Regulation of Cytoplasmic Dynein

描述(由申请人提供)：本研究项目的长期目标是了解基于微管(MT)的运动胞质动力蛋白(“动力蛋白”)是如何调节的。动力蛋白是所有细胞骨架马达中最大和最复杂的；这种&gt；1丙二醛二聚体复合体包含许多机械元件，它们的运动必须在惊人的长分子距离上协调才能实现沿MTS的前进运动。除了其巨大的体积，动力蛋白也是最多功能的分子马达；与人类存在的45个动力蛋白和39个肌球蛋白形成鲜明对比的是，一个动力蛋白基因产物负责在神经元内运输大分子，构建有丝分裂纺锤体，极化细胞，并在发育过程中锚定mRNA。为了赋予dynein执行其多种功能所必需的功能可塑性，几个普遍存在的辅助因子与dynein相互作用，包括Lis1 和动力蛋白复合体。这个项目将应用我们在生物化学、单分子生物物理学和冷冻电子显微镜方面的综合专业知识来研究Dynein与MTS相互作用的结构和机制基础，以及它被Lis1和Dynactin调节的结构和机制。我们最近发现，Dynein与MTS的结合伴随着其MT结合域的构象变化，而Lis1则扮演着解偶联MT结合并从ATP水解中释放的“离合器”的角色，促进了一种强烈的MT结合状态。动力蛋白是一种1.2丙二醛复合体，可增强动力蛋白的加工能力，是细胞中几乎所有动力蛋白功能所必需的，但其作用机制尚不清楚。这笔拨款将解决有关动力蛋白及其调控的主要机制问题。MT结合(目标1)以及Lis1(目标2)和dynactin(目标3)调节的结构和机制基础是什么？

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand how the microtubule (MT)-based motor cytoplasmic dynein ("dynein") is regulated. Dynein is the largest and most complex of all cytoskeletal motors; this >1 MDa dimeric complex contains numerous mechanical elements whose movements must be coordinated over strikingly long molecular distances to achieve processive motility along MTs. In addition to its enormous size, dynein is also the most versatile of the molecular motors; in sharp contrast to the 45 kinesins and 39 myosins present in humans, a single dynein gene product is responsible for transporting macromolecules within neurons, constructing the mitotic spindle, polarizing cells, and anchoring mRNAs during development. To give dynein the functional plasticity necessary for carrying out its many roles, several ubiquitous co-factors interact with dynein, including Lis1 and the dynactin complex. This project will apply our combined expertise in biochemistry, single-molecule biophysics and cryo-electron microscopy to address the structural and mechanistic bases of dynein's interaction with MTs and its regulation by Lis1 and dynactin. We recently showed that binding of dynein to MTs is accompanied by conformational changes in its MT- binding domain and that Lis1 acts as a "clutch" to uncouple MT binding and release from ATP hydrolysis, promoting a strongly MT-attached state. Dynactin, a 1.2 MDa complex, enhances dynein's processivity and is required for nearly all dynein functions in cells, but its mechanism o action is poorly understood. This grant will address major mechanistic questions about dynein and its regulation. What are the structural and mechanistic bases of MT binding (Aim 1), and of regulation by Lis1 (Aim 2) and dynactin (Aim 3)?