Date of Degree

6-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Amy Ikui

Committee Members

Mara Schvarzstein

Hualin Zhong

Frederick Cross

Xiaolan Zhao

Subject Categories

Biology | Cell Biology | Laboratory and Basic Science Research

Keywords

cell cycle, phosphatase, budding yeast, mitosis

Abstract

Eukaryotic cell division is an essential process that is carried out by the cell cycle, a tightly controlled process that has been extensively studied in the budding yeast Saccharomyces cerevisiae. The cell cycle is driven by Cyclin Dependent Kinase (Cdk1) activity. Protein phosphatase 2A-Cdc55 (PP2ACdc55) reverses Cdk1 phosphorylation events during late stages of the cell cycle to ensure the correct order of events. This thesis presents evidence that the anaphase inhibitor Pds1 is a PP2ACdc55 target. Pds1 binds to and inhibits separase (Esp1). Esp1 triggers sister chromatid segregation by cleaving the cohesin complex that holds the chromatids together, and by promoting spindle elongation through a poorly understood mechanism. The Pds1-Esp1 physical interaction is dependent on Cdk1 phosphorylation at the Pds1 C-terminus. In addition to preventing Esp1 proteolytic activity, Pds1-Esp1 binding is also necessary for Esp1 accumulation in the nucleus. The findings presented in this study demonstrate that PP2ACdc55 directly dephosphorylates Pds1 at Cdk1 phosphorylation sites. Nuclear exclusion of PP2ACdc55 resulted in strengthened Pds1-Esp1 interaction and premature Pds1 nuclear accumulation. Findings from this study also showed that exclusion of PP2ACdc55 resulted in accelerated spindle elongation, and conversely, hypophosphorylated Pds1 was associated with unstable spindles. Thus, these findings show novel evidence that Pds1 phosphorylation status is linked to spindle elongation rate and stability. The significance of Pds1 phospho-regulation by PP2ACdc55 in stress conditions is also examined. The Pds1-Esp1 interaction is crucial for maintaining genome integrity, which can be compromised by genotoxic stress such as DNA replication stress. This study shows that both nuclear and cytoplasmic PP2ACdc55 are involved in the cell cycle response to DNA replication stress. PP2A-dependent Pds1 dephosphorylation and spindle inhibition is enhanced during replication stress. This study also examines the possibility that PP2ACdc55 acts independently of known replication stress response pathways. Lastly, this thesis explores a role for PP2ACdc55 in the cell’s response to a loss of DNA replication control, as well as a functional interaction between PP2ACdc55 and the replication protein Cdc6. DNA re-replication triggers cell cycle arrest, which is shown to be dependent on nuclear PP2ACdc55 function. In summary, this study enhances the current understanding of how PP2ACdc55 controls cell cycle processes, including anaphase spindle elongation and checkpoint responses to threats to genome stability.

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