Regulation of Cytoplasmic Dynein

细胞质动力蛋白的调节

基本信息

批准号：
10224220
负责人：
Andres Leschziner
金额：
$ 30.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-10 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10224220
关键词：
3-Dimensional Affect Binding Binding Proteins Binding Sites Biochemical Biological Assay Biological Models Biotinylation Cells Collaborations Communication Complex Cryoelectron Microscopy Data Defect Disease Dynein ATPase Family Funding Genes Human In Vitro Lead Link Measures Mediating Microtubules Molds Molecular Molecular Conformation Molecular Motors Motor Negative Staining Neurodegenerative Disorders Neurons Organelles Organism Pathway interactions Physiological Play Process Production Property Proteins Proteome Regulation Research Resolution Role Saccharomyces cerevisiae Sampling Structure Testing Yeasts base cell motility dimer dynactin experimental study functional plasticity fungus in vivo insight laser tweezer live cell imaging member mutant nervous system disorder novel optical traps particle reconstitution recruit response single molecule stoichiometry three dimensional structure

项目摘要

PROJECT SUMMARY The cellular contents of eukaryotic organisms are highly dynamic, yet organized spatially and temporally. Microtubules and their motors play central roles in these processes and defects in this machinery cause neurological diseases. We focus on cytoplasmic dynein-1 (“dynein”), the motor responsible for nearly all minus- end-directed (typically towards the cell interior) transport along microtubules. The basic dynein machine consists of a dimer of motor subunits and 5 additional subunits that are each present in two copies. Mammalian dynein exists in a closed “Phi” conformation that converts to an “Open” conformation. Binding to dynactin, a large regulatory complex, and a coiled coil-containing activating adaptor stabilizes the Open conformation. This DDX (Dynein, Dynactin, X = an activating adaptor) complex moves processively on microtubules. There are about a dozen activating adaptors, which also link dynein to its cargo and some activating adaptors recruit two dynein dimers (D2DX). S. cerevisiae dynein does not form a stable Phi particle, and as a result is processive on its own, making it an ideal model system for studying basic questions about dynein regulation. In this proposal we focus on how two dynein regulators, Lis1 and Nudel, which are conserved from yeast to human alter the activity of yeast and human dynein. Building on our finding in the previous funding cycle that Lis1 regulates yeast dynein in opposing ways depending on the stoichiometry of its interaction with dynein, we will determine the mechanism of this unique form of regulation. We will also determine how Lis1 affects dynein’s response to load using wild-type dynein and mutants that can’t bind Lis1 at one of its two binding sites. We will then turn our focus to regulation of human dynein by Lis1, NDE1 and NDEL1, the two human Nudel genes. Based on our preliminary findings, we will test the hypothesis that Lis1 and Nudel regulate the Phi to Open transition of dynein. Next we will determine how Lis1 and Nudel regulate active DDX or D2DX complexes, measuring parameters such as stabilization of the active complex, its motile properties, and its response to load. For all of these experiments we will use a combination of cryo-electron microscopy to solve structures, single-molecule motility assays, optical trapping, and biochemical reconstitutions and live-cell imaging to test our hypotheses. Finally, we have identified the human Lis1 and Nudel protein interactomes and we will determine how novel protein interactions we identified affect Lis1 and Nudel regulation of human dynein.

项目概要真核生物的细胞内容物是高度动态的，但在空间和时间上是有组织的。微管及其马达在这些过程中发挥着核心作用，而这种机制的缺陷导致我们关注细胞质动力蛋白-1（“动力蛋白”），它是几乎所有神经系统疾病的运动机制。沿着微管的末端定向（通常朝向细胞内部）运输基本的动力蛋白机器。由一个运动亚基二聚体和 5 个附加亚基组成，每个亚基都有两个副本。哺乳动物动力蛋白以封闭的“Phi”构象存在，可转换为“开放”构象。 dynactin（一种大型调节复合物）和含有卷曲线圈的激活适配器可稳定开放该 DDX（动力蛋白、Dynactin，X = 激活接头）复合物逐渐移动。大约有十几个激活适配器，它们也将动力蛋白与其货物和一些连接起来。激活接头招募两个动力蛋白二聚体 (D2DX) 酿酒酵母动力蛋白不形成稳定的 Phi 颗粒，其结果是其自身具有持续性，使其成为研究有关基本问题的理想模型系统动力蛋白调节。在本提案中，我们重点关注两个动力蛋白调节因子 Lis1 和 Nudel，它们是如何从基于我们在之前的资助中的发现，酵母对人类的影响改变了酵母和人类动力蛋白的活性。 Lis1 以相反的方式调节酵母动力蛋白，具体取决于其与酵母动力蛋白相互作用的化学计量动力蛋白，我们将确定这种独特的调节形式的机制，我们还将确定 Lis1 如何进行。使用野生型动力蛋白和不能在其两个位置之一结合 Lis1 的突变体影响动力蛋白对负载的反应然后我们将把重点转向 Lis1、NDE1 和 NDEL1 这两个蛋白对人类动力蛋白的调节。根据我们的初步发现，我们将检验 Lis1 和 Nudel 基因的假设。接下来我们将确定 Lis1 和 Nudel 如何调节活性 DDX。或 D2DX 复合物，测量活性复合物的稳定性、其运动特性等参数，对于所有这些实验，我们将结合使用冷冻电子显微镜来观察。解析结构、单分子运动测定、光学捕获、生化重建和活细胞最后，我们鉴定了人类 Lis1 和 Nudel 蛋白相互作用组。我们将确定我们发现的新型蛋白质相互作用如何影响人类的 Lis1 和 Nudel 调节动力蛋白。