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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2016

Month Degree Awarded

February

First Advisor

Thomas J. Maresca

Second Advisor

Wei-Lih Lee

Third Advisor

Jennifer L. Ross

Fourth Advisor

Patricia Wadsworth

Subject Categories

Cell Biology

Abstract

Kinetochores are large multiprotein structures through which chromosomes engage with the microtubules of the mitotic spindle. All kinetochore pairs must ultimately adopt a bioriented configuration, with their associated sister chromatids linked to opposite spindle poles and poised to segregate equally between two daughter cells. Erroneous, non-bioriented attachments that are left uncorrected lead to chromosome mis-segregation, producing aneuploid daughter cells with unequal numbers of chromosomes. Before anaphase onset, bioriented attachments are selectively stabilized whereas non-bioriented attachments remain unstable and are eliminated. This error correction process relies heavily on the extent of outer kinetochore phosphorylation by an Aurora B kinase activity centered at the inner centromere, between the two members of a kinetochore pair. Phosphorylation of Aurora B substrates at the outer kinetochore tends to destabilize kinetochore-microtubule attachments by lowering the affinity with which outer kinetochore proteins bind to spindle microtubules. Reduced phosphorylation, and attachment stabilization, are associated with the kinetochore’s capacity to stretch when placed under tension by the pulling force exerted by microtubule plus end dynamics. Such intrakinetochore stretch in response to mechanical tension generally occurs only at bioriented attachments.

This Dissertation examines kinetochore-microtubule attachment stabilization and error correction in Drosophila melanogaster. Experimental deletion of an extendible, structurally disordered segment of the Drosophila kinetochore protein CENP-C is found to be associated with attachment instability and an increased incidence of chromosome misalignment. That association can be explained in terms of reduced intrakinetochore stretch and an inability of the metaphase spindle to pull the outer kinetochore away from inner centromere-based Aurora B. On that view, selective stabilization of bioriented attachments in wild type cells depends on changes in the distance between the inner centromere and the kinetochore-microtubule binding interface. Separately, it is shown that erroneous, syntelic kinetochore-microtubule attachments can be stabilized, and the phosphorylation-based error correction system overridden, by overexpressing the Drosophila chromokinesin motor NOD. NOD overexpression artificially increases the magnitude of the polar ejection force that pushes chromosome arms away from the spindle poles. In turn, the elevated polar ejection force increases the tension experienced by syntelically attached kinetochore pairs, stabilizing what would ordinarily be a highly unstable form of attachment.

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Cell Biology Commons

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