Significance of Usher Protein Dynamics in Hair Cell Function and Deafness

PROJECT SUMMARY/ABSTRACT Our long-term goal is to elucidate the molecular mechanisms that govern the trafficking, assembly, and function of stereociliar proteins and identify the defects in these processes that lead to deafness. This proposal specifically focuses on the Usher-1 proteins, crucial components of the tip link apparatus in hair cell stereocilia. Myosin-7a (USH1B), sans (USH1G), and harmonin-b (USH1C) localizes at the upper end of the tip link. They bind to the intracellular domain of cadherin-23 (USH1D), pulling on the tip link and regulating the mechanosensitivity of hair cells. Mutations in their genes cause Usher syndrome type 1, the leading genetic cause for combined hearing and vision loss in humans. Our published work reveals the molecular processes of how single-headed myosin- 7a assembles into a dimeric motor complex to transport cargoes. Our preliminary results also show that purified sans and harmonin-b co-migrate with myosin-7a along actin, suggesting an active transport mechanism for stereociliar targeting. We further show that harmonin-b exhibits a remarkable phase-transition behavior, driving the assembly of higher-order Usher-1 interactome and the formation of the upper tip link density (UTLD) within hair cell stereocilia. In light of these findings, we propose our central hypothesis: Myosin-7a, sans, and harmonin- b co-assemble as a transport complex to localize to stereociliar tip-ends. Within the stereocilia, myosin- 7a/sans/harmonin-b complexes bind to cadherin-23 and are confined at the upper end of the tip link, leading to increased local concentration and high-order Usher-1 interactome assembly. This ultimately results in a matured UTLD that couples force transmission from the myosin motors to the tip link. To test this model, we will use integrated biochemical, structural, and state-of-the-art single molecule techniques to 1): determine the molecular mechanisms enabling USH1 proteins to localize to stereocilia; 2) elucidate the mechanisms driving the high- order USH1 network assembly. We will also study the effects of Usher syndrome mutations on these processes. Completion of these studies will answer longstanding questions regarding myosin-based cargo transport in hair cell stereocilia, and provide new insights into the mechanisms of human syndromic and non-syndromic deafness arising from defective Usher molecules.