Hair Bundle Structure and Dynamics

项目摘要 这个实验室的长期目标是揭示毛细胞的机械敏感细胞器的结构 并确定发束的结构特征是如何影响其 机械转导功能。在这项提案中，我们将重点放在肌动蛋白核心的未被重视的过程上。 加宽，在维管束的发育过程中发生在立体纤毛中。我们利用小鼠的内毛细胞 耳蜗作为我们的模型系统；它们的立体纤毛不仅显示出独特的直径和长度，而且所有 我们在研究捆绑功能时部署的工具可以与这些单元一起部署。在目标1中，我们将扩展我们的 冷冻电子断层扫描程序，用于发育内毛细胞，具体询问时间和地点 在加宽过程中，将外围肌动蛋白细丝添加到肌动蛋白核心。在目标2中，我们将分离 用Fgf8-GFP标记GFP的内毛细胞；Atoh1-Cre小鼠，然后将它们置于蛋白质量中 光谱分析。与我们在太平洋西北国家实验室的合作者一起，我们有 确定了通过荧光激活细胞分选分离的单个内毛细胞的蛋白质组。虽然我们不会 在本项目中，我们将利用新技术的敏感性来分析单个细胞 在精确的发育时间内从特定的耳蜗区分离出来的细胞池。此外，我们还将 用吸管抽吸法在同一时间点分离内毛细胞立体纤毛，使我们还可以确定 立体纤毛蛋白质组发育过度。通过比较全细胞和立体纤毛蛋白质组学数据，我们 将跟踪每种蛋白质何时进入立体纤毛，并挖掘这些数据以确定新的复合体候选 控制着立体纤毛的扩大。最后，在目标3中，我们将研究三个突变小鼠品系(ESPN，Capzb， Grxcr1)，使用为AIMS 1和2开发的技术来表征 立体纤毛的结构和时间特征。总之，在这个项目中开发的实验将使我们能够 确定毛细胞如何扩大其立体纤毛，这是毛发发育的关键步骤之一 捆绑。

Project Summary 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 focus on the under-appreciated process of actin-core widening, which occurs in stereocilia during development of the bundle. We utilize inner hair cells of the mouse cochlea as our model system; not only do their stereocilia rows show unique diameters and lengths, but all of the tools we deploy in studying bundle function can be deployed with these cells. In Aim 1, we will extend our cryo-electron tomography program to developing inner hair cells, asking specifically when and where peripheral actin filaments are added to the actin core during the widening process. In Aim 2, we will isolate inner hair cells marked with GFP using Fgf8-Gfp;Atoh1-Cre mice, and then subject them to protein mass spectrometry. In conjunction with our collaborators at the Pacific Northwest National Laboratory, we have defined the proteomes of single inner hair cells isolated by fluorescence-activated cell sorting. While we will not analyze single cells in the present project, we will exploit the sensitivity of the new techniques to analyze small pools of cells isolated from a specific region of the cochlea at precise development times. In addition, we will isolate inner hair cell stereocilia at the same time points using pipette aspiration, allowing us to also determine the stereocilia proteome over development. By comparing the whole-cell and stereocilia proteomics data, we will track when each protein enters stereocilia, and mine these data to identify new candidates for complexes that control stereocilia widening. Finally, in Aim 3, we will study three mutant mouse lines (Espn, Capzb, Grxcr1) that have thin stereocilia, using the techniques developed for Aims 1 and 2 to characterize the structural and temporal features of stereocilia. Together, the experiments developed in this project will allow us to determine how the hair cell widens its stereocilia, which is one of the critical steps in development of the hair bundle.