Molecular Analysis of Flagellar Dynein Function

描述（由申请人提供）：动力蛋白是ATP酶AAA+家族的成员，其作为基于微管的分子马达，参与多种基本细胞功能，包括逆行囊泡运输、核膜破裂、纤毛/鞭毛运动和细胞分裂。来自衣原体鞭毛的~ 2.0MDa外动力蛋白臂提供了一个研究动力蛋白结构、功能和调节的极好模型系统，因为它含有与胞质同工酶中的组分密切相关的组分，适合于经典/分子遗传学，并且可以大量纯化用于生化分析。现在很清楚，动力蛋白是受到广泛的监管输入，如氧化还原平衡，钙离子水平，磷酸化等的反应，然而，动力蛋白电机活动的调节机制，在分子水平上，以及如何这些不同的输入集成仍然知之甚少。本申请提出了四个具体的调查领域。1)我们将研究外臂动力蛋白的调节机制，以响应鞭毛氧化还原平衡的改变。这将涉及识别和随后的功能分析的五个（或更多）的蛋白质，形成混合二硫化物与动力蛋白相关的硫氧还蛋白和氧化还原敏感的Ca 2+结合蛋白。2)我们将确定如何与动力蛋白重链的ATP结合模块相关的富含亮氨酸的重复蛋白调节运动活性。该轻链也与微管相关，我们将定义重链/轻链/微管蛋白三元复合物的轴丝几何形状。结合诱变方法来破坏个体的相互作用，我们将测试几个相互竞争的假设，这种调节途径是如何激活的。3)无脑畸形蛋白是一种已知的细胞质动力蛋白调节因子。我们现在已经发现，这种蛋白存在于纤毛和鞭毛，并与外部动力蛋白arm. Further，在鞭毛内的Lis 1水平调制的鞭毛内信号通路。因此，我们将测试的假设，Lis 1代表一个额外的鞭毛动力蛋白的监管系统，涉及改变动力蛋白的四级结构。4)鞭毛内运输（IFT）是纤毛/鞭毛在其远端组装所必需的。这一目标将集中在详细分析的神秘动力蛋白，被认为是负责逆行运动从睫状体尖端的细胞体。使用生物化学方法，我们将纯化这种动力蛋白，定义其组成，并研究以前未描述的组件的功能贡献，逆行运输使用RNAi方法和/或遗传筛选，以确定突变体。此外，我们已经设计了一种纯化方案，产生一个多兆道尔顿复杂的动力蛋白和驱动蛋白负责顺行IFT。我们将使用体外试验来定义运动功能，并测试这两个相反的运动的活动是协调的潜在机制。

DESCRIPTION (provided by applicant): Dyneins are members of the AAA+ family of ATPases that act as microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicle trafficking, nuclear envelope breakdown, ciliary/flagellar motility and cell division. The ~2.0 MDa outer dynein arm from flagella of Chlamydomonas offers an excellent model system in which to study dynein structure, function and regulation as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetics and can be purified in large amounts for biochemical analysis. It is now clear that dyneins are subject to a wide array of regulatory inputs such as responses to redox poise, Ca2+ levels, phosphorylation etc. However, the mechanisms by which dynein motor activity is regulated at the molecular level and how these different inputs are integrated remain very poorly understood. This application proposes four specific areas of investigation. 1) We will investigate the mechanism by which outer arm dynein is regulated in response to alterations in flagellar redox poise. This will involve identification and subsequent functional analysis of the five (or more) proteins that form mixed disulfides with dynein-associated thioredoxins and a redox-sensitive Ca2+-binding protein. 2) We will determine how a leucine-rich repeat protein associated with the ATP-binding modules of the dynein heavy chain regulates motor activity. This light chain also associates with microtubules and we will define the axonemal geometry of the heavy chain / light chain / tubulin ternary complex. Combined with mutagenesis approaches to disrupt individual interactions, we will test several competing hypotheses for how this regulatory pathway is activated. 3) The lissencephaly protein is a known regulator of cytoplasmic dynein. We have now found that this protein is present in cilia and flagella, and associates with the outer dynein arm. Furthermore, Lis1 levels within the flagellum are modulated by an intraflagellar signaling pathway. Thus, we will test the hypothesis that Lis1 represents an additional flagellar dynein regulatory system that involves alteration in dynein quaternary structure. 4) Intraflagellar transport (IFT) is required for the assembly of cilia/flagella at their distal tip. This aim will focus on the detailed analysis of the enigmatic dynein that is thought to be responsible for retrograde movement from the ciliary tip to the cell body. Using biochemical methods, we will purify this dynein, define its composition and investigate the functional contribution of previously undescribed components to retrograde transport using RNAi methods and/or genetic screens to identify mutants. Furthermore, we have devised a purification scheme that yields a multi-megadalton complex containing this dynein and the kinesin responsible for anterograde IFT. We will use in vitro assays to define motor function and to test potential mechanisms by which the activity of these two opposing motors is coordinated.