Biophysical Principles of Microtubule Dynamics

项目总结 微管细胞骨架的动态重塑对多种细胞过程至关重要，包括细胞分裂、细胞 运动性和差异性。微管细胞骨架重组依赖于单个微管聚合物的控制， 它通过一个称为微管动态不稳定的过程在生长和收缩阶段之间切换。 尽管动态不稳定性在几十年前就被发现了，但微管灾难背后的分子机制 和救援，在增长和收缩阶段之间的过渡，以及通过无数集体效应对它们的控制 的监管机构仍在瓦解中。这个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，增长时所经历的条件具有长期影响。 对随后的微管行为的影响，包括灾难、收缩和救援。为了验证这一假设，我们将使用 高度可控的体外重建实验，结合纯化的蛋白质成分、微流控和高密度 时空分辨率光学显微镜方法。我们将确定不同增长条件的不同影响 在两个微管末端，产生了它们独特的动态行为。我们将阐明单独的和综合的影响 微管调节器及其潜在机制的研究。我们将特别关注结合微管调节器的 微管蛋白既有可溶性的，也有聚合性的。在正端，我们将研究TOG结构域蛋白XMAP215和CLASP 为了阐明它们在正端动力学中的不同作用机制的异同。 在负端，我们将研究稳定调节因子(包括kinesin-14 HSET)和不稳定调节因子的相互作用 调节因子，包括微管蛋白隔离蛋白OP18/stathmin和一个研究较少的微管切断蛋白 坐立不安。由于所有这些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，并包含每一端调节器的集体效应。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电子计算机研究结果，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 在细胞方面，我们将扩大我们的细胞研究，重点放在CLAP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说，以在体外和电子实验中进行对照检验。 体外和细胞方法之间的持续反馈最终将提供对 微管细胞骨架动力学，与基础科学和人类健康都具有关键的相关性。

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.