Mechanisms of microtubule-based transport

项目总结 真核细胞的内容是高度动态的，但在空间和时间上是有组织的。这是实现的 主要由微管细胞骨架和相关的运输机械组成，其基本性质是 突出的是由它们的突变引起的许多神经疾病。我的首要目标是 研究计划是为了了解这个系统在分子、细胞和生物体中是如何工作的 比例。我的团队是高度跨学科的，我们使用体外生化重建，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。穿过 我们使用冷冻电子显微镜(Cryo-EM)和冷冻电子断层扫描(Cryo-ET)的合作项目 结合结构指导的方法来理解细胞内的运输，我们开发了可测试的 运输的定量物理模型。我们在确定动力如何发挥作用方面做出了重大贡献 马达工作并受到监管，以开发工具和筛选策略来研究双向运动 了解微管上的货物，并了解细胞内运输的调节。基本原理 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？我们早期的工作揭示了 Lis1是一种在神经发育疾病无脑中突变的蛋白质，它是如何与动力蛋白和 调节其机械力化学循环。在这里，我们将重点确定以下机制的基础 Lis1促进激活的dynein/dynactin复合体的形成。我们还将探索一个新的方向-- RNA编辑的作用--作为一种以前未描述过的调节动力蛋白和动蛋白马达的机制。 基于微管的马达可以搬运数十甚至数百件货物。(2)如何实现货物专用性？我们的 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 负责指定动力蛋白的多种功能的分子。我们过去的工作揭示的一个机制是 细胞器搭便车，在这种情况下，货物通过连接到其他货物而间接连接到发动机，这些货物是 直接绑定到发动机上。实现货物专一性的第二个战略是扩大动力蛋白 激活脊椎动物的适配基因。然而，大多数激活的适配器之间的分子连接 动力蛋白的货物还不得而知。在这里，我们将确定搭便车的潜在机制和 Hook和Ninein系列激活适配器及其货物之间的联系。作为另一种方法 为了了解动力蛋白和运动蛋白是如何与它们的货物相联系的，我们将在三个细胞中可视化这些联系 利用冷冻电子断层扫描技术研究非洲爪哇黑曲霉和黑素小体的大小 Levis黑素载体，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- 基于机动性的。

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.