Hair Bundle Structure and Dynamics

项目摘要 该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， 毛束，并确定如何束的结构特征是负责其 机械传导功能在这个建议中，我们关注的是肌动蛋白核心的未被充分认识的过程， 变宽，这发生在静纤毛在发展过程中的束。我们利用老鼠的内毛细胞 耳蜗作为我们的模型系统;不仅他们的静纤毛行显示独特的直径和长度，但所有的 我们在研究束功能时使用的工具可以与这些细胞一起使用。在目标1中，我们将扩展 冷冻电子断层扫描程序，以发展内毛细胞，问具体何时何地 在加宽过程中，周边肌动蛋白丝被添加到肌动蛋白核心。在目标2中，我们将隔离 使用Fgf 8-Gfp; Atoh 1-Cre小鼠，用GFP标记内毛细胞，然后使它们经受蛋白质质量 光谱法我们与太平洋西北国家实验室的合作者一起， 定义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 分析单细胞在本项目中，我们将利用新技术的灵敏度来分析小 在精确的发育时间从耳蜗的特定区域分离的细胞池。此外，我们将 分离内毛细胞静纤毛在同一时间点使用吸管抽吸，使我们也可以确定 静纤毛蛋白质组在发育过程中的作用。通过比较全细胞和静纤毛蛋白质组学数据， 将跟踪每种蛋白质进入静纤毛的时间，并挖掘这些数据以识别复合物的新候选者 控制静纤毛变宽最后，在目标3中，我们将研究三种突变小鼠系（Espn，Capzb， Grxcr 1），使用为目标1和2开发的技术来表征 静纤毛结构和时间特征。总之，在这个项目中开发的实验将使我们能够 以确定毛细胞如何扩大其静纤毛，这是头发发育的关键步骤之一 捆。

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.