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各种细胞过程至关重要，包括细胞 分裂，细胞运动和分化。微管细胞骨架重组取决于控制 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。尽管几十年前发现了动态不稳定，但 基于微管灾难和救援的分子机制，在 成长和收缩，以及通过无数监管机构的集体影响控制的控制 解开。该项目的目的是阐明微管基础的基本机制 动力学。我们的核心假设是，增长时经历的条件具有长期影响 在随后的微管行为上，包括灾难，收缩和救援。为了检验这一假设，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的轻微分辨率方法。我们将确定不同的 在两个微管末端的不同生长条件的影响，从而产生了它们独特的动态行为。 我们将阐明微管调节剂及其潜在机制的个体和联合效应。 我们将特别关注微管调节剂，这些调节剂均结合微管蛋白的可溶性和聚合物形式。在 另外，我们将研究tog-domain蛋白XMAP215并扣紧以阐明相似性和 其机制的差异是其对加号动力学影响的不同影响的基础。在负端， 我们将研究稳定调节剂的相互作用，包括驱动蛋白14 HSET，并破坏稳定 调节剂，包括微管蛋白序列蛋白OP18/Stathmin和研究不良的微管 切断的蛋白质坐立素。由于这些微管调节剂中的每一个都与人类有关 疾病，尤其是癌症和神经发育障碍，揭示其作用机制是 直接健康相关性。我们的定量体外测量将使我们能够发展数学和 计算模型调解了两个微管末端的动力学，并包括 监管机构两端的集体影响。我们将根据我们的体外直接测试开发的模型 以及使用最先进的快速超分辨率定量的在生理上与生理相关的环境中的硅酸盐发现中 活细胞成像。除了发现细胞中微管动力学的基本机制外， 我们将扩展我们的细胞研究，重点是扣子在细胞迁移和神经元中的作用 发展。我们的细胞研究将不可避免地产生新的假设，可以通过受控的体外进行测试 并在计算机实验中。体外和细胞方法之间的连续反馈将 最终为微管细胞骨架动力学提供基本见解，与 基础科学和人类健康。