Molecular Analysis of Flagellar Dynein Function

鞭毛动力蛋白功能的分子分析

• 批准号: 7886090
• 负责人: Stephen M King
• 金额: $ 10.67万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-13 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7886090
• 关键词: ATP phosphohydrolase; Active Sites; Address; Area; Arginine; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological Models; C-terminal; Calmodulin; Cell division; Cell physiology; Chlamydomonas; Cilia; Complex; Consensus; Distal; Disulfides; Docking; Dynein ATPase; Family; Fingers; Flagella; Frequencies; Genetic Screening; Glycine; Goals; Head; Homologous Gene; Hydrolysis; Individual; Investigation; Isoenzymes; Kinesin; LIS1 protein; Length; Light; Methods; Microtubules; Modeling; Molecular; Molecular Analysis; Molecular Genetics; Molecular Motors; Motor; Motor Activity; Movement; Mutagenesis; Mutation; Nuclear Envelope; Nucleotides; Oxidation-Reduction; Pathway interactions; Phenotype; Phosphorylation; Physiological; Play; Power stroke; Proteins; RNA Interference; Regulation; Regulatory Pathway; Role; Rotation; Scheme; Series; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Structural Models; Structure; System; Testing; Thioredoxin; Tubulin; Upper arm; Variant; Vesicle; base; design; flexibility; in vitro Assay; insight; leucine-rich repeat protein; member; mutant; neuronal cell body; oxidation; particle; response; retrograde transport; rho; stem; trafficking

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.

描述（由申请人提供）：动力蛋白是 ATP 酶 AAA+ 家族的成员，充当基于微管的分子马达，参与​​多种重要的细胞功能，包括逆行囊泡运输、核膜破裂、纤毛/鞭毛运动和细胞分裂。来自衣藻鞭毛的约 2.0 MDa 外动力蛋白臂提供了一个极好的模型系统，用于研究动力蛋白结构、功能和调节，因为它包含与细胞质同工酶密切相关的成分，适合经典/分子遗传学，并且可以大量纯化用于生化分析。现在已经清楚，动力蛋白受到多种调节输入的影响，例如对氧化还原平衡、Ca2+水平、磷酸化等的反应。然而，在分子水平上调节动力蛋白运动活动的机制以及这些不同输入如何整合仍然知之甚少。该申请提出了四个具体的研究领域。 1）我们将研究外臂动力蛋白响应鞭毛氧化还原平衡变化进行调节的机制。这将涉及对与动力蛋白相关硫氧还蛋白和氧化还原敏感 Ca2+ 结合蛋白形成混合二硫化物的五种（或更多）蛋白质进行鉴定和随后的功能分析。 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。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。结合破坏个体相互作用的诱变方法，我们将测试几个关于如何激活该调节途径的相互竞争的假设。 3) 无脑畸形蛋白是已知的细胞质动力蛋白调节因子。我们现在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，我们将检验这样的假设：Lis1 代表一个额外的鞭毛动力蛋白调节系统，涉及动力蛋白四级结构的改变。 4) 纤毛/鞭毛在远端的组装需​​要鞭毛内运输 (IFT)。这一目标将集中于对神秘动力蛋白的详细分析，该动力蛋白被认为负责从睫状体尖端到细胞体的逆行运动。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/或遗传筛选来研究以前未描述的成分对逆行运输的功能贡献，以识别突变体。此外，我们设计了一种纯化方案，产生包含这种动力蛋白和负责顺行 IFT 的驱动蛋白的多兆道尔顿复合物。我们将使用体外测定来定义运动功能并测试协调这两个相反运动的活动的潜在机制。