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。动力蛋白是微管电机中普遍存在的重要家族，也是 参与许多重要的生物过程，如发育过程中的左右构型、染色体 分离，囊泡运输，纤毛和鞭毛运动。关于动力蛋白的主要问题之一 仍然没有答案的是：动力蛋白亚型靶向和锚定到货物上的机制是什么？ 这项建议侧重于动力蛋白中间链在将动力蛋白靶向货物、摄取 鞭毛内臂动力蛋白I1在衣藻中的优势I1动力蛋白被靶向和锚定到 A-微管上的一个独特位置。此外，化学交联研究表明，内部 臂动力蛋白I1中间链IC138和IC140各自独立地与第三个90 kDa蛋白相互作用。我推测90 kDa蛋白可能是I1IC97中的第三个中间链，或者更有可能是一种新的蛋白质，介导了I1动力蛋白复合体与微管的对接。为了验证这些想法，提出了三个目标：(1)识别90 kDa的蛋白质。将使用一种新的富集法来纯化交联产物，并进行串联质谱分析来鉴定蛋白质。可能的结果包括相互作用的蛋白质是以前未知的蛋白质，或者相互作用的蛋白质是I1，IC97中的第三中间链动力蛋白。(2)如果90 kDa蛋白是一个新的蛋白，则对该基因进行克隆、定位并产生抗体。(3)如果质谱学数据显示 90 kDa的蛋白质是IC97，那么绘制IC138和IC140上的相互作用部位就很重要了。 然而，如果90 kDa蛋白质是一种新的蛋白质，那么它是对接机制的一部分的假设 将对I1进行测试。这些数据将定义轴丝内臂动力蛋白内的蛋白质相互作用 复合体I1。这些数据还将揭示动力蛋白对接的新原理，从而更好地理解 由动力蛋白组装和调节缺陷引起的人类疾病，如初级纤毛 运动障碍，发育过程中左-右模式缺陷，男性不育，以及女性生育能力受损。

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.