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，负责沿着微管的负端定向运动(MTS)和Play 在货物运输、有丝分裂和纤毛跳动中的基本作用。动力蛋白是目前汽车领域的研究热点 因为与正端导向的动蛋白相比，它的运动机制还不是很清楚。尽管 动力蛋白驱动的货物在细胞中的快速运输，先前的体外研究发现哺乳动物动力蛋白是一种弱的 马达的，表现出缓慢运动的，产生的力量比动蛋白小。最近，体外重建的 动力蛋白-动力蛋白机制表明，哺乳动物的动力蛋白在不运输货物时是自动抑制的，并且 当动力蛋白与辅因子动力蛋白和货物结合形成2.5-丙二醛三元络合物时，运动性被激活 适配器。因此，以前所有关于哺乳动物动力蛋白的体外工作都使用了非活动的运动和它们的 结论没有反映活跃的动力蛋白-动力蛋白机械如何在细胞内运输货物。 我们未来的目标是剖析活跃的细胞质动力蛋白复合体的机制并确定如何 使用单分子成像、光学技术在多个尺度上调节动力蛋白的激活和运动 捕获、MD模拟和冷冻EM。具体地说，我们将确定Lis1如何在激活 和对动力蛋白运动的调节。我们还将研究在生理相关条件下的动力蛋白运动，并询问 MT相关蛋白MAP7和Tau是否通过立体阻断其微管蛋白结合来抑制动力蛋白的运动 或通过液-液分离排除其MT结合。我们还将描述动力蛋白的运动性和 动力蛋白疾病突变，以揭示与这些突变相关的神经疾病的分子机制。 最后，我们将使用活体研究确定的货物适配器来重建整个MT运输机械 线粒体、自噬小体和囊泡运输，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来控制这些货物的双向运输。 我们还将研究纤毛动力蛋白，即滑动平行排列的轴丝MTS来推动纤毛跳动。几个 已经提出了模型来解释动力蛋白的滑动活动是如何自我调节来协调纤毛的 震荡。这些模型对纤毛动力蛋白机制的预测并不是直接的 测试过。最近建立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研究。与细胞质动力蛋白不同， OAD形成异二聚体，不是进行性的。利用这个系统，我们将对OAD的机制进行表征 动力和力量的产生。然后，我们将通过构建体外模型来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相互作用的几何结构。最后，我们将确定结构组件 这导致了OAD的非进行性运动、曲率感知和自振荡行为。 我们研究项目的成功将揭示动力蛋白的基本机械力化学以及它是如何 实现细胞内货物的逆行运输，并驱动运动纤毛的自我协调振荡。