Activity-Dependent Plasticity In The Cochlear Hair Cell Stereocilia Cytoskeleton And Its Effect On Mechanotransduction

The sensory organelles of the inner ear hair cells are actin-filled projections termed stereocilia. In the cochlea, sound-induced vibrations lead to the deflection of stereocilia and the consequent gating of mechanotransducer channels. Stereocilia in the mammalian auditory hair cells are typically organized in three rows of increasing height in a staircase manner.The actin cytoskeleton within these stereocilia is remarkably stable and it only seems to undergo active remodeling at the stereocilia tips. We have recently identified that such stability depends on the standing current through mechanotransducer channels. Using scanning electron microscopy, we found that a reduction in mechanotransduction current leads to the shortening of stereocilia from the rows that harbor mechanotransducer channels (i.e. middle and short rows). These results were observed either through the pharmacological blockage of mechanotransducer channels or after the removal of the extracellular links that gate these channels (i.e. tip links). Stereocilia were able to regrow upon blocker washout or tip link regeneration, which indicates the presence of a previously-unknown form of stereocilia morphology plasticity that is dependent on the mechanotransducer current. Specific Aim 1: To examine the initial morphological changes in live hair cell stereocilia in response to a decrease or increase in MET current. Given the inability of SEM to image a live cell over time, we were unable to identify any nanoscale changes that could have occurred at shorter time scales (e.g. minutes) after the blockage of the MET current. Hopping probe ion conductance microscopy (HPICM) is a non-contact type of probe microscopy technique that can obtain 3D images of cell topography at nanoscale resolution. Recently, we have optimized the resolution and imaging speed of the HPICM setup in the laboratory, and we can now obtain nanoscale time-lapse images of live hair cell bundles for several hours without disrupting the cohesiveness of the bundle. HPICM will be used to determine the time course of stereocilia retraction immediately after the blockage of MET channels, and to examine the morphological changes in stereocilia after an increase in Ca2+ influx (driven by bundle overstimulation with a fluid jet or with Ca2+ ionophores). The HPICM setup will be equiped with a temperature controller to be able to perform these experiments at 37°C. Specific Aim 2: To determine the effect of transducing stereocilia height changes on bundle sensitivity. It is unclear how changes in the staircase arrangement of the stereocilia bundle could AHRF Regular Grant Application – Principal Investigator: A. Catalina Vélez-Ortega Page 2 alter its sensitivity to sound-induced deflections. The blockage of MET channels for 24 hours did not appear to disturb the tip links. Therefore, the preferential retraction of transducing stereocilia could potentially impact the angle and/or resting tension of the tip links, which should result in changes to the displacement-current relationship in hair cells. Patch-clamp recordings will be performed to examine any displacement-current relationship changes after stereocilia retraction. Significance: The ability of stereocilia to retract and regrow in response to changes in the mechanotransduction current, points to a novel type of activity-dependent plasticity in the stereocilia actin core. In physiological conditions, this type of cytoskeleton remodeling could occur after an event of noise exposure that leads to the breakage of tip links. Changes in stereocilia morphology could impact the overall sensitivity of the bundle to sound-induced vibrations and, therefore, could represent a novel mechanism of hearing sensitivity regulation at the level of the hair cell sensory machinery. Elucidating the physiological relevance of this activity-dependent plasticity in transducing stereocilia would fuel research aimed at finding the molecular machinery responsible for this type of actin remodeling, which might expose genes that confer susceptibility to noise-induced hearing loss.