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.

项目概要 动力蛋白是 AAA+ 马达，负责沿着微管 (MT) 进行负端定向运动和玩耍 在货物运输、有丝分裂和纤毛跳动中发挥重要作用。动力蛋白是目前电机领域的焦点 因为与正端定向驱动蛋白相比，其运动机制尚不清楚。尽管 由于动力蛋白驱动的货物在细胞内快速运输，之前的体外研究发现哺乳动物动力蛋白是一种弱蛋白 马达，表现出缓慢的运动性并产生比驱动蛋白更低的力。最近，体外重构 动力蛋白-动力蛋白机制表明，哺乳动物动力蛋白在不运输货物时会受到自身抑制，并且 当动力蛋白与其辅因子动力蛋白和货物结合形成 2.5 MDa 三元复合物时，运动性被激活 适配器。因此，之前所有关于哺乳动物动力蛋白的体外研究都使用非活动运动及其 结论并不能反映活性动力蛋白-动力蛋白机制如何在细胞中运输货物。 我们未来的目标是剖析活性细胞质动力蛋白复合物的机制并确定如何 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 模拟和冷冻电镜。具体来说，我们将确定 Lis1 在激活中如何发挥作用 和动力蛋白运动的调节。我们还将研究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 位点或通过液-液分层排除其 MT 结合。我们还将表征动力蛋白的运动性和 dynactin 疾病突变体揭示与这些突变相关的神经病的分子机制。 最后，我们将使用体内研究确定的货物适配器重建整个 MT 运输机械 线粒体、自噬体和囊泡运输的作用，但尚未在体外进行表征。使用这种方法， 我们将剖析货物适配器如何调节电机来控制这些货物的双向运输。 我们还将研究纤毛动力蛋白，它使轴丝 MT 平行阵列滑动，为纤毛跳动提供动力。一些 已提出模型来解释动力蛋白的滑动活动如何自我调节以协调纤毛 振荡。这些模型对纤毛动力蛋白机制的预测尚未直接得到证实。 已测试。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研究。与细胞质动力蛋白不同， OAD 形成异二聚体并且不是进行性的。使用该系统，我们将描述 OAD 的机制 动力和力量的产生。然后我们将通过构建体外直接测试每个模型的预测 模仿跳动纤毛中动力蛋白/MT相互作用的几何形状。最后，我们将确定结构组件 引起 OAD 的非过程运动、曲率传感和自振荡行为。 我们的研究计划的成功将揭示动力蛋白的基本机械化学及其如何 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。