PROJECT SUMMARY/ABSTRACT Our ability to hear relies on a small population of specialized sensory ‘hair’ cells in the inner ear that cannot regenerate upon damage. Variants of over 100 human genes have been associated with deafness, many of which alter the structure of the sensory organelle in hair cells, called the hair bundle. The hair bundle consists of organized rows of graded-height membrane protrusions, or stereocilia, on the surface of hair cells. Hair bundle formation during development and its precise architecture during life are vulnerable to a variety of genetic, environmental, and age-related insults, all of which result in the decline or complete loss of hearing ability. My overarching goal is to characterize protein interactions that are vital to the hair bundle’s development and lifelong maintenance. In this proposal, I identify a new role for a calcium-binding protein enriched in the mouse hair bundle, specifically, at the tip of the tallest stereocilia. A group of five proteins, referred to as the Elongation Complex, was previously reported at this same compartment. Loss of any member of this complex prevents proper stereocilia elongation, blurs the distinct identity of stereocilia across rows, and results in profound deafness in both humans and mice. Interestingly, similar defects have been reported in mouse mutants lacking key components of the mechanoelectrical transduction channel. This suggests that, via transduction, active hair bundles somehow influence elongation factors, and thus stereocilia dimensions. I propose that the calcium-binding protein studied in this proposal is a new binding partner and regulator for MYO15A, the myosin motor that transports other Elongation Complex proteins to stereocilia tips. MYO15A was one of the first proteins associated with hearing loss, and MYO15A mutations are the third most common origin of heredity deafness in humans. Aim 1 of this proposal will determine the relationship between MYO15A and our calcium-binding protein, and confirm preliminary results suggesting that this new protein is essential for auditory function. Aim 2 will investigate how changes in calcium levels in the hair bundle upon transduction affect the dynamic localization of our protein and its Elongation Complex partners. Together, these aims identify and investigate this new stereocilia protein as an additional member of the crucial Elongation Complex, and as a mediator that reads hair cell transduction activity to influence stereocilia growth. This project employs cutting- edge mouse models, advanced techniques to culture the auditory organ, and high-resolution imaging of preserved and live hair cells. Completion of the project will contribute to my long-term goal of informing therapeutic strategies to ensure the proper development and lifelong preservation of hair cells.