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.

项目摘要 微管细胞骨架的动态重塑对于多种细胞过程至关重要，包括细胞凋亡， 分裂、细胞运动和分化。微管细胞骨架重组依赖于 单个的微管聚合物，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。虽然动力不稳定性在几十年前就被发现了， 微管灾难和拯救的分子机制，微管阶段之间的转换， 增长和收缩，以及通过无数监管机构的集体效应对其进行控制， 解开。本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，在增长时经历的条件具有长期影响 对随后的微管行为，包括灾难，收缩和救援。为了验证这个假设，我们 将采用高度受控的体外重建实验，结合纯化的蛋白质组分， 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 在两个微管末端不同的生长条件的影响，引起他们独特的动态行为。 我们将阐明微管调节剂的单独和联合作用及其潜在机制。 我们将特别关注结合微管蛋白的可溶性和聚合形式的微管调节剂。在 另外，我们将研究TOG结构域蛋白XMAP 215和CLASP，以阐明其相似性， 它们对加端动态的不同影响的机制差异。在负端， 我们将研究包括驱动蛋白-14 HSET在内的稳定调节因子与不稳定调节因子之间的相互作用。 调节剂，包括微管蛋白螯合蛋白Op 18/Stathmin和一种研究不足的微管 切断Fidgetin蛋白。由于这些微管调节因子中的每一种都与人类的 疾病，特别是癌症和神经发育障碍，揭示其作用机制是 与健康直接相关。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，并涵盖了 监管机构在每一端的集体效应。我们将直接测试基于我们的体外模型开发的模型 以及使用最先进的快速超分辨率定量 活细胞成像除了揭示细胞中微管动力学的基本机制外， 我们将扩大我们的细胞研究，重点是CLASP在细胞迁移和神经元迁移中的作用。 发展我们的细胞研究总是会产生新的假设，并通过体外对照进行测试 和计算机实验。体外和细胞方法之间的持续反馈将 最终提供微管细胞骨架动力学的基本见解， 基础科学和人类健康。