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，包括细胞分裂、细胞周期、细胞周期和细胞周期。 运动和分化。微管细胞骨架重组依赖于对单个微管聚合物的控制， 其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。 尽管动力学不稳定性在几十年前就被发现了，但微管灾难的分子机制 和拯救，增长和收缩阶段之间的过渡，以及通过无数的集体效应对它们的控制。 监管机构的问题仍在解开。这个项目的目标是阐明基本的机制， 微管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。为了验证这一假设，我们将使用 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。我们将确定不同的生长条件的不同影响 在两个微管末端，引起它们独特的动力学行为。我们将阐明单个和组合效应 微管调节器及其潜在机制的研究。我们将特别关注微管调节剂， 微管蛋白的可溶和聚合形式。最后，我们将研究TOG结构域蛋白XMAP 215和CLASP 阐明其机制的相似性和差异性，其机制对正端动力学的不同影响。 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和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蛋白 烦躁不安。由于这些微管调节剂中的每一种都与人类疾病有关，特别是癌症， 神经发育障碍，揭示其作用机制是直接的健康相关性。我们的定量体外 测量将使我们能够开发数学和计算模型， 微管末端，并包括在每个末端的调节剂的集体效应。我们将直接测试模型 基于我们在生理学相关背景下的体外和计算机研究结果，使用最先进的快速超 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，重点是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，通过控制在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最终将为以下方面提供基本见解： 微管细胞骨架动力学与基础科学和人类健康密切相关。