Taperin-Based Macromolecular Complex at the Base of Stereocilia

SPECIFIC AIMS Many genes associated with congenital deafness encode proteins that are essential for formation of the actin- based mechanosensory stereocilia bundles of the inner ear hair cells. Although many molecules responsible for stereocilia growth and establishing their perfect staircase-like arrangement have been identified, less was known about the proteins shaping the base of stereocilia. However, this is exactly the point, around which the rod-like stiff stereocilium rotates during sound-induced vibrations. Therefore, the molecules located there determine both the mechanical properties of the hair bundle (and hence its overall sensitivity) and its susceptibility to excessive deflections (acoustic trauma). Only in the last two decades the identity of these molecules started to emerge. We were the first to identify TRIOBP4/5, Fam65b/RIPOR2, and taperin as the proteins essential for formation of the rootlets of the auditory hair cell stereocilia, shaping stereocilia “taper”, and determining mechanical properties of the hair bundle {refs}. Here, we combine our expertise and propose an idea that, in addition to the already known distinct actin compartments inside stereocilium - at the very tip (stereocilia growth), shaft (widening and stiffening), and rootlets (resilient deflections) - there is a distinct compartment of F-actin that is cross-linked by taperin at the base of stereocilium. This compartment anchors mechanically the rootlet and prevents pulling the stereocilium out of cuticular plate. It also prevents disassembly or “pruning” of stereocilium during postnatal development. These hypotheses are based on our preliminary data and will be tested in following specific aims: AIM 1: Determine how taperin organizes F-actin in vitro. In the preliminary experiments, we have demonstrated that: i) taperin induces formation of higher-order F-actin structures in vitro; ii) taperin N-terminus is essential for actin binding; and iii) taperin is able to dimerize. Furthermore. AlphaFold2 modeling predicts taperin binding to both barbed and pointed ends of an actin monomer. Here, we will further characterize the domains and residues essential for all these interactions in biochemical and ultrastructural assays. Using Total Internal Reflection Fluorescence (TIRF) Microscopy imaging of F-actin assembly in vitro, we will determine whether taperin promotes side-by-side bundling, interconnecting, and/or end-by-end annealing of actin filaments. AIM 2: Determine the effects of taperin deficiency on stereocilia F-actin in vivo. Using various electron microscopy techniques, we will compare the abnormalities in the auditory hair cell stereocilia and their rootlets and F-actin core in Tprn knockout mice (TprnΔ43) and in mice (TprnΔ260) expressing truncated variant of taperin (taperin1-259) that is still able to cross-link F-actin. By immunostaining of these mutants, we will also explore potential mis-localization of key proteins that are known to be essential for rootlet and stereocilia formation. AIM 3: Test the hypothesis that taperin complex is essential for mechanical “anchoring” of F-actin at the base of stereocilia. Our preliminary recordings of the mechano-electrical transduction (MET) currents in Tprn-/- and TprnΔ260 auditory hair cells revealed very unusual “dips” in the MET current during ramp-like positive deflections of the hair bundles with fluid-jet. These “dips” were highly repeatable in consecutive MET current traces at negative but not positive holding potentials, indicating potential modulation by Ca2+ influx through MET channels. We hypothesized that this phenomenon may be caused by elastic pulling of stereocilia from cuticular plate, which would transiently release the tension in the MET apparatus. Consistent with this hypothesis, electron microscopy revealed evidence of such pulling at the base of stereocilia, while high-speed video recordings showed transient disruptions of the movements of the tips of stereocilia during ramp deflections of the hair bundles. Here, we will test this hypothesis by recording simultaneously stereocilia movements at the tips and the base of the hair bundle. Although RIPOR2 proteins do not interact directly with taperin, they encircle taperin and may tighten taperin-linked actin filaments at the stereocilia base. Electron microscopy demonstrates similar “pulling” of stereocilia in Ripor2-/- hair cells, while our preliminary data indicate that RIPOR2 polymerization is Ca2+-dependent. Therefore, we will also compare MET current recordings, video recordings of hair bundle deflections by fluid jet, and stereocilia base abnormalities in Tprn-/- and Ripor2-/- hair cells. AIM 4: Test the hypothesis that taperin/PPP1CB complex determines stereocilia "pruning" in postnatal development. In the preliminary experiments, we showed that PPP1CB interacts with taperin, concentrates at the base of stereocilia, and inhibits F-actin cross-linking by taperin in vitro, even though PPP1CB alone (without regulatory subunit) has been reported to have no enzymatic activity. We generated hair cell-specific Ppp1cb-/- mice (Ppp1cbflox/flox;Atoh1-Cre). Surprisingly, the normal developmental “pruning” of supernumerary stereocilia in hair cells of these mice is arrested, even though it is exaggerated in Tprn-/- mice. Therefore, here, we will test the hypothesis that PPP1CB/taperin ratio controls this developmental pruning by: i) examining developmental changes of PPP1CB/taperin expression at the base of stereocilia; ii) overexpressing PPP1CB or taperin in wild- type, TprnIn103 and Ppp1cb-/- mice with AAV-mediated gene transfer; iii) trying to rescue abnormal stereocilia pruning in Ppp1cb-/- and Tprn-/- hair cells by reducing taperin and PPP1CB expression, correspondingly; and iv) determining whether enzyme-dead PPP1CB directly regulates taperin-induced F-actin cross-linking in vitro.