Zebrafish Hair Cell Machines at Molecular Resolution

我们的听觉和平衡感依赖于正确的 毛细胞中肌动蛋白和肌球蛋白的相互作用和组装成分子机器。我们 建议使用斑马鱼毛细胞切片的电子显微镜断层成像，在分子分辨率上研究这些蛋白质复合体在其自然细胞环境中的结构安排。 我们试图通过表达被二砷配体Flash和ReAsH识别的四半胱氨酸标记的β-肌动蛋白异构体，来确定这两种细胞质异构体是否在毛细胞中表达，以及它们各自的作用是什么。Flash/ReAsH标记可以进行活体内荧光成像，因此可以研究活体动物的蛋白质周转率。我们的目标是利用四半胱氨酸和六组氨酸标记的肌球蛋白6b，分别用ReAsH/光转化或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，以建立两种类型的常染色体显性遗传性进行性感音神经性聋斑马鱼动物模型(DFN20/26和DFNA22)。 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-肌球蛋白复合体的三维可视化 2.斑马鱼毛细胞中野生型和突变型β-肌动蛋白-1和2的鉴定、3D定位和特性 3.野生型和突变型肌球蛋白-6b在斑马鱼中的三维定位 我们相信，这项研究将为以分子分辨率研究毛细胞中的分子机器建立一种新的方法，并允许快速可靠地识别和3D亚细胞定位蛋白质组分。此外，它将为细胞质肌动蛋白和肌球蛋白-6相关性耳聋的发病机制提供重要的见解。

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.