Date

2019

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Molecular Biology

First Adviser

Cassimeris, Lynne

Abstract

Microtubules are cytoskeletal polymers that are essential to many fundamental cellular processes, regulating cell shape, transport, and proliferation. During mitosis, microtubules comprise the mitotic spindle, responsible for sister chromatid segregation. Because of their essential role in cell division, microtubules are a common target in cancer therapy, where microtubule-targeting agents (MTAs) prevent cell cycle progression to mitigate cancer cell proliferation. While MTAs are successful in clinical applications, severe side effects and the rise of drug resistant cancers has led to increasing interest in the mechanisms that govern cell cycle-dependent microtubule array organization and how cancer cells evade microtubule-based therapies. Research presented in this dissertation addresses the contributions of microtubule dynamics and microtubule destabilizing proteins in array reorganization during mitotic entry.Microtubule dynamic instability governs microtubule turnover, as well as spatial and temporal organization. The parameters of dynamic instability have been measured in a wide variety of cell types and in response to cellular signals. Canonical wisdom suggests that changes to dynamic instability on the individual microtubule level are sufficient to alter the organization of the whole array. Monte Carlo simulations generated a macroscopic view of the dynamic microtubule cytoskeleton. The model revealed several features impacting interphase microtubule array organization, including the profound impact of total tubulin concentration and position of the cell boundary. Simulation of the interphase-prophase transition demonstrated that measured shifts in dynamics are sufficient to drive rapid interphase array disassembly, through reduced rescue frequency.Paclitaxel, an MTA and anti-cancer treatment, inhibits microtubule dynamics by promoting microtubule stability. Largely successful in clinical settings, paclitaxel blocks cells in mitosis, preventing cell division and inhibiting proliferation. Blocking at mitosis, with spindles of short microtubules, is surprising given paclitaxel’s microtubule stabilizing activity and that long interphase microtubules must depolymerize before spindle formation. We find that paclitaxel-treated cells progress through mitotic entry by clearing interphase microtubules beginning in the cell interior. This spatial pattern of reorganization differs from that of dynamic microtubule arrays. Research presented in this dissertation tests the hypothesis that microtubule severing and depolymerizing proteins function during mitotic entry to clear paclitaxel-stabilized interphase arrays to allow mitotic spindle assembly. We find that depletion of KIF18A, KIF18B, or spastin reduced microtubule loss in paclitaxel-treated cells at mitotic entry. Understanding the combined contribution of these proteins in clearing paclitaxel-stabilized interphase microtubules, as well as how they are specifically activated in the narrow window of time preceding mitotic entry, could yield potential drug targets that act synergistically with paclitaxel.

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