PROJECT SUMMARY The development and function of hair cell stereocilia in the cochlea are essential for our sense of hearing, as they play a pivotal role in detecting sound through their mechanosensory capabilities. Stereocilia, organized into rows of graded height on the hair cell surface, are susceptible to damage caused by loud sounds and the aging process, leading to irreversible hearing loss. The architecture of stereocilia is critical for detecting sound and is regulated by a structural protein called actin that forms a rigid scaffold of filaments within these structures. My overarching goal is to understand the regulatory mechanisms that govern actin filament growth and ensure the correct development of stereocilia. In this proposal, I explore the function of the molecular motor protein myosin 15 (MYO15A) that controls actin filament assembly and stereocilia size. Mutations in MYO15A cause human hereditary hearing loss, DFNB3, underlying the essential activity of this protein in the cochlea. The central focus of my proposal is to discover how MYO15A and its associated proteins, known as the 'elongation complex' (EC), can control actin polymerization and stimulate stereocilia growth. My preliminary data along with the published work of others, have revealed a unique ability of MYO15A and EC proteins to form biomolecular condensate that are hypothesized to form the stereocilia tip density that controls actin filament elongation. The properties of these MYO15A-EC condensates are poorly understood, but highly relevant to stereocilia biology. In this proposal, I will conduct a comprehensive characterization of the biophysical properties of MYO15A-EC tip density condensates and explore their potential as reaction compartments optimized for actin filament growth. In Aim 1, I will measure the material properties and microrheology of purified MYO15A-EC tip density condensates, revealing their structural makeup. In Aim 2, I will test the hypothesis that MYO15A-EC tip-density condensates can potentiate actin filament growth. This work will utilize cutting-edge experimental approaches including optical trapping force spectroscopy and single-molecule microscopy techniques. The results from these experiments will reveal the fundamental properties of MYO15A-EC tip density condensates and advance our understanding of how stereocilia are built and maintained. Completion of this project will advance my long-term goal of manipulating stereocilia biology therapeutically to treat hearing loss in patients.