MOLECULAR ANALYSIS OF FLAGELLAR DYNEIN FUNCTION

动力蛋白是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、纤毛/鞭毛运动和细胞分裂。衣藻鞭毛1.9 MDa外动力蛋白臂为研究动力蛋白结构和功能提供了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，适合经典/分子遗传学研究，并且可以大量纯化用于生物化学。主要的问题仍然是动力蛋白运动功能是如何被控制的，以及这种酶是如何附着在适当的货物上的。本申请提出了我们最近在动力蛋白粒子中发现的四种不同的轻链（lc）的研究。LC4是一种与γ重链相关的Ca2+结合EF-hand蛋白，我们假设它参与Ca2+介导的运动功能控制。所提出的实验将在体外和体内使用具有不同Ca亲和力的LC4突变体来直接验证这一点。第二个项目将分析与伽马重链马达结构域相关的LcI蛋白。这种LC直接在原位与第二种蛋白（p45）相互作用，从而靶向运动单元。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。我们还将研究两类LC （Tctex1/Tctex2和LC7/路障）所起的一般作用，它们位于动力蛋白颗粒的底部，存在于细胞质和鞭毛同工酶中。我们将在odal2零突变背景下表达改变版本的LC2 (Tctex2)，该背景无法组装外臂以确定该LC类的功能。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质的突变形式，并随后对菌株进行详细的生化/细胞生物学分析。因此，该项目将提供有关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.