Kinetochore Structural Plasticity and its Regulation in Saccharomyces cerevisiae

Dhatchinamoorthy, Karthik (2018). Kinetochore Structural Plasticity and its Regulation in Saccharomyces cerevisiae. PhD thesis The Open University.



The kinetochore is a multi-protein complex that connects the chromosome with microtubule and pulls the chromosome during chromosome segregation. In yeast, a single kinetochore assembles on a Cse4 nucleosome and attaches to a single microtubule, making it an ideal system for studying the architecture of kinetochore-microtubule interaction, function, and regulation. The inner kinetochore assembles on the centromere and anchors the outer kinetochore that attaches to the dynamic microtubule. The interplay between the kinetochore structure and microtubule dynamics in a living cell has not been studied.

I have used a combination of in-house developed quantitative microscopy, genetic techniques and mathematical simulation in the budding yeast, S.cerevisiae, to elucidate a novel structural transition of the kinetochore. During anaphase, the microtubule undergoes rapid depolymerization. Kinetochore tracking of microtubule during this phase is critical for chromosome segregation, but how this tracking is achieved is not well understood. A combination of FRAP and photoconversion on kinetochore proteins have revealed that the kinetochore consists of highly dynamic sub-modules and stable sub-modules. I show that microtubule dynamics significantly affect the dynamic kinetochore sub-modules but not the stable ones. Mathematical modeling of microtubule dynamics and kinetochore attachment has revealed the importance of kinetochore structural transition for microtubule attachment. In addition, I have discovered that kinetochore structural transitions are regulated by known metaphase-anaphase specific pathways.

Scm3 is the chaperone responsible for assembling the centromeric nucleosome that contains a specific histone variant Cse4. I helped to identify and characterize a new chaperone for Cse4. I demonstrated that recombinant yCAF-1 can promote the assembly of Cse4 nucleosomes in vitro and at non-centromeric positions, causing problems for chromatin-based processes.

In summary, my thesis work has significantly contributed to our understanding of centromere and kinetochore function in budding yeast. I speculate that many of the findings will inform our perspectives on centromere and kinetochore function in higher eukaryotes.

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