Calcium and Integrin-binding protein 2 (CIB2) controls force sensitivity of the mechanotransducer channels in cochlear outer hair cells.

Calcium and Integrin-Binding Protein 2 (CIB2) is an essential subunit of the mechano-electrical transduction (MET) complex in mammalian auditory hair cells. CIB2 binds to pore-forming subunits of the MET channel, TMC1/2 and is required for their transport and/or retention at the tips of mechanosensory stereocilia. Since genetic ablation of CIB2 results in complete loss of MET currents, the exact role of CIB2 in the MET complex remains elusive. Here, we generated a new mouse strain with deafness-causing p.R186W mutation in Cib2 and recorded small but still measurable MET currents in the cochlear outer hair cells. We found that R186W variant causes increase of the resting open probability of MET channels, steeper MET current dependence on hair bundle deflection (I-X curve), loss of fast adaptation, and increased leftward shifts of I-X curves upon hair cell depolarization. Combined with AlphaFold2 prediction that R186W disrupts one of the multiple interacting sites between CIB2 and TMC1/2, our data suggest that CIB2 mechanically constraints TMC1/2 conformations to ensure proper force sensitivity and dynamic range of the MET channels. Using a custom piezo-driven stiff probe deflecting the hair bundles in less than 10 µs, we also found that R186W variant slows down the activation of MET channels. This phenomenon, however, is unlikely to be due to direct effect on MET channels, since we also observed R186W-evoked disruption of the electron-dense material at the tips of mechanotransducing stereocilia and the loss of membrane-shaping BAIAP2L2 protein from the same location. We concluded that R186W variant of CIB2 disrupts force sensitivity of the MET channels and force transmission to these channels.

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.