Browsing by Subject "microtubule"
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Item Microtubule-based control of glioma cell migration mechanics(2018-08) Prahl, LouisCell migration underlies the extensive tissue invasion that drives brain tumor (glioma) progression. Glioma cell migration involves the coordinated mechanical functions of the actin cytoskeleton, myosin motors, and substrate adhesions through a biophysical motor-clutch model. Although computational forms of the motor-clutch model predict glioma cell migration behaviors as a function of tissue stiffness, less is known about how other cellular structures such as microtubules influence migration. Presently, a number of microtubule-targeting agents (MTAs) are used to treat various cancers (including gliomas) so understanding their mechanism of action is necessary in order to develop better therapies. In this dissertation, I show that two commonly used MTAs (paclitaxel and vinblastine) each have distinct and nearly opposite effects on traction forces that motor-clutch simulations predict, and which correlate with changes to microtubule organization and dynamics. Effects of MTAs are consistent with influencing F-actin assembly and nucleation rates of protrusions, which impairs the ability of glioma cells to spontaneously polarize and migrate. Microtubule-dependent signaling networks that are perturbed in MTA-treated cells support novel roles for receptor tyrosine kinase (RTK) signaling pathways in mediating these effects. In the final study, we use microfabricated channels that replicate geometric and mechanical features of brain tissue alongside simulation-based methods to study confined glioma cell migration. Simulations recapitulate the dynamics of glioma cell migration in microchannels, as well as accurate predictions of the effects of MTAs and other pharmacological inhibitors of motor-clutch system components. This provides novel evidence for motor-clutch-based cell migration in confinement. In summary, this dissertation identifies specific mechanisms by which microtubules regulate motor-clutch based migration of glioma cells, and outlines a systems-level physics-based approach for understanding anti-motility therapy.Item Quantification and Analysis of Tau Protein Effects on Microtubule Dynamics in Mammalian (LLC-PK1) Cells(2020-07) Doersch, AlexandraOverall, we found that multiple biophysical properties of tau protein affect MT dynamics in LLC-PK1 cells; various isoforms exhibit differential effects. 2N4R tau exhibits MT tip avoidance (~200 nm) during growth that is lost for 0N4R tau. 2N4R and 0N4R tau phenocopy MTAs to make MTs less dynamic via different processes. We propose 2N4R tau has at least two binding sites, one of higher and one of lower affinity, resulting in KD=0.31 µM that preferentially associates with GDP-tubulin lattice to enable growing tip avoidance. 0N4R tau loses access to higher but retains lower affinity binding, resulting in KD=3.2 µM. Uniquely, 0N4R P301L tau does not bind to MTs (KD>>10 µM), indicating loss of both higher and lower affinity binding, perhaps due to induced conformation changes. Possible implications for tauopathies include the decreased ability of 0N4R P301L tau to bind to MTs, which may promote disease-associated progression toward tau oligomerization.Item The Role of Microtubule End Structure in Microtubule Acetylation and Dynamics(2016-07) Coombes, CourtneyMicrotubules 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.