The mechanosensitive hair cells of the inner ear consist of a precise array of actin-based stereocilia that insert as rootlets into a dense actin-based meshwork known as the cuticular plate Numerous studies have demonstrated that the proteins that shape the stereocilia are required for normal hearing and balance. On the other hand, little is known about the role the proteins that shape the cuticular plate play in hearing and balance. Indeed, the function that the cuticular plate itself endows to hair bundle development and maintenance remains elusive. Within the cuticular plate, ultrastructural analyses from Tilney’s group in the 1980s revealed numerous types of actin- associated proteins, which presumably control the morphology and the integrity of this unique subcellular structure. Supervillin, a 205 kD actin-crosslinking protein from the villin/gelsolin superfamily, regulates actin dynamics by interacting with non-muscle myosin II. Mutations in MYH9 gene that encodes myosin II are linked to the human hearing loss DFNA17. We showed using RNA in situ hybridization that two isoforms of supervillin; svila and svilc are robustly expressed in zebrafish hair cells of the inner ear. We also demonstrated using our novel Svila rabbit polyclonal antiserum that Svila localizes to the cuticular plate of hair cells in the inner ear of zebrafish. In mice, we showed that Svil is also expressed in the hair cells of the inner ear. In agreement, in mice, in the vestibular type I and type II hair cells, Svil localizes to the cuticular plate at both postnatal day 1 and adult stages. In mice cochlea, Svil localizes to the cuticular plate and near the apical junctional complexes of supporting cells, that surround hair cells, including the head plates of Deiters’ and outer pillar cells and the apicolateral margins of inner phalangeal cells. Here, we identify supervillin role in the development and function of the mechanosensory hair cells is to bundle actin filament networks to shape the cuticular plate. We are utilizing genetic and molecular biological techniques along with physiological experiments to test this hypothesized role in a zebrafish model. In this grant proposal we further study the role of supervillin in hair bundle formation and function.

Our ability to distinguish a remarkable range of sounds and to maintain a balanced posture depends on the ability of the mechanosensory receptors, known as hair cells, in the inner ear to transduce sound energy and head movement into electrical signals then forward these signals to the brain for interpretation. Hair cells are known for their extraordinary structure where they usually have a flask-shaped or a cylindrical cell body that lacks both dendrites and axons. Perhaps the major distinguishing feature of the hair cells is the presence of actin-based staircase-like hair bundle that insert into an actin-rich meshwork known as the cuticular plate. Deviation in this precise morphology, as small as it can be, leads to hearing loss and balance disorders. In this research plan I propose to use standard genetic and molecular biology techniques along with physiological experiments to understand the role of an actin-bundeling protein that is specifically localizes to the cuticular plate in the development and maintenance of these mechanosensory receptors. The proposed work is important because nearly 48 million Americans have significant hearing loss and 1 in 5 American teens experience some level of hearing impairment.