Fig. 8: LOXHD1 and TMC1 are part of protein complexes attached to the tip-link branches in vivo, and molecular interaction models between TMC1 and the tip link, with or without LOXHD1.
a In SUB experiments on P21 IHCs, some row 2 tips are detached from the rest of the stereocilia but still connected to the tip links (arrowheads and high magnification in the lower panels). Anti-HA gold-beads were specifically found at detached row 2 tips for LOXHD1-HA and TMC1-HA but not in the WT sample. Scale bars: 500 nm and 50 nm for the low and high mag pictures, respectively. b Quantification of the percentage of P21 IHC detached row 2 tips containing gold. Loxhd1HA/HA anti-HA: ncells = 17, ndetached tip with gold / total = 31/54; WT anti-HA: ncells = 5, ndetached tip with gold / total = 0/11; Tmc1HA/HA anti HA: ncells = 10, ndetached tip with gold / total = 34/44; WT anti-BAIAP2L2: ncells = 14, ndetached tip with gold / total = 1/42. The experiment was performed once. c Cartoon representing the mature auditory mechanotransduction protein complex in the wild-type (WT). d Model summarizing the molecular connections between TMC1 and PCDH15. e Cartoon representing the mature auditory mechanotransduction protein complexes in the absence of LOXHD1: initially, a population of TMC1 channels still functions while a second population, progressively increasing in proportion, is physically disconnected from the tip-link and cannot receive sound-induced forces. f Model summarizing the molecular connections between TMC1 and PCDH15 persisting upon the absence of LOXHD1: some TMC1/LHFPL5/TMIE proteins are initially sufficient to maintain some TMC1 channels at the tip link. However, with time, more and more TMC1 is found at a distance from the PCDH15, with LHFPL5 remaining attached to PCDH15 along with some TMIE molecules. A subset of TMIE proteins is predicted to remained attached to the mislocalized TMC1 molecules. The predicted localization of CIB2 remains hypothetical. However, considering that CIB2 can interact with BAIAP2L2 and is necessary for proper localization of BAIAP2L2 in hair bundles54, and given that we observed BAIAP2L2 mislocalization similar to TMC1 at P21, it is probable that CIB2 is also associated with the mislocalized TMC1 molecules and absent from the stereocilia tip. Predicted positions are indicated with transparent colors. Source data are provided as a Source Data file.
