The Mechanism and Regulation of Cytoplasmic and Ciliary Dyneins

细胞质和纤毛动力蛋白的机制和调控

• 批准号: 10133096
• 负责人: Ahmet Yildiz
• 金额: $ 61.73万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10133096
• 关键词: Adaptor Signaling Protein; Alzheimer' s Disease; Autophagosome; Behavior; Binding; Binding Sites; Cells; Chemicals; Cilia; Complex; Cryoelectron Microscopy; Development; Disease; Dynein ATPase; Exhibits; Future; Generations; Geometry; Goals; In Vitro; Intracellular Transport; Investigation; Kinesin; Knock-out; Lead; Liquid substance; Male Infertility; Microtubule-Associated Proteins; Microtubules; Mitochondria; Mitosis; Modeling; Molecular; Motor; Movement; Mutation; Neurobiology; Neuropathy; Pathogenesis; Physiological; Play; Primary Ciliary Dyskinesias; Recombinants; Regulation; Research; Role; Schizophrenia; Slide; Structure; System; Testing; Tetrahymena; Tubulin; Work; arm; biophysical analysis; cell motility; cilium motility; cofactor; dynactin; human disease; in vivo; inhibitor/antagonist; lissencephaly; molecular imaging; motor neuron degeneration; mutant; optical traps; predictive modeling; programs; reconstitution; retrograde transport; simulation; single molecule; success; tau Proteins; vesicle transport

Project Summary Dyneins are AAA+ motors responsible for minus-end-directed motility along microtubules (MTs) and play fundamental roles in cargo transport, mitosis, and ciliary beating. Dynein is currently the focus of the motor field as the mechanism of its movement is not well understood in comparison to plus-end-directed kinesins. Despite rapid transport of dynein-driven cargos in cells, previous in vitro studies identified mammalian dynein as a weak motor, exhibiting slow motility and producing lower forces than kinesin. Recently, in vitro reconstitution of the dynein-dynactin machinery revealed that mammalian dynein is autoinhibited when not transporting cargo, and motility is activated when dynein forms a 2.5 MDa ternary complex with its cofactor dynactin and a cargo binding adaptor. Therefore, all of the previous in vitro work on mammalian dynein used inactive motor and their conclusions do not reflect how active dynein-dynactin machinery transports cargos in cells. Our future goals are to dissect the mechanism of active cytoplasmic dynein complexes and determine how dynein activation and motility are regulated across multiple scales using single molecule imaging, optical trapping, MD simulations, and cryoEM. Specifically, we will determine how Lis1 plays a role in the activation of and regulation of dynein motility. We will also study dynein motility in physiologically relevant conditions and ask whether MT-associated proteins, MAP7 and Tau, inhibit dynein motility by sterically blocking its tubulin binding site or by excluding its MT binding via liquid-liquid demixing. We will also characterize the motility of dynein and dynactin disease mutants to reveal the molecular mechanism of neuropathies associated with these mutations. Finally, we will reconstitute the entire MT transport machinery using cargo adaptors identified by in vivo studies of mitochondria, autophagosomes, and vesicle transport, but not yet characterized in vitro. Using this approach, we will dissect how cargo adaptors regulate motors to control the bidirectional transport of these cargos. We will also study ciliary dyneins that slide parallel array of axonemal MTs to power ciliary beating. Several models have been proposed to explain how the sliding activity of dyneins is self-regulated to orchestrate ciliary oscillations. Predictions that these models make about the mechanism of ciliary dyneins have not been directly tested. Recently, a recombinant expression system was developed for Tetrahymena outer-arm dynein (OAD), enabling us to perform in-depth structural and biophysical studies of ciliary dyneins. Unlike cytoplasmic dynein, OAD forms a heterodimer and is not processive. Using this system, we will characterize the mechanism of OAD motility and force generation. We will then directly test the predictions of each model by constructing in vitro geometries that mimic dynein/MT interactions in a beating cilium. Finally, we will identify structural components that give rise to the nonprocessive motility, curvature sensing, and self-oscillatory behavior of OAD. The success of our research program will reveal the fundamental mechanochemistry of dynein and how it achieves retrograde transport of intracellular cargos and drives the self-coordinated oscillations of motile cilia.

项目摘要 动力蛋白是AAA+马达，负责沿着微管（MT）的负末端定向运动， 在货物运输、有丝分裂和纤毛跳动中的基本作用。动力蛋白是目前电机领域研究的热点 因为与正末端导向的驱动蛋白相比，其运动的机制还没有被很好地理解。尽管 在细胞中动力蛋白驱动的货物的快速运输，以前的体外研究确定哺乳动物动力蛋白是一种弱的 运动，表现出缓慢的运动性和产生比驱动蛋白低的力。最近，在体外重建的 动力蛋白-动力蛋白机制揭示了哺乳动物动力蛋白在不运输货物时是自抑制的， 当动力蛋白与其辅因子动力蛋白和货物结合蛋白形成2.5MDa三元复合物时，运动被激活。 适配器因此，之前所有关于哺乳动物动力蛋白的体外工作都使用了失活的马达， 结论并没有反映出主动动力蛋白-动力肌动蛋白机制如何在细胞中转运货物。 我们未来的目标是剖析活跃的细胞质动力蛋白复合物的机制，并确定如何 动力蛋白的激活和运动性使用单分子成像、光学 捕获、MD模拟和冷冻EM。具体来说，我们将确定Lis 1如何在激活 和动力蛋白运动的调节。我们还将研究动力蛋白在生理相关条件下的运动， MT相关蛋白MAP 7和Tau是否通过空间阻断其微管蛋白结合来抑制动力蛋白运动 位点或通过经由液-液分层排除其MT结合。我们还将描述动力蛋白的运动性， dynactin疾病突变体，以揭示与这些突变相关的神经病变的分子机制。 最后，我们将使用通过体内研究鉴定的货物衔接子重建整个MT转运机制 线粒体，自噬体和囊泡运输，但尚未在体外表征。使用这种方法， 我们将剖析货物适配器如何调节马达以控制这些货物的双向运输。 我们还将研究纤毛动力蛋白，滑动轴丝MT的平行阵列，以动力纤毛跳动。几 已经提出了一些模型来解释动力蛋白的滑动活动是如何自我调节的， 振荡这些模型对纤毛动力蛋白机制的预测还没有被直接证实。 测试.最近，开发了四膜虫外臂动力蛋白（OAD）的重组表达系统， 使我们能够对纤毛动力蛋白进行深入的结构和生物物理学研究。与细胞质动力蛋白不同， OAD形成异二聚体并且不进行性。利用这个系统，我们将描述OAD的机制 运动性和力的产生。然后，我们将通过体外构建直接测试每个模型的预测， 在跳动的纤毛中模拟动力蛋白/MT相互作用的几何形状。最后，我们将确定结构组件 引起OAD的非进行性运动、弯曲感测和自振荡行为。 我们的研究计划的成功将揭示动力蛋白的基本机械化学以及它是如何 实现细胞内货物的逆行运输并驱动运动纤毛的自协调振荡。