Regulation of Cytoplasmic Dynein

项目摘要 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。 微管和它们的马达在这些过程中起着核心作用，而这种机制的缺陷会导致 神经系统疾病。我们专注于细胞质动力蛋白-1（“动力蛋白”），负责几乎所有负- 末端定向（通常朝向细胞内部）沿沿着微管运输。基本动力蛋白机器 由运动亚基的二聚体和5个额外的亚基组成，每个亚基存在于两个拷贝中。 哺乳动物动力蛋白以闭合的“Phi”构象存在，其转化为“开放”构象。结合 动力蛋白，一个大的调节复合物，和一个卷曲的线圈含有激活适配器稳定开放 构象这种DDX（Dynein，Dynactin，X =激活衔接子）复合物在细胞内持续移动。 微管大约有十几个激活适配器，它们也将动力蛋白与其货物联系起来， 激活衔接子募集两个动力蛋白二聚体（D2 DX）。S.酿酒酵母动力蛋白不形成稳定的Φ颗粒， 因此，它本身具有过程性，使其成为研究以下基本问题的理想模型系统： 动力蛋白调节 在这个建议中，我们专注于如何两个动力蛋白调节器，Lis 1和Nudel，这是保守的， 改变酵母和人类动力蛋白的活性。基于我们在之前的资助中的发现， Lis 1以相反的方式调节酵母动力蛋白，这取决于其与 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Lis 1 使用野生型动力蛋白和不能在其两个位点之一结合Lis 1的突变体， 结合位点。然后，我们将把我们的重点放在调节人类动力蛋白的Lis 1，NDE 1和NDEL 1，这两个 人类裸基因基于我们的初步发现，我们将检验Lis 1和Nudel 调节动力蛋白的Phi到Open转变。接下来，我们将确定Lis 1和Nudel如何调节活跃的DDX 或D2 DX络合物，测量参数如活性络合物的稳定性，其运动性质， 以及它对负载的响应。对于所有这些实验，我们将使用冷冻电子显微镜的组合， 解决结构，单分子运动分析，光学捕获，生化重组和活细胞 来验证我们的假设最后，我们已经鉴定了人Lis 1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Lis 1和Nudel调节 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.