Mechanisms of microtubule-based transport

基于微管的运输机制

• 批准号: 10205528
• 负责人: SAMARA L RECK-PETERSON
• 金额: $ 36.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-14 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10205528
• 关键词: 3-Dimensional; Address; Aspergillus nidulans; Biochemical; Cells; Chemicals; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cytoskeleton; Defect; Disease; Dynein ATPase; Endosomes; Eukaryotic Cell; Family; Genes; Genetic; Goals; Human; In Vitro; Intracellular Transport; Kinesin; Link; Melanophores; Melanosomes; Microtubules; Molecular; Molecular Machines; Motor; Movement; Mutate; Nature; Nerve Degeneration; Organelles; Protein Engineering; Proteins; Proteomics; RNA Editing; Regulation; Research; Role; Specific qualifier value; Specificity; Structure; System; Vertebrates; Work; Xenopus laevis; base; cell motility; disease-causing mutation; dynactin; fungal genetics; genetic approach; imaging genetics; insight; lissencephaly; live cell imaging; molecular imaging; nervous system disorder; ninein; physical model; programs; reconstitution; screening; single molecule; tool

PROJECT SUMMARY The contents of eukaryotic cells are highly dynamic, yet organized spatially and temporally. This is achieved primarily by the microtubule cytoskeleton and associated transport machinery, whose fundamental nature is highlighted by the many neurological diseases caused by mutations in them. The overarching goal of my research program is to understand how this system works at the molecular, cellular, and organismal scales. My team is highly interdisciplinary and we use in vitro biochemical reconstitution, protein engineering, single-molecule imaging, proteomics, live-cell imaging, and fungal genetics to achieve our goals. Through collaborative projects we use cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to incorporate a structure-guided approach to understanding intracellular transport, and we develop testable quantitative physical models of transport. We have made major contributions to determining how the dynein motor works and is regulated, to developing tools and screening strategies to study bi-directional movement of cargos on microtubules, and to understanding the regulation of intracellular transport in cells. Fundamental questions that we will address here include: (1) How does the dynein motor work? Our earlier work revealed how Lis1, a protein mutated in the neurodevelopmental disease lissencephaly, interacts with dynein and regulates its mechanochemical cycle. Here, we will focus on determining the mechanistic underpinnings for how Lis1 promotes the formation of activated dynein/dynactin complexes. We will also explore a new direction—the role of RNA editing—as a previously undescribed mechanism to regulate dynein and kinesin motors. Microtubule-based motors move dozens if not hundreds of cargos. (2) How is cargo-specificity achieved? Our past work used two complementary discovery-based approaches—genetics and proteomics—to identify molecules responsible for specifying dynein’s many functions. One mechanism revealed by our past work is organelle hitchhiking, where cargos link to motors indirectly, by attaching themselves to other cargos that are directly bound to the motors. A second strategy for achieving cargo specificity is the expansion of dynein activating adaptor genes in vertebrates. However, the molecular connections between most activating adaptors and dynein’s cargo are unknown. Here, we will determine the mechanisms underlying hitchhiking and the linkages between the Hook and Ninein families of activating adaptors and their cargos. As an additional approach to understand how dynein and kinesin link to their cargos, we will visualize these connections in cells in three dimensions using cryo-electron tomography of endosomes in Aspergillus nidulans and melanosomes in Xenopus laevis melanophores, two systems where we can use exquisite genetics or chemical tools to control microtubule- based motility.

项目摘要 真核细胞的含量是高度动态的，但经常且暂时组织。这是实现的 主要的微管细胞骨架和相关的运输机制，其基本性质是 由其中突变引起的许多神经系统疾病强调。我的总体目标 研究计划是了解该系统如何在分子，细胞和有机体上起作用 秤。我的团队是高度跨学科的，我们使用体外生化重构，蛋白质工程， 单分子成像，蛋白质组学，活细胞成像和真菌遗传学，以实现我们的目标。通过 协作项目我们使用冷冻电子显微镜（Cryo-EM）和冷冻电子层析成像（Cryo-ET） 结合了一种结构引导的方法来理解细胞内运输，我们开发可测试的方法 运输的定量物理模型。我们为确定动力蛋白做出了重大贡献 运动工程并受到监管，以制定工具和筛选策略来研究双向运动 微管上的碳，并了解细胞内细胞内转运的调节。基本的 我们将在此处解决的问题包括：（1）Dynein Motor如何工作？我们较早的工作揭示了 LIS1是一种在神经发育疾病中突变的蛋白质，与动力蛋白相互作用 调节其机械化学周期。在这里，我们将专注于确定如何确定机械基础 LIS1促进了活化的动力蛋白/dynactin复合物的形成。我们还将探索一个新的方向 - RNA编辑的作用 - 作为调节动力蛋白和驱动器电动机的先前未描述的机制。 基于微管的电动机会移动数十个（即使不是数百个）。 （2）如何实现货物特异性？我们的 过去的工作使用了两种基于发现的方法（基因和蛋白质组学）来识别 负责指定Dynein的许多功能的分子。我们过去工作揭示的一种机制是 cargos的Organelle Hitchhiking通过将自己固定在其他碳中 直接绑定到电动机。实现货物特异性的第二种策略是动力蛋白的扩展 激活脊椎动物中的衔接基因。但是，大多数激活适配器之间的分子连接 而Dynein的货物是未知的。在这里，我们将确定搭便车的基础机制和 激活适配器的钩子和九型家族之间的联系。作为另一种方法 为了了解动力蛋白和动力蛋白如何与它们的货物链接，我们将可视化三个细胞中的这些连接 使用曲曲霉中内体的冷冻电子层析成像和Xenopus中黑素的尺寸 Laevis黑色素团，两个系统，我们可以使用独家遗传学或化学工具来控制微管 基于运动。