mis-segregation phenotypes. These alleles have been useful in helping us understand the required geometry of proteins in the outer kinetochore, and how protein-protein interactions promote the stable microtubule-kinetochore attachments necessary for high-fidelity chromosome segregation.