Determining the Structure, Function and Regulation of Dynein and Flagella

描述（由申请人提供）： 纤毛和鞭毛是进化上保守的细胞器，在运动、感觉和信号传导中具有重要的生物学作用。它们在大多数真核生物的生命周期中是必不可少的，纤毛几乎存在于每个人类细胞中。本研究的目的是确定调节信号如何改变分子马达蛋白动力蛋白的活性，以及动力蛋白活性如何在运动纤毛和鞭毛中调节以产生适当的跳动。我们的实验室为纤毛和鞭毛的三维组织和蛋白质组成打开了一个新的窗口，最近发现了以前看不见的调节动力蛋白功能和/或对鞭毛组装至关重要的结构。在前一个项目期间收集的初步数据的基础上，该提案直接解决了该领域的三个具体目标中的关键开放问题，这些目标旨在了解纤毛和鞭毛中以下三个主要调控和信号转导复合物的三维结构，亚基组成，蛋白质相互作用和调控功能：（1）连接蛋白-动力蛋白调节复合物，（2）I1内部动力蛋白复合物，和（3）放射状辐条三联体生物体中的放射状辐条3。该项目采用多学科方法和尖端技术，结合冷冻电子断层扫描和图像处理的原位分子成像、生物化学和质谱分析、综合结构遗传学方法和蛋白质标记技术，直接可视化细胞中的基因产物。拟议的研究将有助于基础知识和更深入地了解运动蛋白功能和控制的分子水平上的机制和纤毛和鞭毛的功能组织。 公共卫生相关性：人类几个重要器官的正常功能需要纤毛的活性，纤毛运动和组装的缺陷是导致多种危及生命的先天性疾病的原因，例如多囊肾病、Bardet-Biedl综合征、原发性纤毛运动障碍、内脏逆位、呼吸系统疾病和心脏缺陷。我们的工作解决了运动蛋白动力蛋白如何工作，并调节引起纤毛的正常运动。我们预计，这项研究将提供新的见解纤毛相关疾病在人类的潜在机制，也适用于动力蛋白驱动的运输沿着微管细胞骨架，如果有缺陷，可以引起癌症，脑发育和神经退行性疾病。这种基本的理解是一个先决条件，以发展的治疗方案能够减轻这些疾病的过程。

DESCRIPTION (provided by applicant): Cilia and flagella are evolutionarily conserved organelles with important biological roles in motility, sensation and signaling. They are essential in the life cycles of most eukaryotic organisms, and cilia are found in nearly every human cell. The goal of this research is to determine how regulatory signals change the activity of the molecular motor protein dynein, and how dynein activity is regulated in motile cilia and flagella to generate proper beating. Our laboratory opened a new window into the three-dimensional organization and protein composition of cilia and flagella, and recently discovered previously unseen structures that regulate dynein function and/or are essential for flagellar assembly. Building on a strong base of preliminary data gathered in the preceding project period, this proposal directly addresses key open questions in the field in three specific aims that are directed at understanding the three-dimensional structure, subunit composition, protein interactions and the regulatory functions of the following three major regulatory and signal transduction complexes in cilia and flagella: (1) the nexin-dynein regulatory complex, (2) the I1 inner dynein complex, and (3) radial spoke 3 in organisms with radial spoke triplets. The project uses a multi-disciplinary approach and cutting-edge techniques, combining in situ molecular imaging by cryo-electron tomography and image processing, biochemical and mass spectrometric analyses, integrated structural-genetics approaches and protein labeling techniques to directly visualize gene products in cells. The proposed research will contribute fundamental knowledge and a deeper understanding of the mechanisms underlying motor protein function and control on a molecular level and of the functional organization of cilia and flagella. PUBLIC HEALTH RELEVANCE: The proper function of several vital organs in humans requires the activity of cilia, and defects in ciliary motility and assembly are responsible for a wide variety of life-threatening, congenital disorders, such as polycystic kidney disease, Bardet-Biedl syndrome, primary ciliary dyskinesia, situs inversus, respiratory diseases and heart defects. Our work addresses how the motor protein dynein works and is regulated to give rise to normal movement of cilia. We anticipate that this research will provide new insights into the underlying mechanisms of ciliary-linked disorders in humans, and be applicable also to dynein-driven transport along the microtubule cytoskeleton, which, if defective, can give rise to cancer, brain developmental and neurodegenerative diseases. Such fundamental understanding is a prerequisite to the development of therapeutic protocols capable of attenuating these disease processes.