Regulation of Cytoplasmic Dynein

项目总结 真核生物的细胞内容是高度动态的，但在空间和时间上都是有组织的。 微管和它们的马达在这些过程中起着核心作用，并在这种机械原因中产生缺陷 神经系统疾病。我们把重点放在细胞质动力蛋白-1(“动力蛋白”)上，这是负责几乎所有负- 末端定向(通常朝向细胞内部)沿微管运输。基础动力机 由一个马达亚基和另外5个亚基组成，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，并转化为“开放”构象。绑定到 一个大的调节复合体dynactin和一个包含线圈的激活适配器稳定了开放 构象。这种DDX(Dynein，dynactin，X=激活的接头)复合体连续移动 微管。大约有十几个激活的适配器，它们也将dynein连接到它的货物和一些 激活的适配器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， 因此，它本身就是一个过程性的，使其成为研究 动力蛋白调节。 在这个提案中，我们关注两个dynein调节器，Lis1和Nudel，它们是如何从 酵母对人体的作用可以改变酵母菌和人体动力蛋白的活性。基于我们在上一次资助中的发现 Lis1以相反的方式调节酵母动力蛋白的周期取决于它与酵母动力蛋白相互作用的化学计量比 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 使用野生型动力蛋白和不能结合其中一种的突变体来影响动力蛋白对负荷的反应 结合部位。然后我们将把重点转向对人类动力蛋白的调控，这两个基因分别是Lis1、NDE1和NDEL1 人类的努德尔基因。根据我们的初步发现，我们将检验利斯1和努德尔的假设 调节pHi为动力蛋白的开放转变。接下来，我们将确定Lis1和Nudel如何调节活性DDX 或D2DX络合物，测量参数，如活性络合物的稳定性，其运动性质， 以及它对负载的反应。对于所有这些实验，我们将使用低温电子显微镜的组合来 解决结构、单分子运动分析、光学捕获、生化重组和活细胞 成像来检验我们的假设。最后，我们已经确定了人的Lis1和Nudel蛋白相互作用并 我们将确定我们发现的新的蛋白质相互作用如何影响人类的Lis1和Nudel调控 动力蛋白。

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.