Molecular and Genetic Analysis of Flagellar Dyneins

描述（由申请人提供）：真核生物纤毛和鞭毛的运动性已知依赖于沿着双态微管移动的动力蛋白atp酶马达。鞭毛动力蛋白必须在正常弯曲形成和传播过程中进行调节，以协调它们的活动，此外还必须调节拍打频率和波形，以响应不断变化的生理需求。外行动力蛋白组装所必需的两个基因，ODA7和ODA8，最近被鉴定为LC1的同源物，LC1结合到HC伽马动力蛋白催化结构域，可能作为一个调节轻链。Oda7p和Oda8p作为HC α和HC β调节轻链的假设功能将进行测试。支架蛋白被假设为多个动力蛋白异构体之间的连接，沿着每个双态微管。两个候选支架蛋白基因（PF13和ODA16）已经克隆基于插入突变破坏动力蛋白组装和功能。我们提出了实验来确定每个基因产物在将动力蛋白与双态微管表面的调节蛋白对接中的作用。运动蛋白是鞭毛中发现的另一个微管相关运动蛋白家族，但它们在鞭毛运动中的作用尚不清楚。鞭毛运动的全局调控涉及双相关动力蛋白和中央对微管相关复合物之间的径向辐条传递的信号。两个中心对投射在这一过程中的作用将通过一个突变来研究，该突变破坏了一个投射（cpcl）和rnai介导的在另一个投射（Klp1）中发现的激酶的敲低。中心对激酶的工作将扩展到包括所有被鉴定为中心对蛋白的激酶，敲低菌株将用于筛选中心对激酶基因的零突变。

DESCRIPTION (provided by applicant): The motility of eukaryotic cilia and flagella is known to depend on dynein ATPase motors that move along doublet microtubules. Flagellar dyneins must be regulated to coordinate their activity during normal bend formation and propagation, and additionally to modulate beat frequency and waveform in response to changing physiological requirements. Two genes essential for outer row dynein assembly, ODA7 and ODA8, have recently been identified as homologs of LC1, which binds to the HC gamma dynein catalytic domain and may act as a regulatory light chain. Hypothesized functions of Oda7p and Oda8p as HC alpha and HC beta regulatory light chains will be tested. Scaffold proteins have been hypothesized to function as links between multiple dynein isoforms along each doublet microtubule. Two candidate scaffold protein genes (PF13 and ODA16) have been cloned based on insertional mutations that disrupt dynein assembly and function. Experiments are proposed to define the role of each gene product in docking dyneins to regulatory proteins on the doublet microtubule surface. Kinesins represent another family of microtubule associated motor proteins found in flagella, but their role in flagellar motility is not known. Global regulation of flagellar motility involves signals transmitted by radial spokes between doublet-associated dyneins and the central pair microtubule-associated complex. The role of two central pair projections in this process will be studied using a mutation that disrupts one projection (cpcl) and RNAi-mediated knockdown of a kinesin found in another projection (Klp1). Work on central pair kinesins will be extended to include all kinesins identified as central pair proteins, and knockdown strains will be used to screen for null mutations in central pair kinesin genes.