Mechanotransduction-Dependent Remodeling of the Stereocilia Cytoskeleton

Stereocilia are the sensory machinery of the inner ear sensory hair cells. We recently found that the stability of these actin-filled projections depends on the constant influx of calcium through the mechano-electrical transduction (MET) channels that are located at the tips of stereocilia (Velez-Ortega, et al., eLife, 2017). We found that the blockage of MET channels or the breakage of their gating tip links leads to the shortening of stereocilia; but once the blockage is removed or the tip links regenerate, stereocilia are able to regrow. Based on the changes in stereocilia height in response to variations in the resting MET current, I hypothesize that the stereocilia actin cytoskeleton exhibits activity-dependent plasticity. This project constitutes the first step in the search for the molecular players involved in the MET-dependent remodeling of the stereocilia cytoskeleton. Aim 1 will directly test for changes in the actin cytoskeleton after manipulations that increase or decrease the calcium influx through MET channels. Mouse cochlear hair cells will be transfected with fluorescently-labeled actin isoforms to allow for the quantification of actin incorporation (fluorescence recovery after photobleaching), or the renewal of actin filaments via treadmilling (tracking of discrete photoconverted regions along the stereocilia length). In addition, changes in the ratio of â-actin to ã-actin in the stereocilia core will be assessed via immunohistochemistry. Aim 2 will test for the contribution of myosin 15 (MYO15) isoforms in the MET-dependent regulation of the stereocilia height. MYO15 is a non-conventional myosin motor that delivers other proteins to the tips of stereocilia. Mouse mutants carrying defects in one (Myo15ÄN/ÄN) or two (Myo15sh2/sh2) MYO15 isoforms exhibit mechanotransduction currents, but impaired stereocilia maintenance and growth. Therefore, it is plausible that MYO15 delivers some of the key proteins involved in the MET-dependent regulation of the stereocilia cytoskeleton. In this aim, we will use electron microscopy to test the effects of variations in the calcium influx through MET channels on mouse cochlear hair cells from Myo15ÄN/ÄN and Myo15sh2/sh2 mice. MET-dependent changes to stereocilia morphology could have an impact on the sensitivity of hair cells. Moreover, stereocilia shortening—and perhaps eventual stereocilia disappearance—could occur after noise exposure (if the gating tip links of MET channels break) or in certain cases of congenital deafness (due to impaired MET currents). Therefore, the results of this project will expand our knowledge of the molecular machinery involved in the control of hearing sensitivity and the prevention of hearing loss.