Inner Ear Hair Cells: Bundle Development and Regeneration

项目B -内耳毛细胞：束发育和再生 曼弗雷德奥尔 项目总结/摘要 毛束是脊椎动物中负责机械电传导的精密细胞器 动物内耳毛细胞和侧线感觉系统。它由静纤毛组成， 肌动蛋白束，其通过非常规肌球蛋白连接到周围的质膜（并且还 radixin）。鸡卵囊组织树脂包埋的相关定量蛋白质组学和三维断层成像研究 部分已经揭示了大分子库存和仅部分有序的3D组织。这 建议的重点是使用冷冻断层扫描法对玻璃化的整体静纤毛样本进行冷冻，这确保了最近的- 原生样本保存并消除了对适当切片几何形状的冗长搜索，例如 适应机制的可视化。 我们建议完成未染色肌动蛋白核心的3D分析，定位和识别 非常规肌球蛋白通过正重金属染色冷冻断层扫描和模板匹配， 以及透化或脱膜玻璃化整片静纤毛的免疫细胞化学标记。我们 建议使用已知的大分子差异来识别不同的局部非常规肌球蛋白， 尺寸、二聚化状态和优选的区域分布。最后，我们将研究 斑马鱼侧线神经肥大、小鸡椭圆囊感觉上皮和斑点静纤毛中的再生毛细胞 以及胚胎和诱导多能干细胞（ESC/iPSC）衍生的毛细胞样细胞。 我们将测试以下假设：（1）肌动蛋白核心的组织更像凝胶，而不是凝胶。 （2）预测的基于MYO 1C的适应存在明确的超微结构相关性 （3）位于毛束中的不同非常规肌球蛋白占据不同的区域， 使用整体包埋免疫亲和标记和重金属染色或在玻璃化之前观察静纤毛， （4）再生毛细胞的毛束和静纤毛结构与再生毛细胞的相似。 未处理的小鸡胞果和斑马鱼侧线毛束/静纤毛。 我们相信，我们的超微结构和亲和标记方法将有助于解决一些问题， 毛细胞领域的问题，例如关于肌动蛋白组织和周转，以及非常规肌球蛋白 定位和功能。此外，它将提供一个超微结构的框架，在其中解释其他类型的 关于毛细胞再生的数据。最后但并非最不重要的是，我们的建议提供了一个强大的技术平台 用于未来的超微结构毛细胞研究以及结构细胞生物学领域。

Project B - Inner Ear Hair Cells: Bundle Development and Regeneration Manfred Auer PROJECT SUMMARY/ABSTRACT The hair bundle is a precision organelle responsible for mechanoelectrical transduction in vertebrate animal hair cells in inner ear and lateral line sensory system. It consists of stereocilia that contain a crosslinked actin bundle, which is connected to the surrounding plasma membrane via unconventional myosins (and also radixin). Correlative quantitative proteomics and 3D tomographic studies of resin-embedded chick utricle tissue sections have revealed the macromolecular inventory and an only partially ordered 3D organization. This proposal focuses on vitrified whole-mount stereocilia samples using cryo-tomography, which ensures nearest- to-native sample preservation and eliminates the lengthy search for adequate section geometry e.g. for visualization of the adaptation machinery. We propose to complete our 3D analysis of the unstained actin core, localize and identify unconventional myosins through positive heavy metal staining cryo-tomography and template matching, as well as immunocytochemical labeling of permeabilized or demembranated vitrified whole-mount stereocilia. We propose to identify the different localized unconventional myosin using known differences in macromolecular dimensions, dimerization state, and preferred regional distribution. Last we will study hair bundles of regenerated hair cells in zebrafish lateral line neuromast, chick utricle sensory epithelia and blotted stereocilia and embryonic and induced pluripotent stem cell (ESC/iPSC)-derived hair cell-like cells. We will test the hypotheses that (1) the actin core is organized more like a gel rather than a paracrystal; (2) there is a defined ultrastructural correlate of the predicted MYO1C-based adaptation machinery; (3) the different unconventional myosins residing in the hair bundle occupy different territories in stereocilia using whole-mount immuno-affinity labeling and heavy metal staining or prior to vitrification and cryo-EM imaging; (4) the hair bundle and stereocilia organization of regenerated hair cells ressembles those of untreated chick utricle and zebrafish lateral line hair bundles/stereocilia. We believe that our ultrastructural and affinity-labeling approaches will help settle a number of questions in the hair cell field, e.g. regarding actin organization and turnover, as well as unconventional myosin localization and function. Also, it will provide an ultrastructural framework in which to interpret other types of data with respect to hair cell regeneration. Last but not least our proposal provides a strong technical platform for future ultrastructural hair cell studies and beyond for the field of structural cell biology.