Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Mark Winey

Second Advisor

Ken Krauter

Third Advisor

Gia Voeltz

Fourth Advisor

Tin Tin Su

Fifth Advisor

Jeffrey Moore

Abstract

Centrosomes are essential components of the cell-cycle machinery of eukaryotic cells; dysfunction of centrosomes is thus linked to cell-cycle misregulation, aneuploidy and tumorigenesis. A critical role of centrosomes is microtubule nucleation, and is mediated by γ-tubulin complexes to form the mitotic spindle required for chromosome segregation. The conserved yeast γ-tubulin linker, Spc110 (pericentrin/kendrin in humans) is essential to tether γ-tubulin complexes to the nuclear plaque of the SPB to nucleate microtubules necessary for spindle formation. Despite the clear importance of the γ-tubulin tether Spc110 in the nucleation of microtubules, its assembly to SPBs remains unclear.

The aim of this study is to determine the mechanism by which the C-terminus of Spc110 localizes and assembles into the nuclear side of the SPB where it functions to tether γ-tubulin complexes that nucleate spindle microtubules. In the current work, I have identified the Spc110 C-terminal domain residues required for localization to SPBs by live-cell fluorescence microscopy. I also have determined direct interaction between Spc110 and central plaque components, Spc29 and Spc42 by identifying the necessary C-terminal residues required for binding via yeast two-hybrid analysis. Additionally, I have established that overexpression of Spc110 C-terminus is toxic and impedes SPB duplication by sequestering Spc42, a component of the satellite essential for duplication. I propose a model for Spc110 assembly whereby Spc110 is recruited to the central plaque of the SPB by binding Spc29 through the highly conserved internal region in Spc110 C-terminus. In addition, I propose the far C-terminal domain of Spc110 binds Spc42 at the SPB central plaque, acting as a spacer to tightly lock Spc110 to the SPB. Spc110 C-terminus interactions with Spc29 and Spc42 thus function collaboratively to assemble and localize Spc110 into the SPB central plaque. This in turn allows Spc110 to be securely locked at the SPB to tether γ-tubulin complexes necessary for mitotic spindle formation and chromosome segregation.

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