Coombes, Courtney2018-09-212018-09-212016-07https://hdl.handle.net/11299/200213University of Minnesota Ph.D. dissertation July 2016. Major: Biology. Advisor: Melissa Gardner. 1 computer file (PDF); vii, 116 pages.Microtubules are long hollow filaments that have the striking ability to restructure themselves. This is accomplished via a behavior termed “dynamic instability”, which involves stochastic transitions between growing and shortening at microtubule ends. They are also subject to post-translational modifications including acetylation. By performing biochemical assays, fluorescence and electron microscopy experiments, and computational simulations, we found that both acetylation and changes in dynamics are dependent on the microtubule structure itself. Acetylation is unique from other post-translational modifications in that it is the only modification which takes place in the hollow lumen of the microtubule. It is known that the α-tubulin acetyltransferase αTAT1 is responsible for the majority of the microtubule acetylation that is observed in cells. However, the mechanism for how αTAT1 accesses the Lys40 acetylation site inside of the microtubule lumen is not fully understood. We found that the microtubule α-tubulin acetylation rate is limited by accessibility of the enzyme to the lumen, and that αTAT1 preferentially targets tapered microtubule ends with exposed Lys40 acetylation sites. These results provide important insights into the mechanism for αTAT1 microtubule acetylation and its dependence on the microtubule structure. Tapering at microtubule ends may also be playing a role in microtubule dynamics. The dynamics at microtubule ends are characterized by periods of slow growth, followed by stochastic switching events termed “catastrophes”, in which microtubules suddenly undergo rapid shortening. The mechanistic basis of catastrophe is not known. To investigate microtubule catastrophe events, we performed 3D mechanochemical simulations that account for interactions between neighboring protofilaments. We found that there are two separate factors which contribute to catastrophe events in the 3D simulation: the GTP-Tubulin cap size and the structure of the microtubule tip. Importantly, 3D simulations predict, and both fluorescence and electron microscopy experiments confirm, that microtubule tips become more tapered as the microtubule grows. This effect destabilizes the tip and ultimately contributes to microtubule catastrophe. Thus, the likelihood of a catastrophe event may be intimately linked to the aging physical structure of the growing microtubule tip.enacetylationmicrotubuleThesis or Dissertation