Biophysical Principles of Microtubule Dynamics

微管动力学的生物物理原理

• 批准号: 9141607
• 负责人: Marija Zanic
• 金额: $ 39.25万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9141607
• 关键词: Address; Architecture; Back; Behavior; Binding; Biochemical; Biological; Biology; Biophysics; Cell division; Cells; Complex; Coupling; Cytoskeleton; Development; Family; Foundations; Goals; Growth; Health; Human; In Vitro; Individual; Interphase; Intracellular Transport; Kinesin; Length; Malignant Neoplasms; Measurement; Medical; Microfluidics; Microtubule-Associated Proteins; Microtubules; Minus End of the Microtubule; Modeling; Molecular; Motor; Neurodegenerative Disorders; Physics; Play; Polymers; Proteins; Regulation; Role; System; Techniques; Testing; Theoretical model; Time; Tubulin; base; cell motility; chemotherapeutic agent; fluorescence imaging; insight; interdisciplinary approach; mathematical model; nervous system disorder; network architecture; predictive modeling; reconstitution; research study; single molecule; theories; tool

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.

项目摘要 微管动力学的生物物理原理 微管是细胞分裂，细胞运动和细胞内转运必不可少的细胞骨架聚合物，并且 与许多癌症和神经系统疾病有关。微管网络的重塑 空间和时间的架构取决于单个微管在之间切换的能力 生长和收缩，被称为“微管动态不稳定性”的行为。虽然发现比 三十年前，微管动态不稳定性及其调节的分子机制在很大程度上保持 未知。微管的复杂且高度相互联系的网络加剧了这个问题 相关蛋白质，该蛋白集体调节细胞内部的微管动力学。这个项目的目标是 提供对微管行为及其调节的基本分子理解。完成 这个目标，我们将使用跨学科的方法，结合生物学和物理学。受概念的启发 使用细胞生物学工具开发的模型，我们将使用纯化的生化体外重构结构 蛋白质成分，单分子总内部荧光（TIRF）成像和微流体技术 获得微管系统行为的定量描述。微管长度分布是 通过微管增长率，收缩率和从 收缩的生长（灾难）和背部（救援）。但是，这四个参数只是表现形式 定义宏观参数值及其相互的基本分子机制 关系。我们将解决微管增长与灾难之间的耦合的关键问题 探测以前无法实现的实验制度，对于对现有和现有的严格测试至关重要 开发新的理论模型。我们将对微管负端进行详细测量 迄今为止的动态仍然很大程度上没有研究，尽管最近被认为是积极调节的 内部单元格。我们将确定微管救援的分子机制，最不理理解 动态不稳定性的参数，在相间微管体系结构中起重要作用。我们的 测量将用纯化的小管蛋白以及微管相关蛋白的集合进行 （包括末端结合EB蛋白，Tog-Domain XMAP215和扣蛋白以及驱动蛋白运动蛋白 从驱动蛋白13和-14家族中），以阐明它们对微管行为的集体影响。基于 在此定量表征上，我们将开发预测性数学模型，以捕获 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。理论和实验的这种结合将提供对 微管系统调节，最终为人类健康的新进步奠定了基础。