MOLECULAR ANALYSIS OF FLAGELLAR DYNEIN FUNCTION

动力蛋白是一种以微管为基础的分子马达，参与多种重要的细胞功能，包括逆行运输囊泡、纤毛/鞭毛运动和细胞分裂。衣藻鞭毛中的1.9个丙二醛动力蛋白外臂是研究动力蛋白结构和功能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，适合于经典/分子遗传学研究，并且可以大量纯化用于生化。主要的问题仍然是动力蛋白运动功能是如何控制的，以及酶是通过什么机制连接到适当的货物上的。这项应用提出了对我们最近在动力蛋白颗粒中发现的四个不同的轻链(LC)的研究。Lc4是一种与钙离子结合的EF-Hand蛋白，与伽马重链相关，我们推测它参与了钙离子介导的运动功能控制。拟议的实验将在体外和体内直接测试这一点，使用表现出不同钙亲和力的LC4突变版本。第二个项目将分析与伽马重链的运动域相关的LCI蛋白。这种LC直接与第二个蛋白质(P45)在原位相互作用，从而以运动单位为靶点。我们建议鉴定P45并确定LC1在重链上的结合位置。随后的实验将集中于确定这个新系统在动力蛋白功能中的作用。我们还将研究位于动力蛋白颗粒底部的两类基本LC(Tctex 1/Tctex 2和Lc7/Roadblock)所起的普通作用，这两类LC在细胞质和鞭毛同工酶中都有发现。我们将在odal2缺失的突变背景中表达Lc2(Tctex 2)的改变版本，该背景无法组装外臂来确定这个LC类的功能。已在果蝇中发现了LC7/路障蛋白的显性负突变。因此，我们将在体内过度表达这种蛋白的突变形式，并随后对这些菌株进行详细的生化/细胞生物学分析。因此，本项目将提供关于这种复杂分子马达的组装、调节和活性的详细信息。

Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicular trafficking, ciliary/flagellar motility and cell division. The 1.9 MDa outer dynein arm from flagella of Chlamydomonas offers an excellent model system in which to study dynein structure and function as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetic study and may be purified in large amount for biochemistry. Major questions remain about how dynein motor function is controlled and the mechanisms by which the enzyme is attached to the appropriate cargo. This application proposes study of four distinct light chains (LCs) that we have recently identified within the dynein particle. LC4 is a Ca2+-binding EF-hand protein associated with the gamma heavy chain that we hypothesize is involved in Ca2+-mediated control of motor function. The proposed experiments will test this directly both in vitro and in vivo using mutant versions of LC4 exhibiting different Ca affinities. The second project will analyze the LcI protein which is associated with the motor domain of the gamma heavy chain. This LC interacts directly in situ with a second protein (p45) that is thereby targeted to the motor unit. We propose to identify p45 and determine where LC1 binds on the heavy chain. Subsequent experiments will focus on defining the role of this novel system in dynein function. We also will examine the generic roles played by two essential classes of LC (Tctex1/Tctex2 and LC7/roadblock) that are located at the base of the dynein particle and are found in both cytoplasmic and flagellar isozymes. we will express altered versions of LC2 (Tctex2) in the odal2 null mutant background which is unable to assemble an outer arm to determine the function of this LC class. Dominant negative mutations for the LC7/roadblock proteins have been identified in Drosophila. Therefore, we will overexpress mutant forms of this protein in vivo and subsequently subject the strains to detailed biochemical/cell biological analysis. Thus, this project will provide detailed information on the assemble, regulation and activity of this complex molecular motor.