Browsing by Subject "Microtubule"

Now showing 1 - 5 of 5
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    Microtubule Sub-Structure and its Role in Protein Binding
    (2018-07) Reid, Taylor
    Microtubules are structural polymers that participate in a wide range of cellular functions. The microtubule binding protein EB1 localizes to the growing ends of microtubules, where it facilitates interactions of key cellular proteins with the microtubule plus-end. Recent work has demonstrated that microtubule plus-ends have open, tapered conformations, which diverge greatly from a closed tube conformation. Thus, in this work we explored whether microtubule structure could impact the binding of EB1 to microtubules. Using quantitative fluorescence and electron microcopy experiments, we found that EB1 preferentially binds structurally disrupted or open structural features of microtubules as compared to the closed microtubule lattice. In corresponding 3D single- molecule diffusion simulations, a 70-fold rise in EB1 on-rates to tapered microtubule tip structures was observed relative to a closed lattice conformation, due to a high steric hindrance barrier that impedes EB1 from binding in its four-tubulin pocket-like lattice site, with greatly increased accessibility on two-tubulin protofilament edges at tapered microtubule ends. Thus, EB1’s four-tubulin pocket-like binding site on the microtubule leads to microtubule structural recognition based on a steric-hindrance-mediated on- rate, which may allow the tapered tip structures that are typical at growing microtubule plus ends to assist in facilitating the rapid arrival of EB1 to the microtubule plus-end.
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    Modeling and analysis of microtubule-mediated chromosome transport during mitosis.
    (2008-08) Gardner, Melissa Klein
    During mitosis, dynamic arrays of kinetochore-associated microtubules (kMTs) and molecular motors are organized into a mitotic spindle that serves to accurately segregate chromosomes into daughter cells. Understanding the dynamics and organization of mitotic spindle components could ultimately apply to clinical applications, such as in cancer treatment, because of the centrality of the mitotic spindle in mediating cell mitosis. Computer simulation can provide a bridge between mitotic spindle phenotypes and the individual dynamic spindle components that produce these phenotypes. I have found that by simulating the dynamics of kMTs mediating chromosome segregation during mitosis, it is possible to build a model for their regulation which results in specific predictions for molecular functions within the mitotic spindle. Specifically, by simulating the dynamics of molecular motors and chromosomes relative to kMT dynamics, and by comparing these simulations to experiments using fluorescent proteins and cryo-electron tomography, major mechanisms regulating proper chromosome congression in yeast have been uncovered. I have shown (1) that tension generated via the stretch of chromosomes between sister kinetochores is important in regulating the proper separation of sister kinetochores during metaphase, and (2) that a molecular motor, specifically the Kinesin-5 molecular motor Cin8p, is responsible for mediating a gradient in kMT catastrophe frequency that is required for proper chromosome congression. Dynamic microtubule plus-ends are responsible for the proper segregation of chromosomes during mitosis, as well as for other critical cellular functions. By performing molecular-level Monte Carlo simulations of microtubule assembly and comparing these simulations to in vitro measurements of microtubule assembly, I have found that microtubule assembly at the nanoscale is highly variable. This result supports a model for microtubule dynamic instability in which there is exists a substantial and dynamic GTP-cap during microtubule assembly that is critical for microtubule growth.
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    Multi-Scale Modeling of Microtubule Dynamics and the Regulation by Microtubule-Targeting Agents
    (2020-01) Hemmat, Mahya
    Microtubules (MTs) serve to facilitate vital cellular functions, such as chromosome segregation during mitosis and synaptic plasticity. MTs self-assemble via “dynamic instability,” in which the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e., what is different between the GTP- and GDP-tubulin states that enables MT growth and shortening, respectively? More generally, MTs are a great example of a complex biological system with spatial and temporal scales ranging from atomistic interactions such as GTP hydrolysis to cell-level behavior such as response to MT dynamics during mitotic progression. To understand a complex biological system behavior, a key challenge is connecting together the vast range of theoretical frameworks across length- and time scales. At the same time, MT interactions with associated proteins and binding agents, such as chemotherapy drugs, can strongly affect this dynamic process through molecular mechanisms that remain to be elucidated. The work in this dissertation integrates multiscale computational modeling with high resolution experimental observations to understand the molecular mechanism underlying MT dynamic instability and the regulation of dynamics by a well-established microtubule-targeting agent (MTA), colchicine. First, we develop a multi-scale modeling framework in which molecular dynamics (MD) are performed to investigate the interaction potential energies of tubulin-tubulin heterodimers, then, those results will be incorporated into Brownian dynamics (BD) simulations to study the kinetics of dimers assembly into MT lattice, and finally, thermo-kinetic and mechanochemical modeling of MT assembly, with inputs from MD and BD simulations, provide an insight into individual MT dynamics and details about MT tip structures. The model results point to a nucleotide-independent lateral bond of ~4 kBT, a nucleotide-dependent longitudinal bond of ~9 and ~5 kBT (∆∆G_long^0≈ 4 kBT) for GTP- and GDP-dimers, respectively and a radial bending angle preference (~1.5 kBT) for GDP-dimers. Furthermore, the framework informs us on how a well-known MTA, colchicine, affects MT dynamics. We found that colchicine binds mainly to free tubulin and sub-stoichiometrically poisons the end of protofilaments (PFs) through a copolymerization mechanism by which tubulin-colchicine (TC) complexes reduce the affinity of the PF for further tubulin addition and reinforce tubulin-tubulin lateral bond, a mechanism entirely distinct from that of paclitaxel or vinblastine.. In summary, this dissertation advances our knowledge about the molecular mechanism that drives dynamic instability and its regulation by MTAs within the context of cellular biology through a multi-scale approach and can be used for the development of more effective cancer therapeutic agents.
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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.