Pilot--Assembly and Regulation of Dynein: Role of the Intermediate Chains

我研究的总体目标是确定动力蛋白的机制和调节，特别是 强调鞭毛动力蛋白。动力蛋白是微管马达中普遍存在的重要家族， 参与许多重要的生物过程，如发育过程中的左右模式，染色体 分离，囊泡运输，纤毛和鞭毛运动。动力蛋白的一个主要问题是 动力蛋白异构体靶向和锚定货物的机制是什么？ 这项建议的重点是动力蛋白中间链的作用，针对动力蛋白的货物， 鞭毛内臂动力蛋白I1的优势。I1动力蛋白靶向并锚定于 A微管上的一个独特位置此外，化学交联研究表明， 臂动力蛋白I1中间链IC 138和IC 140各自独立地与第三个90 kDa蛋白相互作用。我假设，90 kDa的蛋白质是I1 IC97中的第三个中间链，或者更有可能是介导I1动力蛋白复合物与微管对接的一种新蛋白质。为了验证这些想法，提出了三个目标：（1）鉴定90 kDa蛋白。将使用一种新的富集程序来纯化交联产物，并进行串联质谱法来鉴定蛋白质。可能的结果包括相互作用蛋白质是以前未鉴定的蛋白质，或者相互作用蛋白质是I1中的第三中间链动力蛋白，IC 97。(2)如果90 kDa蛋白质是一种新的蛋白质，则将克隆该基因，绘制图谱并产生抗体。(3)如果质谱数据显示 如果90 kDa蛋白质是IC97，那么在IC138和IC140上定位相互作用位点将是重要的。 然而，如果90 kDa蛋白是一种新蛋白，那么它是对接机制的一部分的假设 将测试I1。这些数据将确定蛋白质相互作用的轴丝内臂动力蛋白 复合物I1。数据还将揭示动力蛋白对接的新原理，从而更好地理解 由于动力蛋白组装和调节的缺陷而导致的人类疾病，如初级纤毛 运动障碍、发育过程中的左右模式缺陷、男性不育和女性生育能力受损。

The overall goal of my research is to determine the mechanism and regulation of dynein with particular emphasis on flagellar dyneins. Dynein is ubiquitous and essential family of microtubule motors that is involved in many vital biological processes such as left-right patterning during development, chromosome segregation, vesicular trafficking, and ciliary and flagellar motility. One of the major questions about dyneins that remains unanswered is: What is the mechanism of dynein isoform targeting and anchoring to cargo? This proposal focuses on the role of dynein intermediate chains in targeting dynein to cargo, taking advantage of the flagella inner arm dynein I1 in Chlamydomonas. The I1 dynein is targeted and anchored to a unique position on the A-microtubule. Additionally, chemical cross-linking studies revealed that the inner arm dynein I1 intermediate chains IC138 and IC140 each independently interact with a third 90 kDa protein. I postulate that the 90 kDa protein is either the third intermediate chain in I1 IC97,or more likely, a novel protein that mediated the docking of the I1 dynein complex to the microtubule. To test these ideas three aims are proposed: (1) Identify the 90 kDa protein. A novel enrichment procedure will be used to purify the cross-linked product and tandem mass spectrometry will be performed to identify the protein. Possible outcomes include the interacting protein is a previously unidentified protein, or the interacting protein is the third intermediate chain dynein in I1, IC97. (2) If the 90 kDa protein is a novel protein the gene will be cloned, mapped and antibodies generated. (3) If the mass spectrometry data reveal that the 90 kDa protein is IC97 then it will be important to map the interaction sites on IC138 and IC140. However, if the 90 kDa protein is a novel protein then the hypothesis that it is part of the docking mechanism for I1 will be tested. These data will define protein interactions within the axonemal inner arm dynein complex I1. The data will also reveal new principles for docking of dynein, leading to a better understanding of human diseases that result from defects in dynein assembly and regulation, such as primary cilia dyskinesia, defective left-right patterning during development, male infertility, and impaired female fertility.