Biophysical Principles of Microtubule Dynamics

微管动力学的生物物理原理

• 批准号: 10630506
• 负责人: Marija Zanic
• 金额: $ 3.36万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630506
• 关键词: Basic Science; Behavior; Binding; Biochemical; Biophysics; Cell Differentiation process; Cell division; Cell physiology; Cells; Computer Models; Cytoskeleton; Feedback; Goals; Growth; Health; Human; In Vitro; Individual; Investigation; Kinesin; Long-Term Effects; Malignant Neoplasms; Measurement; Microfluidics; Microtubules; Modeling; Molecular; Neurodegenerative Disorders; Neurodevelopmental Disorder; Phase; Physiological; Play; Polymers; Process; Proteins; Research; Resolution; Role; Tertiary Protein Structure; Testing; Time; Tubulin; Ursidae Family; base; cell motility; chemotherapeutic agent; experience; experimental study; human disease; in silico; insight; interdisciplinary approach; light microscopy; live cell imaging; mathematical model; neuron development; reconstitution; spatiotemporal; stathmin

PROJECT SUMMARY Dynamic remodeling of the microtubule cytoskeleton is crucial for a variety of cellular processes, including cell division, cell motility and differentiation. Microtubule cytoskeleton reorganization relies on the control of individual microtubule polymers, which switch between phases of growth and shrinkage through a process known as microtubule dynamic instability. Although dynamic instability was discovered decades ago, the molecular mechanisms that underlie microtubule catastrophe and rescue, the transitions between phases of growth and shrinkage, and their control through collective effects of a myriad of regulators are still being unraveled. The goal of this project is to elucidate the fundamental mechanisms underlying microtubule dynamics. Our central hypothesis is that conditions experienced at the time of growth have long-term effects on subsequent microtubule behavior, including catastrophe, shrinkage and rescue. To test this hypothesis, we will employ highly-controlled in vitro reconstitution experiments, combining purified protein components, microfluidics and high spatiotemporal resolution light-microscopy approaches. We will determine the different impacts of distinct growth conditions at the two microtubule ends, giving rise to their unique dynamic behaviors. We will elucidate individual and combined effects of microtubule regulators and their underlying mechanisms. We will particularly focus on microtubule regulators that bind both soluble and polymeric form of tubulin. At the plus end, we will investigate TOG-domain proteins XMAP215 and CLASP to elucidate the similarities and differences in their mechanisms underlying their differential effects on plus-end dynamics. At the minus end, we will investigate the interplay of stabilizing regulators, including Kinesin-14 HSET, and destabilizing regulators, including tubulin-sequestering protein Op18/Stathmin and a poorly-studied microtubule severing protein Fidgetin. Since every one of these microtubule regulators has been implicated in human disease, particularly cancer and neurodevelopmental disorders, revealing their mechanisms of action is of direct health relevance. Our quantitative in vitro measurements will enable us to develop mathematical and computational models reconciling the dynamics of both microtubule ends, and encompassing the collective effects of regulators at each end. We will directly test the models developed based on our in vitro and in silico findings in physiologically-relevant contexts using state-of-the-art fast super-resolution quantitative live cell imaging. Beyond uncovering the fundamental mechanisms underlying microtubule dynamics in cells, we will expand our cellular studies with a focus on the role of CLASP in cell migration and neuronal development. Our cellular investigations will invariably yield new hypotheses to be tested by controlled in vitro and in silico experiments. The continuous feedback between in vitro and cellular approaches will ultimately provide fundamental insights into microtubule cytoskeleton dynamics, bearing critical relevance to both basic science and human health.

项目概要 微管细胞骨架的动态重塑对于多种细胞过程至关重要，包括细胞 分裂、细胞运动和分化。微管细胞骨架重组依赖于 单个微管聚合物，通过一个过程在生长和收缩阶段之间切换 称为微管动态不稳定性。尽管动态不稳定性在几十年前就被发现了， 微管灾难和救援的分子机制，阶段之间的转变 增长和萎缩，以及通过无数监管机构的集体效应对其进行控制仍在进行中。 解开。该项目的目标是阐明微管的基本机制 动力学。我们的中心假设是，成长时经历的条件会产生长期影响 影响随后的微管行为，包括灾难、收缩和救援。为了检验这个假设，我们 将采用高度控制的体外重构实验，结合纯化的蛋白质成分， 微流体和高时空分辨率光学显微镜方法。我们将确定不同的 两个微管末端不同生长条件的影响，导致其独特的动态行为。 我们将阐明微管调节剂的个体和组合效应及其潜在机制。 我们将特别关注结合可溶性和聚合形式的微管蛋白的微管调节剂。在 最后，我们将研究 TOG 结构域蛋白 XMAP215 和 CLASP，以阐明它们的相似性和 它们对正端动态产生不同影响的机制差异。在负端， 我们将研究稳定调节因子（包括 Kinesin-14 HSET）和不稳定调节因子之间的相互作用 调节因子，包括微管蛋白隔离蛋白 Op18/Stathmin 和研究较少的微管 切断蛋白质 Fidgetin。由于这些微管调节因子中的每一个都与人类的 疾病，特别是癌症和神经发育障碍，揭示其作用机制具有重要意义 直接的健康相关性。我们的定量体外测量将使我们能够开发数学和 计算模型协调微管两端的动力学，并涵盖 两端监管机构的集体效应。我们将直接测试基于我们的体外开发的模型 并使用最先进的快速超分辨率定量技术在生理相关环境中进行计算机模拟研究 活细胞成像。除了揭示细胞微管动力学的基本机制之外， 我们将扩大我们的细胞研究，重点关注 CLASP 在细胞迁移和神经元中的作用 发展。我们的细胞研究总是会产生新的假设，并通过受控的体外测试进行测试 以及计算机模拟实验。体外和细胞方法之间的持续反馈将 最终提供对微管细胞骨架动力学的基本见解，与 基础科学和人类健康。