Browsing by Subject "Paclitaxel"

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    Multiscale modeling and analysis of microtubule self-assembly dynamics
    (2014-08) Castle, Brian Thomas
    Microtubules are dynamic biopolymers that self-assemble from individual subunits of αβ-tubulin. Self-assembly dynamics are characterized by stochastic switching between extended phases of growth and shortening, termed dynamic instability. Cellular processes, including the chromosome segregation during mitosis and the proper partitioning of intracellular proteins, are dependent on the dynamic nature of microtubule assembly, which facilitates rapid reorganization and efficient exploration of cellular volume. Microtubule-targeting chemotherapeutic agents, used to treat a wide range of cancer types, bind directly to tubulin subunits and suppress dynamic instability, ultimately impeding the capacity to complete cellular processes. Microscale length changes observed during dynamic instability are the net-effect of the addition and loss of individual subunits, dictated by the interdimer molecular interactions. Therefore, a multiscale approach is necessary to extrapolate submolecular level effects of microtubule-targeting agents to dynamic instability. The work presented in this dissertation integrates multiscale computational modeling and experimental observations with the goal of better understanding the functional mechanisms of microtubule-targeting agents. First, we develop a computational model for the association and dissociation of tubulin subunits, in which the interdimer interaction potentials are specifically simulated. Simulation results indicate that the local polymer end structure sterically inhibits subunit association as much as an order of magnitude. Additionally, the model informs how microtubule-targeting agents could alter assembly dynamics through the properties of the interdimer interactions. Second, the mechanisms of kinetic stabilization by microtubule-targeting agents are tested and constrained by combining predictions from a computational model for microtubule self-assembly and experimental observations in mammalian cells. We find that assembly- and disassembly-promoting agents induce kinetic stabilization via separate mechanisms. One is a true kinetic stabilization, in which the kinetic rates of subunit addition and loss are reduced 10- to 100-fold, while the other is a pseudo-kinetic stabilization, dependent upon mass action of tubulin subunits between polymer and solution. Overall, this work advances our knowledge of the basic physical principles underlying multistranded polymer self-assembly and can inform the future design and development of more effective and tolerable microtubule-targeting drugs.
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    UNC-45A is a Novel Microtubule Destabilizing Protein and Regulator of Paclitaxel Sensitivity in Ovarian Cancer
    (2019-05) Mooneyham, Ashley
    UNC-45A is a highly-conserved member of the UCS protein family that has important roles in regulating cytoskeletal-associated functions in invertebrates and mammalian cells including cytokinesis, exocytosis, cell motility, and neuronal development. Here we show for the first time that UNC-45A is a microtubule-associated protein (MAP) with microtubule (MT) destabilizing activity. Using in vitro biophysical reconstitution and TIRF microscopy analysis, we show that UNC-45A directly binds to taxol-stabilized microtubules in absence of any additional cellular cofactors and acts as an ATP-independent microtubule destabilizer. In cells, we show that UNC-45A binds to and destabilizes microtubules and its depletion causes severe defects in chromosome congression, segregation, and spindle polarity. We also show that UNC-45A is overexpressed in human specimens of chemoresistant ovarian cancer and that UNC-45A overexpressing, chemoresistant cells resist chromosome mis-segregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel. Lastly, we show that UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and increases sensitivity to paclitaxel. Taken together, our studies support the role of UNC-45A as a novel member of the MT destabilizing protein family and as a molecular target for paclitaxel-resistant human cancers.