Thursday, November 21, 2024
11/21/2024
Project Summary Congenital hearing loss affects about 2 to 3 out of every 1,000 children in the USA. Many genes associated with congenital deafness encode proteins that are essential for formation of the actin-based mechanosensory stereocilia in the inner ear hair cells. A number of molecules responsible for growth of stereocilia and establishing their perfect staircase-like arrangement within a hair bundle have been identified. However, less is known about the proteins shaping the base of stereocilia. Yet, this is exactly the point, around which the rod-like stiff stereocilium rotates during sound-induced vibrations. Therefore, the molecules located at stereocilia base 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, we and others identified TRIOBP4/5, Fam65b/RIPOR2, and taperin (TPRN) 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. Here, we propose a central hypothesis 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 TPRN at the base of stereocilium. This compartment anchors mechanically the rootlet and prevents pulling the stereocilium out of cuticular plate. It also controls disassembly of supernumerary stereocilia during postnatal development. This central hypothesis is well supported by our preliminary data. We have found that TPRN crosslinks actin filaments in vitro and disrupts stereocilia actin core when overexpressed in vivo. We have also identified interacting partners of TPRN that form protein complex at the base of stereocilia. We have generated several mouse models with genomic truncations or deletions in Tprn gene, including deletion that does not disrupt F-actin crosslinking in vitro. Preliminary studies revealed structural and functional abnormalities in the auditory hair cell stereocilia of these mice. To test our hypotheses further, we will: i) determine how exactly TPRN organizes F-actin in vitro; ii) determine the effects of taperin deficiency on stereocilia F-actin in vivo; iii) elucidate the effects of TPRN deficiency on mechanical properties of stereocilia bundles; and iv) determine how TPRN-based complex affects normal disassembly of supernumerary stereocilia in postnatal development. Overall, our study will elucidate the molecular machinery that shapes stereocilia at their base, thereby determining their optimal mechanical properties that are essential for normal hearing.
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