Molecular Analysis of Flagellar Dynein Function

描述（由申请人提供）：动力蛋白是AAA+ atp酶家族的成员，作为基于微管的分子马达，参与各种基本细胞功能，包括逆行囊泡运输，核膜破裂，纤毛/鞭毛运动和细胞分裂。衣藻鞭毛~2.0 MDa外动力蛋白臂是研究动力蛋白结构、功能和调控的一个很好的模型系统，因为它含有与细胞质同工酶密切相关的成分，符合经典/分子遗传学，可以大量纯化用于生化分析。现在很清楚，动力蛋白受到广泛的调控输入，如对氧化还原平衡的反应，Ca2+水平，磷酸化等。然而，动力蛋白运动活动在分子水平上受到调节的机制以及这些不同的输入是如何整合的仍然知之甚少。该应用程序提出了四个具体的调查领域。1)我们将研究外臂动力蛋白受鞭毛氧化还原平衡改变调控的机制。这将包括鉴定和随后的功能分析五种（或更多）蛋白质，这些蛋白质与动力蛋白相关的硫氧还毒素和氧化还原敏感的Ca2+结合蛋白形成混合二硫化物。2)我们将确定与动力蛋白重链的atp结合模块相关的富含亮氨酸的重复蛋白如何调节运动活动。这条轻链也与微管相关，我们将定义重链/轻链/微管蛋白三元复合物的轴突几何结构。结合突变破坏个体相互作用的方法，我们将测试几种相互竞争的假设，以了解这种调节途径是如何被激活的。3)无裂脑蛋白是已知的细胞质动力蛋白的调节因子。我们现在已经发现这种蛋白质存在于纤毛和鞭毛中，并与外动力蛋白臂相关。此外，鞭毛内的Lis1水平受到鞭毛内信号通路的调节。因此，我们将验证Lis1代表一个额外的鞭毛动力蛋白调控系统的假设，该系统涉及动力蛋白四级结构的改变。4)纤毛/鞭毛远端组装需要鞭毛内运输（IFT）。这一目标将集中在神秘的动力蛋白的详细分析，被认为是负责从纤毛尖端逆行运动到细胞体。使用生化方法，我们将纯化这种动力蛋白，定义其组成，并使用RNAi方法和/或基因筛选来鉴定突变体，研究先前未描述的成分对逆行运输的功能贡献。此外，我们还设计了一种纯化方案，产生含有该动力蛋白和负责顺行IFT的动力蛋白的多兆道尔顿复合物。我们将使用体外实验来定义运动功能，并测试这两个相反的运动协调的潜在机制。

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.