Abstract
Stereocilia tip links of inner ear hair cells are subjected to constant stretching during hair-bundle deflection, and accordingly are well designed to prevent from being broken by mechanical tensions. The roots of tip links, which couple tip links with the cytoskeleton, supposedly play important roles in withstanding large forces under stimulated conditions. The upper root of the tip link is mainly formed by the cytoplasmic tail of cadherin23 and its actin-anchoring protein harmonin. However, the detailed organization mode of the two proteins that gives rise to a strong upper root remains unclear. Here we show that the exon68-encoded peptide of cadherin23 can either interact with the N-terminal domain (NTD) of harmonin or form a homodimer. We demonstrate that the three harmonin binding sites of cadherin23, namely the NTD-binding motif, the exon68 peptide, and the C-terminal PDZ binding motif, do not synergize with each other in binding to harmonin, instead they facilitate formation of polymeric cadherin23/harmonin complexes. The exon68 peptide can promote the cadherin23/harmonin polymer formation via either binding to harmonin NTD or self-dimerization. We propose that the polymeric cadherin23/harmonin complex formed beneath the upper tip link membranes may serve as part of the stable rootlet structure for anchoring the tip links of stereocilia.
Highlights
How the root of stereocilia tip link is designed to withstand mechanical stretching is poorly understood
Exon68 Binds to the N-terminal Domain of Harmonin—Previously we found that the ␣-helix encoded by cadherin23 NBM binds to the solvent-exposed pocket of harmonin N-terminal domain (NTD) through hydrophobic interactions involving Leu-3188, Ile-3192, and Tyr-3195 of NBM [16]
Deletion of this NBM did not abolish the specific interaction between the cytoplasmic tail of cadherin23(ϩ68) and harmonin NTD, indicating the existence of an additional NTD binding site in the cadherin23 tail
Summary
How the root of stereocilia tip link is designed to withstand mechanical stretching is poorly understood. The upper root of the tip link is mainly formed by the cytoplasmic tail of cadherin and its actin-anchoring protein harmonin. Cadherin assembles the upper part of tip link, and its cytoplasmic tail is anchored to the actin filaments of stereocilia via binding to the actin-binding protein harmonin [9]. The three harmonin binding sites of cadherin, the exon peptide, NBM, and PBM, do not synergize with each other in binding to harmonin Instead these three regions facilitate formation of concentration-dependent, polymeric cadherin23/harmonin complexes. Considering the special role of tip link as the mechanoelectrical transduction channel gating spring, as well as the specific expression of exon in the stereocilia bundle [20], we propose that exon68-facilitated cadherin23/harmonin polymer formation may provide a mechanistic explanation for the formation of the sturdy upper tip link root organized by the cdherin23/harmonin/actin filaments network
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