Investigation of the functions of the NDC80 kinetochore complex in budding yeast

• Main Author: Ma, Lina
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/29565

Kinetochores are multi protein complexes comprised of inner kinetochore proteins that assemble on centromeric DNA sequences and outer kinetochore proteins that bind to microtubules. Kinetochores are responsible for accurate transmission of genetic information during each cell division. In the budding yeast Saccharomyces cerevisiae, the highly conserved Ndc80 kinetochore complex has been well characterized in terms of its roles in chromosome-microtubule attachment and spindle checkpoint. The work presented in this thesis suggests that the Ndc80 complex has additional cellular roles. The kinetochore is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle in S phase is not well understood. In Chapter 2, I demonstrate that a mutation in Spc24, a component of the Ndc80 complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. I propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. In Chapter 3 and Chapter 4, I present data suggesting that the Ndc80 complex has a role in the cAMP/PKA glucose signaling pathway and actin regulation. Firstly, I identified genetic interactions between Spc24 and the components of cAMP/PKA pathway and demonstrated that spc24 mutants have defects in PKA signaling in response to glucose depletion. Interestingly, most of the temperature sensitive mutant alleles of the Ndc80 complex are rescued by non-glucose carbon sources. Finally, I present actin defects in spc24 mutants and genetic interactions between spc24 mutants and mutants of proteins involved in actin turnover, nucleation or polarity establishment. Further investigation on the mechanism and rationale behind the kinetochore-cytoskeleton interactions will be an intriguing avenue for future research.