Fig 1: CIB2 isoforms and hearing sensitivity in CIB2 −/− mice ADomain structure of the CIB2 protein, indicating the positions of the CIB2 mutations in USH1J (red) and DFNB48 (green) patients. The CIB2‐floxed mice, CIB2 fl/fl, were engineered by adding LoxP sites on either side of exon 4, which is common to all four known CIB2 transcripts.BRT–PCR analysis confirming the loss of CIB2 exon 4‐containing transcripts in the inner ear, eye, brain, muscle, kidney and testis of CIB2 −/− mice. β‐Actin was used as an endogenous control.C, DABR thresholds (C) and DPOAE amplitudes (D) in CIB2 +/− (dark, n = 6) and CIB2 −/− (red, n = 9) P20‐P25 mice. ABR thresholds in CIB2 −/− mice exceeded 100 dB SPL (mean ± SD), indicating profound deafness. (D) DPOAE amplitudes were absent in CIB2 −/− P20 mice at 10 and 15 kHz (red) (Mann–Whitney, ***P = 0.002 for both 10 and 15 kHz).EMET responses in OHCs from CIB2 +/− and CIB2 −/− P7 mice. The left panels show the mechanical stimulation protocol, with examples of MET currents for each genotype. In the right panels, the MET current values and mean amplitude‐displacement relationships (I(X)) (mean ± SD) in CIB2 +/− (black, n = 3 cells) and CIB2 −/− (red, n = 5) mice highlight the absence of a MET response in CIB2 −/− OHCs (Welch's unpaired t‐test, ****P = 0.0007).
Fig 2: Abnormal architecture of cochlear hair bundles in CIB2 −/− miceRepresentative scanning electron microscopy micrographs of the hair bundles in the hair cells of CIB2 +/− and CIB2 −/− P9 and P18 mice. A, BUnlike the cohesive and highly organized V‐shaped hair bundle in CIB2 +/− mice (A), various structural abnormalities are observed in the absence of CIB2, for example horseshoe‐like shape, stereociliary shortening, total loss of short row of stereocilia or split bundles (B).CSeveral examples of IHC hair bundles in CIB2 −/− P18 mice illustrating the heterogeneous lengths within IHC stereocilia rows and the persistence in some IHC bundles of the kinocilium (artificially coloured in green).Data information: Scale bars: 2 μm.
Fig 3: CIB2 in the cochlear hair bundles and the apical surface of hair cells A–CLocation of CIB2 in F‐actin‐labelled (red) IHCs and OHCs. (A) On P7, specific labelling (absent from CIB2 −/− mice, right panels) was observed in the basal body of the kinocilium (open arrowheads) at the apical surface of hair cells, and in the stereocilia, mostly in the basolateral region (arrows). Some unspecific labelling is observed in the supporting cells (asterisk in A). (B, C) On P10 (B) and P20 (C), CIB2 immunostaining was detected in different regions of the stereocilia, including their tips (arrowheads), and at the apical surface of hair cells (arrows), around the cuticular plate.DRegardless of the stage analysed (P7, P10 or P20), no significant immunostaining was observed in the vestibular hair cells.Data information: Scale bars: 2 μm.
Fig 4: The distribution of myosin VIIa, harmonin, protocadherin‐15, integrins β1 and CIB1 in CIB2 −/− mice A–CExamples of outer hair cells (OHCs) from CIB2 −/− and CIB2 +/− P7 mice. There is no evidence for a change in the stereocilia‐immunostaining of myosin VIIa (A) harmonin (B) and protocadherin‐15 (C) immunostaining (green) in the absence of CIB2.DFor integrin β1, a very minor change could be observed, visible mainly in IHCs (see close‐up view). Rather than a restricted staining at the base (arrowheads on the IHC bundle) of the stereocilia, the immunostaining is slightly diffuse, extending along the stereocilia in the absence of CIB2 (arrows). No such change is observed in the hair bundles of vestibular hair cells.EIn CIB2 +/− P20 mice, CIB1 is located at the apical surface of the hair cells, concentrated at the cell periphery nearby the basal body of the kinocilium. In CIB2 −/− P20 mice, the CIB1 immunostaining is, instead, found at the apical cell centre of the hair cell.Data information: Scale bars: 2 μm.
Fig 5: Schematic representation illustrating the key steps during the morphogenesis of the hair bundle and the role of CIB2 in the apical region of the cochlear hair cells AIn the mouse differentiating hair cells, the migration of the kinocilium from the cell centre to the cell periphery between E14.5 and E17.5 initiates a differential growth of the stereocilia; those closest to the kinocilium grow faster and longer forming the tallest stereocilia row. The organization into a highly arranged staircase pattern then takes place and is stabilized during the first postnatal week, when the kinocilium and extra‐numerary stereocilia disappear and the proper shape of the hair bundle is refined. The molecular and structural organization is set by P12–P14 at hearing onset and is maintained over time to ensure proper transduction of sound.B, CCIB proteins have been proposed to function as broad regulators of integrin function. (B) At the stereocilia tips, it is yet unknown how lack of CIB2 results in total loss of MET current responses. Possible direct link between CIB2 and the TMC1/2 MET channel complex has been proposed (Giese et al, 2017). (C) Because lack of CIB2 has been shown to alter whirlin and integrin α8 staining at the basolateral regions of the stereocilia, it is likely that this protein is part of a signalling platform, probably through multimers of CIB2, influencing the integrin‐mediated essential link between the extracellular matrix and the intracellular cytoskeleton of the hair bundle. It has been suggested that CIB proteins activate integrin (1) (Hynes, 2002; Hager et al, 2008), in a CIB2‐mediated “inside‐out” signalling (2) process promoting integrin ligand binding, which in turn would affect potential “outside‐in” signalling (3) coupling the extracellular matrix (e.g. the α8 subunit) and intracellular responses in the hair bundle (e.g. whirlin and/or FERM proteins). Such a crosstalk might be essential to maintain the shape and stereocilia integrity of the mature functioning hair bundles.
Supplier Page from Abcam for Anti-CIB2 antibody