Biophysical Principles of Microtubule Dynamics

微管动力学的生物物理原理

• 批准号: 10796513
• 负责人: Marija Zanic
• 金额: $ 10.17万
• 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/10796513
• 关键词: Basic Science; Behavior; Binding; Biochemical; Biophysics; Cell Differentiation process; Cell division; Cell physiology; Cells; Computer Models; Cytoskeletal Modeling; Cytoskeleton; Feedback; Goals; Growth; Health; Human; In Vitro; Individual; Investigation; Kinesin; Long-Term Effects; Malignant Neoplasms; Measurement; Microfluidics; Microtubule Polymerization; Microtubules; Modeling; Molecular; Neurodegenerative Disorders; Neurodevelopmental Disorder; Phase; Physiological; Play; Polymers; Process; Proteins; Research; Resolution; Role; Tertiary Protein Structure; Testing; Time; Tubulin; cell motility; chemotherapeutic agent; experience; experimental study; human disease; in silico; insight; interdisciplinary approach; light microscopy; live cell imaging; mathematical model; neuron development; protein purification; reconstitution; spatiotemporal; stathmin; ultra high resolution

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 运动性和分化性。微管细胞骨架重组依赖于单个微管聚合物的控制， 它通过称为微管动态不稳定性的过程在生长和收缩阶段之间切换。 尽管几十年前就发现了动态不稳定性，但微管灾难背后的分子机制 和救援、增长和收缩阶段之间的转变，以及通过无数因素的集体效应对其进行控制 监管机构的监管仍有待厘清。该项目的目标是阐明潜在的基本机制 微管动力学。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。我们将确定不同生长条件的不同影响 在两个微管末端，产生其独特的动态行为。我们将阐明个体和综合效应 微管调节因子及其潜在机制。我们将特别关注结合的微管调节剂 微管蛋白的可溶性和聚合形式。最后，我们将研究 TOG 结构域蛋白 XMAP215 和 CLASP 阐明它们对正端动态产生不同影响的机制的相似性和差异。 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 和一种研究较少的微管切断蛋白 坐立不安。由于每一种微管调节因子都与人类疾病有关，特别是癌症和 神经发育障碍，揭示其作用机制与健康直接相关。我们的体外定量 测量将使我们能够开发数学和计算模型来协调两者的动态 微管末端，并包含两端调节因子的集体效应。我们将直接测试模型 基于我们在生理相关环境中的体外和计算机研究结果，使用最先进的快速超级技术开发 分辨率定量活细胞成像。除了揭示微管动力学的基本机制之外 在细胞中，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发育中的作用。 我们的细胞研究总是会产生新的假设，并通过受控的体外和计算机实验进行测试。 体外和细胞方法之间的持续反馈将最终提供基本的见解 微管细胞骨架动力学，与基础科学和人类健康密切相关。