Mechanisms of microtubule-based transport

项目摘要 真核细胞的内容物是高度动态的，但在空间和时间上是有组织的。实现这一点 主要是通过微管细胞骨架和相关的运输机制，其基本性质是 由它们的突变引起的许多神经系统疾病突出显示。我的首要目标是 研究计划是了解这个系统如何在分子，细胞和有机体中工作。 鳞片我的团队是高度跨学科的，我们使用体外生化重组，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来实现我们的目标。通过 合作项目我们使用冷冻电子显微镜（cryo-EM）和冷冻电子断层扫描（cryo-ET）， 结合结构引导的方法来理解细胞内转运，我们开发了可测试的 运输的定量物理模型。我们对动力蛋白的研究做出了重大贡献 电机工程和调节，开发工具和筛选策略，以研究双向运动的 微管上的货物，并了解细胞内运输的调节。基本 我们将在这里解决的问题包括：（1）动力蛋白马达是如何工作的？我们之前的研究显示 Lis 1是一种在神经发育疾病无脑畸形中突变的蛋白质，它如何与动力蛋白相互作用， 调节机械化学循环。在这里，我们将重点关注如何确定机械基础， Lis 1促进激活的动力蛋白/动力肌动蛋白复合物的形成。我们还将探索一个新的方向， RNA编辑的作用-作为一个以前未描述的机制，以调节动力蛋白和驱动蛋白马达。 基于微管的发动机可以移动几十个甚至几百个货物。(2)如何实现货物专属性？我们 过去的工作使用了两种互补的基于发现的方法-遗传学和蛋白质组学-来识别 这些分子负责指定动力蛋白的许多功能。我们过去的工作揭示了一个机制， 细胞器搭便车，其中货物间接连接到马达，通过将自己附着到其他货物， 直接绑定到电机上。实现货物特异性的第二个策略是动力蛋白的扩增 激活脊椎动物的适配基因。然而，大多数激活接头之间的分子连接 和动力蛋白的货物都是未知的在这里，我们将确定搭便车的潜在机制， Hook和Ninein家族的激活衔接子和它们的货物之间的联系。作为一种补充办法， 为了理解动力蛋白和驱动蛋白如何与它们的货物连接，我们将在三个细胞中观察这些连接。 构巢曲霉核内体和非洲爪蟾黑素体的冷冻电子断层扫描 黑色素细胞，这两个系统，我们可以使用精致的遗传学或化学工具来控制微管- 基于运动性。

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.