Biophysical Principles of Microtubule Dynamics

项目摘要 微管生物学的生物物理学原理 微管是细胞分裂、细胞运动和细胞内运输所必需的细胞骨架聚合物， 与许多癌症和神经系统疾病有关。微管网络的重塑 空间和时间的结构依赖于单个微管在不同时间段之间切换的能力。 生长和收缩，称为“微管动态不稳定性”的行为。虽然发现了超过 30年前，微管动力学不稳定性及其调控的分子机制仍在很大程度上 未知这个问题被一个复杂且高度互连的微管网络所加剧- 相关蛋白，共同调节细胞内微管动力学。这个项目的目标是 提供微管行为及其调节的基本分子理解。完成 为了实现这一目标，我们将采用生物学和物理学相结合的跨学科方法。灵感来自概念 使用细胞生物学工具开发的模型，我们将采用纯化的生物化学体外重建， 蛋白质组分、单分子全内荧光（TIRF）成像和微流体技术 以获得微管系统行为的定量描述。微管长度分布为 由微管的生长速率、收缩速率和从微管的收缩到微管的转变速率的组合决定。 从增长到收缩（灾难）再回到（拯救）。然而，这四个参数只是一种表现形式 定义宏观参数值及其相互作用的潜在分子机制 关系。我们将解决微管生长和灾难之间的耦合的关键问题， 探索以前无法实现的实验制度，对于严格测试现有和 建立新的理论模型。我们将对微管负端进行详细的测量， 动力学，迄今为止在很大程度上仍然没有研究，尽管最近被认为是积极的监管 在细胞内。我们将确定微管拯救的分子机制， 动态不稳定性的参数，这在相间微管结构中起着重要作用。我们 将用纯化的微管蛋白以及微管相关蛋白的集合进行测量 （包括末端结合EB蛋白、TOG结构域XMAP 215和CLASP蛋白以及驱动蛋白马达蛋白） 来自驱动蛋白-13和-14家族）以阐明它们对微管行为的集体作用。基于 在这种定量表征的基础上，我们将开发预测性数学模型， 微管动力学及其调节的基本原理。我们的定量模型的预测 将在细胞内进行测试。这种理论和实验的结合将提供基本的洞察力， 微管系统的调节，最终奠定了人类健康的新进展的基础。

PROJECT SUMMARY BIOPHYSICAL PRINCIPLES OF MICROTUBULE DYNAMICS Microtubules are cytoskeletal polymers essential for cell division, cell motility and intracellular transport, and are implicated in many cancers and neurological disorders. Remodeling of the microtubule network architecture in space and time relies on the ability of individual microtubules to switch between periods of growth and shrinkage, behavior known as `microtubule dynamic instability'. Although discovered more than thirty years ago, the molecular mechanisms of microtubule dynamic instability and its regulation remain largely unknown. This problem is exacerbated by a complex and highly interconnected network of microtubule- associated proteins, which collectively regulate microtubule dynamics inside of cells. The goal of this project is to provide a fundamental molecular understanding of microtubule behavior and its regulation. To accomplish this goal, we will use an interdisciplinary approach, combining biology and physics. Inspired by conceptual models developed using cell biological tools, we will employ biochemical in vitro reconstitution with purified protein components, single-molecule total-internal-fluorescence (TIRF) imaging, and microfluidics techniques to obtain a quantitative description of the microtubule system behavior. Microtubule length distributions are determined by the combination of microtubule growth rates, shrinkage rates and the rates of transitions from growth to shrinkage (catastrophe) and back (rescue). However, these four parameters are only a manifestation of the underlying molecular mechanisms that define the macroscopic parameter values and their mutual relationships. We will address the key question of the coupling between microtubule growth and catastrophe by probing previously unattainable experimental regimes, critical for rigorous testing of existing and the development of new theoretical models. We will perform detailed measurements of the microtubule minus end dynamics, which to date remains largely unstudied, although recently recognized to be actively regulated inside cells. We will determine the molecular mechanisms of microtubule rescue, least well understood parameter of dynamic instability, which plays an important role in interphase microtubule architecture. Our measurements will be performed with purified tubulin, as well as ensembles of microtubule-associated proteins (including end-binding EB proteins, TOG-domain XMAP215 and CLASP proteins, and kinesin motor proteins from Kinesin-13 and -14 families) in order to elucidate their collective effects on microtubule behavior. Based on this quantitative characterization, we will develop predictive mathematical models that will capture the fundamental principles of microtubule dynamics and its regulation. The predictions of our quantitative models will be tested inside cells. This combination of theory and experiment will provide fundamental insight into microtubule system regulation, ultimately laying the foundation for new advances in human health.