Hair Bundle Structure and Dynamics

 描述（由申请人提供）：本实验室的长期目标是揭示毛细胞的机械敏感细胞器（毛束）的结构，并确定毛束的结构特征如何负责其机械转导功能。在这个提案中，我们扩展了前一个资助期的成就，在那里我们开发了一个发束的部件清单，并在这里确定了在发育过程中束蛋白表达的顺序，以及控制其表达和功能的机制。在目标1中，我们专注于转录的~175关键束蛋白，使用多重RT-qPCR测定来量化它们在整个感觉上皮和分离的毛细胞池中的动态。我们还研究了相同的转录在孤立的单细胞，允许更详细地了解基因表达的时间和随机性。在目标2中，我们通过靶向蛋白质组学测定来定量这些蛋白质中的许多或大部分，并且还确定它们从索马转移到束的时间过程。在目标3中，我们专注于结构;使用冷冻电子断层扫描，我们将获得静纤毛结构的精细理解，而我们将使用免疫荧光和结构化照明显微镜来确定哪些蛋白质靶向哪些结构。使用常规条件等位基因或CRISPR/Cas9敲除，目的1和3中的实验将分别测试特定转录因子和束蛋白在控制束转录物表达和束结构中的作用。我们预计，我们正在研究的一些束蛋白将在导致听力障碍的遗传疾病中受到影响。

 DESCRIPTION (provided by applicant): The long-term goal of this laboratory is reveal the structure of the hair cell's mechanosensitive organelle, the hair bundle, and to determine how structural features of the bundle are responsible for its mechanotransduction function. In this proposal, we extend on the accomplishments of the previous funding period-where we developed a parts list for hair bundles-and here determine the sequence in which bundle proteins are expressed during development, as well as the mechanisms that control their expression and function. In Aim 1, we focus on transcripts for ~175 key bundle proteins, using multiplex RT-qPCR assays to quantify their dynamics in whole sensory epithelium and in pools of isolated hair cells. We also examine the same transcript in isolated single cells, allowing a more detailed understanding of the timing and stochastic nature of gene expression. In Aim 2, we quantify many or most of these proteins by targeted proteomics assays, and also determine their time course for transfer from the soma to the bundle. In Aim 3, we focus on structure; using cryo-electron tomography we will gain a fine understanding of stereocilium structure, while we will use immunofluorescence and structured illumination microscopy to determine which proteins target to which structures. Using conventional conditional alleles or CRISPR/Cas9 knockouts, experiments in both Aims 1 and 3 will test the roles of specific transcription factors and bundle proteins in, respectively, control of bundle transcript expression and bundle structure. We anticipate that some of the bundle proteins we are examining will be affected in genetic diseases that cause hearing impairment.