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The Kinetochore Protein Mif2p is Targeted by Cdk1p and Development of a Selection for Regulators of Centromere/Kinetochore Structure/Function

Abstract

ABSTRACT OF THE DISSERTATION

The Kinetochore Protein Mif2p is Targeted by Cdk1p and Development of a Selection for Regulators of Centromere/Kinetochore Structure/Function

by

Isha Kimisha Wallace

Doctor of Philosophy, Graduate Program in Cell, Molecular and Developmental Biology

University of California, Riverside, March 2010

Dr. Jeffrey Bachant, Chairperson

Chromosome segregation is a critical determinant of cell survival and genome stability. Defects in chromosome segregation lead to aneuploidy, a hallmark of cancer cells. Kinetochores, built specifically on a unique centromeric region on each chromosome, are large protein complexes that mediate chromosome attachment to the mitotic spindle. Interactions between kinetochores and spindle microtubules power chromosome movements during chromosome segregation. Kinetochore-spindle interactions are also monitored by checkpoint and error correction pathways that ensure replicated chromosomes attach and align on the spindle properly. Genetic screens and biochemical assays have revealed that in the budding yeast S. cerevisiae, the kinetochore is comprised of 80+ proteins grouped into several subcomplexes. Collectively, these proteins function together to assemble the kinetochore, to mediate chromosome attachment and force generation, and to control spindle checkpoints.

The central focus of the work in this dissertation is the conserved, essential kinetochore protein Mif2p, which is homologous to the mammalian protein CENP-C. Previous studies suggest that Mif2p may help build the foundation of the kinetochore and serve as a linker between inner and central kinetochore complexes. Here, we describe a novel Cdk1p-dependent phosphorylation event on Mif2p; Cdk1p is the master cell cycle regulatory kinase that catalyzes entry into and progression through mitosis. My evidence that Mif2p is a novel Cdk1p substrate includes the following observations: (1) Mif2p displays an electrophoretic mobility shift on western blots that can be collapsed with phosphatase treatment. This indicates that the electrophoretic mobility shift represents a phosphorylated Mif2p species; (2) the Mif2p phosphorylation shift is cell cycle regulated and coincides with periods in which mitotic forms of Cdk1p are known to be active; (3) the Mif2p phosphorylation shift is dependent on Cdk1p activity. The mutant Cdk1p alleles cdc28-4 and cdc28-1N, and inhibition of the analogue-sensitive cdc28-as1 allele, all abolished the Mif2p phosphorylation shift observed on western blots; (4) mutation of the six Cdk1p consensus phosphorylation sites in Mif2p to non-phosphorylatable alanine residues abolished the phosphorylation shift as well; and (5) a Mif2 peptide containing five of the six Cdk1p consensus phosphorylation sites can serve as in vitro substrate of Clb2-Cdk1. Interestingly, I observed that interactions between the outer kinetochore Dam1/DASH complex and spindle microtubules are required for Cdk1p to target Mif2p. In the context of previous observations, this leads me to hypothesize that Cdk1p targeting of Mif2p may be part of a regulatory mechanism that converts initial interactions between kinetochores and microtubules into stable, end-on attachments.

The secondary focus of the work presented here is the development of a genetic selection for factors influencing centromere/kinetochore chromatin structure. This strategy is conceptually distinct from the genetic approaches used to identify most other kinetochore proteins, which have relied on chromosome loss as a primary screening criterion. To accomplish this, I describe the modification a technique, originally developed by Phil Hieter and colleagues, called the centromere transcription read-through assay. In this assay, a centromere is placed between the strongly inducible GAL promoter and a LacZ reporter gene. In the presence of galactose, the centromere/kinetochore protein complex impedes LacZ transcription. In contrast, mutants defective in centromere/kinetochore structure allow increased LacZ expression. We have modified this assay by replacing LacZ with yeast nutritional markers, permitting selection of strains that allow transcription through the centromere region. Thus, the rationale is that by selecting for a centromere transcription read-though phenotype one can potentially identify a broad range of factors that are controlling centromere chromatin structure and function. I describe my success in establishing this assay as a genetic selection technique as well as some problems I encountered. Potential strategies to overcome these problems and move forward in implementing this promising approach will also be discussed. Unexpectedly, this approach also revealed a possible epigenetic aspect to centromere specification. While epigenetics plays a well-defined role in the specification of centromeres of higher eukaryotes, there is only limited evidence to suggest that budding yeast centromere specification also involves an epigenetic aspect.

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