Zebrafish Hair Cell Machines at Molecular Resolution

分子分辨率的斑马鱼毛细胞机器

• 批准号: 7091830
• 负责人: MANFRED AUER
• 金额: $ 10万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-18 至 2007-07-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7091830
• 关键词: actins; biological signal transduction; cryoelectron microscopy; deafness; ear hair cell; electron microscopy; genetically modified animals; hearing; hearing disorders; molecular shape; myosins; pathologic process; protein isoforms; protein localization; protein protein interaction; protein structure function; three dimensional imaging /topography; tomography; zebrafish

Our senses of hearing and balance rely on the correct interactions and assembly of actin and myosin proteins into molecular machines in hair cells. We propose to study at molecular resolution the structural arrangement of these protein complexes in their native cellular context using electron microscopy tomographic imaging of zebrafish hair cell sections. We seek to determine whether both cytoplasmic isoforms are expressed in hair cells, and what their respective roles may be, by expressing tetracysteine-tagged beta-actin isoforms that are recognized by the biarsenical ligands FlAsH and ReAsH. FlAsH/ReAsH labeling allows intravital fluorescence imaging, and therefore the study of protein turnover rates in live animals. We will aim to determine the role of myosin 6b in zebrafish hair cells using tetracysteine- and hexahistidine- tagged myosin 6b, labeling with ReAsH/photoconversion or Ni-NTA-nanogold, respectively, and electron microscope imaging. We believe that subcellular localization at molecular resolution will reveal its function in hair cells as an anchoring and/or motor protein. We will further express mutated beta-actin and myosin 6b proteins in order to generate zebrafish animal models of two types of autosomal dominant progressive sensineural hearing loss (DFN20/26 and DFNA22). We therefore propose the following specific aims: 1. 3D visualization of zebrafish hair bundle actin-myosin complexes at molecular resolution 2. Identification, 3D localization and characterization of wildtype and mutant cytoplasmic beta-actin-1 and 2 in zebrafish hair cells 3. 3D localization of wildtype and mutant myosin-6b in zebrafish We believe that the proposed research will establish a new approach for studying molecular machines in hair cells at molecular resolution, and permit fast and reliable identification and 3D subcellular localization of protein components. In addition, it will provide important insight into the pathogenesis of cytoplasmic actins and myosin-6-associated deafness.

我们的听力和平衡感取决于正确的 肌动蛋白和肌球蛋白蛋白的相互作用和组装到毛细胞中的分子机中。我们 建议在分子分辨率上研究这些蛋白质复合物在其天然细胞环境中使用斑马鱼毛细胞切片的电子显微镜层析成像的结构排列。 我们试图通过表达由Biarsenical Grigands Flash和Reash识别的四酰胺标记的β-肌动蛋白，确定是否在毛细胞中表达两种细胞质同工型，以及它们各自的作用。 Flash/reash标记允许插入式荧光成像，因此可以研究活动物的蛋白质更新率。我们将旨在使用四环素体系氨酸和标记的肌球蛋白6B来确定肌球蛋白6b在斑马鱼毛细胞中的作用，分别用ROESH/Spotoconversion或Ni-Ni-Nta-Nanogold标记，以及电子显微镜成像。我们认为，分子分辨率下的亚细胞定位将揭示其在毛细胞中作为锚定和/或运动蛋白的功能。我们将进一步表达突变的β-肌动蛋白和肌球蛋白6B蛋白，以生成两种类型的常染色体显性渐进性感觉性听力损失的斑马鱼动物模型（DFN20/26和DFNA22）。 因此，我们提出以下具体目标： 1。3D可视化斑马鱼束肌动蛋白肌球蛋白复合物在分子分辨率下 2。鉴定，野生型和突变型细胞质β-肌动蛋白1和2在斑马鱼毛细胞中的鉴定和表征 斑马鱼中的野生型和突变体肌球蛋白6B的3。3d定位 我们认为，拟议的研究将建立一种新的方法，以分子分辨率研究毛细胞中的分子机器，并允许快速可靠的鉴定以及蛋白质成分的3D亚细胞定位。此外，它将为细胞质肌动蛋白和肌球蛋白6相关的耳聋的发病机理提供重要的见解。