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.