Role of class III myosins in governing inner ear hair cell stereocilia length and width - Project Summary The stereocilia of inner ear hair cells are parallel actin bundle based protrusions that must be precisely formed from microvilli structures during development and maintained throughout a lifetime to allow for normal hearing and vestibular function. The stereocilia form a staircase pattern with precise length and width required for normal function in mechanotransduction. Class III myosins (MYO3A and MYO3B) are crucial for forming proper stereocilia length and width, while mutations in MYO3A lead to delayed onset deafness and stereocilia degeneration. The knockout of both MYO3A and MYO3B leads to long and thin stereocilia and profound deafness in a mouse model. We propose that MYO3A is essential because it functions as a tension sensor at the tips of actin protrusions, which is crucial for mediating the balance of forces that govern stereocilia length regulation. We also proposed that MYO3B can only partially compensate for the lack of MYO3A because it lacks specific motor properties, such as the ability to change its motor properties in response to assistive and resistive loads. Indeed, our preliminary data suggests MYO3A has a unique load sensitivity different from any other myosin characterized to date. In addition, our preliminary data and published studies demonstrate that MYO3A can regulate a dynamic population of actin at the stereocilia tips, which we call tip filaments. MYO3A may stabilize the tip filaments which is crucial for the stereocilia widening process. In Aim 1 we will investigate the key differences between MYO3A and MYO3B motors at the biochemical and biophysical level to determine mechanistically why MYO3B cannot compensate of the loss of functional MYO3A. Thus, ensemble and single molecule biophysical studies will characterize the most important mechanical properties of MYO3A&B. In Aim 2 we will investigate the impact of deafness-associated mutations in MYO3A motor domain, which will lay the foundation for understanding the key aspects of MYO3A motor function required for its function in stereocilia. In Aim 3 we will investigate the impact of MYO3B and MYO3A deafness mutations on stereocilia length, width, and tip filament actin turnover. We will also investigate the role of MYO3A binding partners, ESPIN and MORN4, in mediating the stability of the tip filaments and stereocilia length/width. Overall, our comprehensive approach will reveal novel insights into MYO3A function in actin protrusions and lay the foundation for the development of therapies to treat inner ear hair cell stereocilia degeneration.
