Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Mark Winey

Second Advisor

Tin Tin Su

Third Advisor

Gia Voeltz

Fourth Advisor

Greg Odorizzi

Fifth Advisor

Robert Sclafani

Abstract

Centrosomes serve as the main microtubule–organizing centers in many eukaryotic cells. Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and requires control by protein kinases, including cyclin–dependent kinases (Cdks). However, an understanding of the mechanisms by which centrosome duplication is regulated and, in particular, restricted to once per cell cycle is still lacking. Sfi1, a conserved component of centrosomes, is a phospho–protein that is a key candidate for establishing the site of new centrosome assembly in the budding yeast Saccharomyces cerevisiae, likely via domain–specific functions. I have examined the role of phosphorylation of the Sfi1 amino (N) and carboxy (C) termini at the spindle pole body (SPB), the budding yeast centrosome. I have established that a nonphosphorylatable sfi1 N–terminal allele shows genetic interactions with protein–encoding genes of the new SPB precursor. This suggests that these proteins are promising candidates as physical interactors of the Sfi1 N terminus and may be recruited by the Sfi1 N terminus for assembly of the new SPB during duplication. I additionally propose that this step of new SPB assembly requires the protein kinase Mps1. Importantly, I also have identified Sfi1 as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. I found that reducing Cdk1 phosphorylation by changing Sfi1 C–terminal phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated SPBs and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. My findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, I provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1. This work could provide crucial insight into the initiation of centrosome duplication.

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