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.

我们的听觉和平衡感依赖于正确的 肌动蛋白和肌球蛋白相互作用并组装成毛细胞中的分子机器。我们 建议使用斑马鱼毛细胞切片的电子显微镜断层成像以分子分辨率研究这些蛋白质复合物在其天然细胞环境中的结构排列。 我们试图通过表达被双砷配体 FlAsH 和 ReAsH 识别的四半胱氨酸标记的 β-肌动蛋白同工型来确定这两种细胞质同工型是否在毛细胞中表达，以及它们各自的作用。 FlaAsH/ReAsH 标记可实现活体荧光成像，从而研究活体动物中的蛋白质周转率。我们的目标是使用四半胱氨酸和六组氨酸标记的肌球蛋白 6b、分别用 ReAsH/光转换或 Ni-NTA-纳米金标记以及电子显微镜成像来确定肌球蛋白 6b 在斑马鱼毛细胞中的作用。我们相信，分子分辨率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，以生成两种常染色体显性进行性感性听力损失（DFN20/26和DFNA22）的斑马鱼动物模型。 因此，我们提出以下具体目标： 1. 分子分辨率下斑马鱼毛束肌动蛋白-肌球蛋白复合物的 3D 可视化 2. 斑马鱼毛细胞中野生型和突变型细胞质 β-actin-1 和 2 的鉴定、3D 定位和表征 3. 斑马鱼野生型和突变型 myosin-6b 的 3D 定位 我们相信，拟议的研究将建立一种以分子分辨率研究毛细胞中分子机器的新方法，并允许快速可靠地识别和蛋白质成分的 3D 亚细胞定位。此外，它将为细胞质肌动蛋白和肌球蛋白 6 相关耳聋的发病机制提供重要的见解。