Regulation of Cytoplasmic Dynein

项目总结 这项研究项目的长期目标是了解基于微管(MT)的马达 细胞质动力蛋白(“动力蛋白”)是受调节的。动力蛋白是所有细胞骨架马达中最大、最复杂的； 这种&gt；1丙二醛二聚体包含许多机械元件，它们的运动必须是 在惊人的长分子距离上进行协调，以实现沿MTS的前进运动。除了它的 巨大的体型，动力蛋白也是最通用的分子马达；与45个运动蛋白形成鲜明对比 和39个肌球蛋白存在于人类中，一个动力蛋白基因产物负责运输 神经元内的大分子，构建有丝分裂纺锤体，极化细胞，并在 发展。为了赋予动力蛋白发挥其多种作用所必需的功能可塑性， 无处不在的辅助因子与动力蛋白相互作用，包括Lis1和动力蛋白复合体。这个项目将应用我们的 结合生物化学、单分子生物物理学和冷冻电子显微镜的专业知识来解决 Dynein与MTS相互作用的结构和机制基础及其受Lis1和Dynactin的调节。 我们最近发现，Dynein与MTS的结合伴随着其MT-1的构象变化。 结合结构域和Lis1起到一个“离合器”的作用，使MT结合解离并从ATP水解中释放， 推动一种强烈依附于MT的状态。动力蛋白，一种1.2丙二醛复合体，增强动力蛋白的处理能力，是 几乎是细胞中所有动力蛋白功能所必需的，但其作用机制尚不清楚。这笔赠款将 解决有关动力蛋白及其调节的主要机制问题。什么是结构性和机械性的 MT结合的基础(目标1)，以及由Lis1(目标2)和动力蛋白(目标3)调节的基础？

PROJECT SUMMARY 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 of 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)?