Expression and generation of Cib2-mutant mouse strains

To determine the role of CIB2 in the inner ear, we generated a p.F91S missense mutation knockin mouse (Cib2 F91S) (Fig. 1a), which corresponds to the most prevalent CIB2 allele found in human families with nonsyndromic deafness19. We also used Cib2 tm1a mutant mice. These mice carry a gene trap cassette with a lacZ reporter between exons 3 and 4 (Fig. 1a). The gene trap leads to the translation of a truncated protein consisting of only the first 66 amino-acids of CIB2. Homozygous Cib2 tm1a/tm1a and Cib2 F91S/F91S mutant mice are fertile and appear healthy. Cib2 tm1a mice were crossed with ubiquitous Cre expressers (C57BL/6NTac-Tg(ACTB-cre)3Mrt/H) to delete the neomycin cassette and exon 4 of Cib2 (Cib2 tm1b; Fig. 1a). Cib2 is ubiquitously expressed (Fig. 1b, Supplementary Fig. 1a)19. Phenotyping of multiple organs from Cib2 tm1b mice, missing the neo cassette (Fig. 1a), revealed abnormal voluntary movements, circulating high-density lipoprotein-cholesterol level, and heart left ventricle morphology, in addition to the loss of the startle response and elevated auditory thresholds (Supplementary Table 1). X-gal staining and β-gal immunostaining revealed that Cib2 is highly expressed in sensory hair cells of both the organ of Corti and the vestibular system. We did not observe any changes of Cib2 expression during the first postnatal week or differences in Cib2 expression along the length of the cochlea (Fig. 1b, Supplementary Fig. 1b).

Fig. 1 CIB2 is localized in the auditory hair cell stereocilia. a Structure of the wild-type, the Cib2 F91S and the Cib2 tm1a(EUCOMM)Wtsi (Cib2 tm1a, EUCOMM) alleles. b X-gal staining to document the Cib2 locus activity in the organ of Corti of P12 Cib2 tm1a/+ mice (left). Immunolocalization of β-galactosidase (green) in Cib 2tm1a/+ mouse cochlea at P12 (right). The cochlear actin is labeled with rhodamine–phalloidin (red). c, d Confocal images of IHC (c) and OHC (d) stereocilia bundles immunostained with CIB2 antibody (middle panels in c and red in the merge panels), and actin was labeled with phalloidin (left panels in c and green in the merge panels) in the control Cib2 tm1a/+, Cib2 tm1a/tm1a, and Cib2 F91S/F91S mice at P12. Note that Cib2 truncation causes disappearance of CIB2 protein from stereocilia, while p.F91S mutation does not. e CIB2 localization in the rat IHC stereocilia at P10. f CIB2 localization at P12 in Cib2 F91S/+ OHCs. Scale bars: b 10 μm, c–f 5 μm Full size image

Using an antibody directed against the N-terminus, wild-type CIB2 was observed in the stereocilia and at the tips of surrounding microvilli of the auditory hair cells of control heterozygous mice (Fig. 1c, top). We detected CIB2 in the stereocilia of cochlear hair cells at P12 and as early as P5 (Fig. 1c, Supplementary Fig. 2a, b). Similar labeling was observed in rat auditory hair cells (Fig. 1e). We were not able to detect CIB2 immunolabeling in the stereocilia at earlier ages, probably due to relatively low affinity of our antibodies. According to the SHIELD28 database, Cib2 is expressed in the organ of Corti hair cells as early as embryonic day 16. We also detected prominent lacZ reporter expression driven by Cib2 promoter at postnatal day 0. Furthermore, we demonstrated that exogenous CIB2 is properly targeted to the auditory hair cell stereocilia as early as P2-P319. In the inner hair cells (IHCs) of control heterozygous animals, CIB2 was distributed along the length of stereocilia and accumulated at the tips of the shortest (but still mechanotransducing3) stereocilia (Fig. 1c, top). In the outer hair cells (OHCs), we observed punctate labeling along the length of stereocilia, including labeling at the very tips of OHC stereocilia (Fig. 1d, top and Fig. 1f). CIB2 immuno-labeling was specific, because it completely disappeared from stereocilia bundles of Cib2 tm1a/tm1a mice (Fig. 1c, d, middle). Therefore, our new immunolabeling data are consistent with the previously hypothesized role of CIB2 in mechanotransduction19, although its additional role outside the MET complex cannot be excluded.

Since our antibodies were raised against N-terminus (amino acid 1–99), they may recognize not only wild-type CIB2 but also truncated CIB2 in Cib2 tm1a/tm1a mice and mutated CIB2 in Cib2 F91S/F91S mice. In contrast to the absence of truncated CIB2 in Cib2 tm1a/tm1a stereocilia (Fig. 1c, d, middle), the p.F91S point mutation does not affect CIB2 labeling in the stereocilia of Cib2 F91S/F91S mice (Fig. 1c, d, bottom). This is consistent with our previous observation that exogenous CIB2 protein with p.F91S variant is properly targeted to the stereocilia in the cultured vestibular sensory epithelia29. Thus, we concluded that p.F91S mutation does not disrupt localization of CIB2 in hair cell stereocilia.

Cib2 mutant mice are deaf

To characterize hearing function, we measured auditory-evoked brainstem responses (ABR) in Cib2 mutant and control littermate mice at P16. Normal ABR waveforms and thresholds were observed in wild-type and heterozygous mice. However, both Cib2 tm1a/tm1a and Cib2 F91S/F91S homozygous mice did not respond to click or tone burst stimuli of 100 dB sound pressure level (SPL), indicating that they are profoundly deaf (Fig. 2a, b), recapitulating human DFNB48/USH1J deafness. We also recorded distortion product otoacoustic emission (DPOAE), a by-product of cochlear amplification that depends on the integrity of OHCs. At P16-P18, wild-type and Cib2 tm1a/+ and Cib2 F91S/+ heterozygous mice generated apparently normal and similar DPOAEs. However, in both Cib2 mutant mice, DPOAEs were indiscernible from the noise floor (Fig. 2c). Taken together with ABRs, these results suggest that hearing loss in CIB2 mutant mice is caused by peripheral (cochlear) deficiencies.

Fig. 2 CIB2 deficiency leads to profound sensorineural hearing loss and degeneration of the organ of Corti. a, b ABR thresholds to broadband clicks (a) and tone-pips with frequencies of 8 kHz, 16 kHz, and 32 kHz (b) in wild-type (open bar), Cib2 tm1a/+, Cib2 F91S/+ (black), Cib2 tm1a/tm1a (red), and Cib2 F91S/F91S (green) mice at P16. Number of animals in each group is shown in parentheses. The same animals were tested with clicks and tone pips. c DPOAEs of control (black; n = 13), Cib2 tm1a/tm1a (red; n = 10), and Cib2 F91S/F91S (green; n = 5) mice at P16-P18. Noise floor is shown in grey. All data in a–c are shown as Mean ± SEM (***p < 0.001). d SEM images of the organ of Corti at the apical, medial and basal turns of the cochlea in the control, and Cib2 tm1b/tm1b mice at P14, P21 and P110. Scale bar: 10 μm Full size image

Despite prominent hearing loss, both Cib2 tm1a/tm1a and Cib2 F91S/F91S mice display no obvious indications of vestibular dysfunction, such as circling, hyperactivity, or head bobbing. The absence of an overt vestibular phenotype in both Cib2 mutants indicates that in contrast to humans, CIB2 function is likely to be redundant in mouse vestibular hair cells. To test this possibility, we used quantitative reverse transcription polymerase chain reaction to investigate the expression of the Cib gene family (Cib1-4) in cochlear and vestibular sensory epithelia at P12. In the control mice, the most dramatic difference between vestibular and cochlear tissues was observed for Cib3, which showed an almost 8-fold higher expression in the vestibular samples (Supplementary Fig. 3a). In Cib2 tm1a/tm1a mice, CIB2 deficiency resulted in significant upregulation of Cib1 in the cochlea (Supplementary Fig. 3b) and slight, not statistically significant, upregulation of Cib3 in the vestibular periphery (Supplementary Fig. 3b). Mouse CIB2 is 61% identical and 78% similar to CIB3 and both proteins share identical domain structure with three EF hand domains. Therefore, functional redundancy between these proteins is plausible. Substantially larger expression of Cib3 in the vestibular periphery (Supplementary Fig. 3b) may account for the relative insensitivity of the vestibular end organs to the loss of Cib2 compared to the cochlea.

Surprisingly, we did not observe any differences in ABR or DPOAE between Cib2 tm1a/tm1a mice that have no CIB2 in the stereocilia and Cib2 F91S/F91S mice that still have mutant CIB2 at the stereocilia tips. We conclude that, while the F91S mutation does not affect protein localization, it does cause loss of CIB2 function. As such, the Cib2 F91S/F91S mice represent a good experimental model to study functional effects of CIB2 interactions with other stereocilia proteins.

Morphological changes in the organ of Corti of cib2 mutants

Gross morphology and cytoarchitecture of the organs of Corti in both Cib2 tm1a/tm1a and Cib2 F91S/F91S mice appear normal at postnatal day 0 (Fig. 2d, Supplementary Fig. 4a–c). However, by P16-18, OHCs of both Cib2 mutant mice started to degenerate in the mid-basal part of the cochlea, which was exacerbated by P27 (Fig. 2d, Supplementary Fig. 4b, c). IHCs remain largely intact, though some of them were missing around P27 (Fig. 2d, Supplementary Fig. 4b, c). In P110 mice, OHCs completely degenerated and only few IHCs survived (Fig. 2d). The loss of sensory hair cells was followed by the progressive degeneration of spiral ganglion neurons (Supplementary Fig. 5).

Stereocilia bundle morphology in the auditory hair cells of Cib2 mutants

Next, we evaluated the morphology of the stereocilia bundles by scanning electron microscopy (SEM). Although there was no loss of either OHCs or IHCs at P12-18 in the middle of the cochlea (Fig. 3a–c), we observed identical abnormalities in the stereocilia bundles of both Cib2 tm1a/tm1a and Cib2 F91S/F91S mice. The mutant OHC bundles had a horseshoe shape and, most importantly, a disrupted staircase architecture. Instead of building precise 2nd and 3rd rows of stereocilia in the bundle (Fig. 3d), these cells often over-grew the second row of stereocilia, while the third rows were either over-grown (Fig. 3e, f, arrows up) or retracted (Fig. 3e, f, arrows down). In contrast to OHCs, the staircase arrangement of IHC stereocilia was still present in both Cib2 tm1a/tm1a and Cib2 F91S/F91S mice (Fig. 3g–i). However, in mutant IHCs, the 3rd and 4th row stereocilia were abnormally thick and the kinocilium failed to regress properly (Fig. 3h, i), which was confirmed with tubulin labeling (Supplementary Fig. 6a).

Fig. 3 CIB2 deficiency disrupts stereocilia bundle morphology of the auditory hair cells. SEM images of the organs of Corti (a–c), as well as OHC (d–f) and IHC (g–i) stereocilia bundles from control heterozygous (a, d, g), Cib2 tm1a/tm1a (b, e, h), and Cib2 F91S/F91S (c, f, i) mice at the mid-cochlear location. Over-grown and retracted stereocilia are shown by arrows up and down, respectively. The IHC kinocilium does not regress properly in Cib2 mutant mice (pink). Scale bars: a–c 5 µm, (d–i) 1 µm Full size image

To explore the mechanisms of stereocilia growth dysregulation, we quantified the morphology of the OHC and IHC bundles in wild-type and Cib2 tm1a/tm1a littermates at the same mid-apical location. The staircase morphology of the Cib2 tm1a/tm1a OHC bundles showed severe disruption. At P6, this disruption was evident as an increased variability in heights of the shorter row stereocilia in Cib2 tm1a/tm1a OHCs compared to wild-type OHCs, especially in the 3rd row, where individual stereocilium either over-grew or disassembled (Fig. 4a). At P6, we observed almost exclusively an overgrowth of the remaining shorter row stereocilia in the Cib2 tm1a/tm1a OHCs (Fig. 4a). Similarly, as early as P6, Cib2 tm1a/tm1a IHCs exhibited abnormally elongated tips of the 2nd and 3rd row stereocilia (Fig. 4b). A similar stereocilia phenotype with elongated tips was also observed in Cib2 F91S/F91S IHCs (Fig. 5c). Using SEM images from the “back” side of the bundle, we measured the total height of individual IHC stereocilia in the tallest (1st) row of the bundle and found no differences between wild-type and Cib2 tm1a/tm1a mice (2.08 ± 0.04 μm, number of analyzed stereocilia/cells n = 51/6; and 2.16 ± 0.14 μm, n = 41/6, correspondingly). However, measurements of “staircase steps” in the same bundles, i.e. the distance between different stereocilia rows, showed an over-growth of the majority of the 2nd and 3rd row stereocilia (Fig. 4d). In contrast to IHCs, neither row of OHC stereocilia in Cib2 tm1a/tm1a mice exhibited elongated tips (Fig. 4c). To determine the effects of CIB2 deficiency on the known proteins responsible for stereocilia elongation, we explored localization of whirlin, myosin XVa, Eps8, and Eps8L2 in Cib2 tm1a/tm1a mice. All four proteins were localized at the stereocilia tips in Cib2 mutant mice (Supplementary Fig. 6b), suggesting that CIB2 is not essential for transporting them to the stereocilia tips. We concluded that, despite some differences between IHCs and OHCs, a common effect of CIB2 deficiency is the disruption of length regulation in the shorter row stereocilia and an over-elongation of the majority of them. It is worth mentioning that the shorter row stereocilia are the ones that harbor MET channels in mammalian auditory hair cells3, which may imply a link between CIB2 and mechanotransduction. We previously hypothesized this link based on the importance of CIB2 for hair cell function in zebrafish19.

Fig. 4 CIB2 deficiency leads to over-elongation of the transducing shorter row stereocilia in the auditory hair cells. a, b SEM images of OHC (a) and IHC (b) bundles from wild-type (left) and Cib2 tm1a/tm1a (right) littermate mice at P6 at the same cochlear location in the middle of the apical turn. Insets show magnified images of stereocilia tips and stereocilia links in the areas indicated by dashed boxes. c, d Quantification of the step height differences between the 1st (longest) and 2nd and between the 1st and 3rd rows of stereocilia (illustrated on the inset cartoons) in the OHC (c) and IHC (d) bundles at P6 (c, left and d) and P18 (c, right). Each dot represents one pair of stereocilia. Mean ± SEM values are also shown. We were not able to quantify the staircase morphology in the IHCs at P18 because mouse IHCs are very fragile at this age and their bundles lose cohesiveness after dissection. Number of cells: IHCs, wild-type, n = 6; Cib2 tm1a/tm1a, n = 6; OHCs at P6, wild-type, n = 9; Cib2 tm1a/tm1a, n = 18; OHCs at P18, wild-type, n = 10; Cib2 tm1a/tm1a, n = 10. In all panels, asterisks indicate statistical significance of the difference between the data from mutant and wild-type cells. NS, not significant; ****p < 0.0001 (ANOVA). Scale bars: 1 μm Full size image

Fig. 5 CIB2 is essential for mechanotransduction in the auditory hair cells. a, b Maximum intensity projections of Z-stacks of confocal fluorescent images (left) and corresponding DIC images (right) of control (a) and Cib2 tm1a/tm1a (b) cultured organ of Corti explants imaged after exposure to 3 μM of FM1-43 for 10 s. The samples were dissected at P5 and kept 2 days in vitro (P5 + 2div). Scale bar: 20 µm. c SEM images of IHCs in acutely isolated organ of Corti explants from wild-type (left), Cib2 F91S/F91S (middle), and Cib2 tm1a/tm1a (right) mice. Insets show the tip links at high magnification in the areas indicated by dashed boxes. Scale bars are 0.5 µm and 200 nm in the insets (d). Experimental setup for MET current recordings. Positive pressure in fluid-jet deflects hair bundle toward kinocilium and activates “conventional” MET channels gated by tip link tension, while negative pressure closes these channels but may activate “reverse-polarity” currents at certain conditions51, 52. Scale bar is 10 µm (e, f) MET current traces (e) and average MET current (Mean ± SEM) (f) to the graded deflections of the hair bundles with fluid-jet (bottom traces in e) in wild-type (black and grey), Cib2 F91S/F91S (red), and Cib2 tm1a/tm1a (magenta) IHCs. Statistical significance is indicated with asterisks: **p < 0.01, ***p < 0.001 (Student’s t-test). g MET responses produced by a sinusoidal fluid-jet stimulus (bottom) in control Cib2 F91S/+ (top traces) and Cib2 F91S/F91S (middle traces) IHCs at different holding potential ranging from −104 to +76 mV (indicated by the traces). Age of the IHCs in e–g: P4–P7. h Reverse-polarity current in the OHCs of Cib2 tm1a/tm1a mice at different developmental ages indicated by the traces. Note that the current is activated by negative but not positive bundle deflections. i Average values (Mean ± SEM) of normal and reverse-polarity currents in Cib2 tm1a/tm1a OHCs at different ages. Number of cells is indicated by each data point. Full size image

CIB2 is essential for auditory hair cell mechanotransduction

We investigated first whether the auditory hair cells of Cib2 tm1a/tm1a mice have functional MET channels that are open at rest. We briefly incubated cultured organ of Corti explants from Cib2 mutants with the MET channel-permeable dye FM1-43. In control explants, this incubation produced strong labeling of both OHCs and IHCs (Fig. 5a, Supplementary Fig. 7a), while no dye uptake was observed in Cib2 tm1a/tm1a and Cib2 F91S/F91S cochleae (Fig. 5b, Supplementary Fig. 7b). These data suggest that the MET channels in CIB2-deficient auditory hair cells are either closed at rest or non-functional.

Using conventional whole-cell patch-clamp recordings, we next studied MET currents in IHCs, because these cells have a less disrupted staircase bundle structure as compared to OHCs in Cib2 mutants (Fig. 4a, b). Examination of IHC bundles with high-resolution SEM in freshly isolated organ of Corti explants revealed shorter but abundant links at the tips of 2nd and 3rd row stereocilia in Cib2 F91S/F91S and Cib2 tm1a/tm1a IHCs (Fig. 5c). We were not able to quantify the number of tip links in the Cib2 tm1a/tm1a IHCs, due to very tight proximity between neighboring stereocilia in the bundle. However, we counted similar numbers of tip links in the IHCs of wild-type and Cib2 F91S/F91S littermates (0.82 ± 0.03 links per stereocilium in wild-type vs. 0.84 ± 0.03 links in Cib2 F91S/F91S; number of cells, n = 12 and n = 10, correspondingly). After few days in culture, the tips of IHC 2nd and 3rd row stereocilia in Cib2 mutants become more rounded and prominent tip links were observed (Supplementary Fig. 7c–e).

To deflect hair bundles, we used a fluid-jet, since it is able to produce not only positive but also very strong negative deflections of the bundle (Fig. 5d). Whole-cell patch-clamp recordings showed prominent MET currents in the control IHCs but not in Cib2 tm1a/tm1a or Cib2 F91S/F91S IHCs in both freshly isolated and cultured preparations (Fig. 5e, Supplementary Fig. 7c–e). In fact, we did not observe any MET current responses in Cib2 mutants, even to the strongest bundle deflections that normally saturate MET current (Fig. 5f). In theory, MET current might be blocked by very large Ca2+ concentration inside stereocilia if, for some reason the intra-pipette Ca2+ buffer (1 mM of EGTA) was not strong enough to overcome the potential loss of Ca2+ buffering in Cib2 mutants. Therefore, we recorded MET responses at large positive potentials that usually eliminate Ca2+ block to the MET channels14, 17. We did not detect any MET responses in all Cib2 F91S/F91S IHCs (n = 6) that were tested at positive intracellular potentials (Fig. 5g). Complete loss of mechanotransduction in IHCs was observed in both strains of CIB2-deficient mice and in both acutely isolated (Fig. 5e–g) and cultured (Supplementary Fig. 7) preparations. We also did not observe any statistically significant differences in the resting concentration of free intracellular Ca2+ ([Ca2+] i ) measured with Fura-2 indicator inside the OHCs of heterozygous and homozygous Cib2 tm1a/tm1a mice (92.7 ± 1.6 nM, n = 15 and 93.0 ± 1.5 nM, n = 44, correspondingly, Mean ± SD, mid-cochlear location). Consistent with recent findings30, 31, IHCs at P4-P7 did not exhibit reverse-polarity mechanosensitive currents, i.e., the currents activated by negative bundle displacements, in both control and Cib2-mutant mice (Fig. 5e–g). However, these currents were observed in young OHCs of Cib2 tm1a/tm1a mice (Fig. 5h, i), indicating that CIB2 deficiency does not affect Piezo2-associated31 reverse-polarity currents. In fact, the reverse polarity current seems to be accentuated by CIB2 deficiency and its developmental downregulation prolonged (Fig. 5i), exactly as observed in Tmc1/Tmc2 double knockout mice31. We conclude that conventional mechanotransduction is impaired in Cib2 tm1a/tm1a and Cib2 F91S/F91S auditory hair cells.

CIB2 interacts with TMC1 and TMC2

Because CIB2 is essential for mechanotransduction, we examined the localization of some known proteins associated with the tip links or the MET apparatus (PCDH15, TMC1, TMC2) in Cib2 tm1a/tm1a mice using confocal imaging and found no differences from heterozygous control (Fig. 6), suggesting CIB2 is not essential for targeting of these proteins in stereocilia.

Fig. 6 CIB2 deficiency does not mislocalize TMC1, TMC2 and Pcdh15 from the auditory hair cell stereocilia. Confocal images showing IHC stereocilia of Cib2 tm1a/+ and Cib2 tm1a/tm1a mice immunostained with TMC1, TMC2, and Pcdh15 antibodies (red) and actin was labeled with phalloidin (green) at P12, P4 and P7 correspondingly. Scale bar: 10 μm Full size image

Next, we examined the potential interaction of CIB2 with known components of the MET machinery including TMC1, TMC2, LHFPL5, and TMIE in heterologous cells using FRET assays. For positive control, we used FRET between CIB2 tagged with green fluorescent protein (GFP) and CIB2 tagged with V5, as our previous studies have shown that CIB2 can multimerize19. Although we cannot rule out the possibility of tags interfering with the interaction of CIB2 with TMIE and LHFPL5, we observed FRET interaction only between CIB2 and TMC1 and TMC2 (Fig. 7a, b), suggesting potential interactions between them. Concurrently, CIB2–TMC1 interaction was also identified in a yeast two-hybrid screening using N-terminus of TMC1 as a bait (Supplementary Fig. 8). These experiments also confirmed that CIB2 can form dimers. However, physiological significance of CIB2 multimerization is yet unclear.

Fig. 7 CIB2 interacts with TMC1 and TMC2. a Fluorescent images of cells co-expressing LHFPL5-GFP, TMIE-GFP, CIB2-GFP (positive control), TMC1-EGFP or TMC2-EGFP (donor) and CIB2-V5 (acceptor) before and after acceptor photobleaching within the indicated region (white box). For negative control, we used TMC1-EGFP (donor) and CIB1-V5 (acceptor). FRET efficiency images are calculated as E FRET = 100 × (I Post −I Pre )/I Post , where I Pre and I Post are the EGFP pixel intensities before and after acceptor bleaching, respectively. Scale bars: 10 µm. b Quantitative analysis of FRET efficiency in CHO-K1 cells co-expressing LHFPL5-GFP, TMIE-GFP, CIB2-GFP, TMC1-EGFP or TMC2-EGFP (donor) and CIB2-V5 (acceptor). Quantitative analysis of negative control [TMC1-EGFP (donor) and CIB1-V5 (acceptor)] is also shown. Asterisks indicate statistical significance of FRET efficiency (p ≤ 0.0001). Error bars represent SEM. NS: not significant Full size image

To further confirm and map interaction domains, we generated deletion constructs of the N-terminus of TMC1 and tested their ability to associate with CIB2 in co-immunoprecipitation assays. Indeed, the N-terminus of TMC1, TMC1N (1–193), does interact and co-immunoprecipitate with CIB2 (Fig. 8a, left, Supplementary Figs. 8 and 11). Further deletion experiments demonstrated several other examples of TMC1-CIB2 interaction (Fig. 8a, right) and showed that a small domain (amino acids 81–130) in the TMC1 cytoplasmic N-terminus is critical for the TMC1-CIB2 interaction (Fig. 8a, bottom left). These results confirm that CIB2 is a binding partner of TMC1 and TMC2.

Fig. 8 Molecular mechanisms of DFNB48/USH1J deafness. a Co-immunoprecipitation assays of differentially truncated N-terminal fragments of TMC1 and full-length CIB2 (top panels). Schematics of truncated N-terminal fragments of TMC1 are shown on the bottom left panel. Specific N-terminal domain of TMC1 (amino acids 81–130) is essential for binding to CIB2 (red dashed lines). b Top: The protein alteration identified in USH1J is shown in red, while the protein alterations identified in DFNB48 are shown in black. Bottom: Quantitative analysis of FRET efficiency in cells co-expressing EGFP tagged human TMC1 (donor) and V5 tagged human CIB2 with deafness causing mutations. Error bars represent SEM. Asterisks indicate statistical significance of FRET efficiency changes relative to the control interaction of TMC1 with wild-type CIB2 (ANOVA with Dunnett’s test) Full size image

Molecular mechanisms of DFNB48/USH1J deafness

We hypothesized that CIB2 may function as an accessary subunit essential for the MET channel activity. Therefore, the loss of hair cell mechanotransduction in Cib2 mutants may result from the loss of TMC1-CIB2 interaction. If indeed CIB2 acts as an auxiliary subunit of MET channel complex, one would expect that CIB2 alleles, which do not affect targeting of CIB2 in stereocilia (e.g. p.F91S) might affect the interaction of CIB2 with TMC1, resulting in loss of MET current and hearing loss. To date, six CIB2 missense mutations (Fig. 8b, top) causing Usher syndrome type 1 (p.E64D) and non-syndromic hearing loss (p.R66W, p.F91S, p.C99W, p.I123T, p.R186W) have been reported in humans19, 29, 32. None of these six missense mutations resulted in notable changes in CIB2 intracellular localization in CHO-K1 cells (Supplementary Fig. 9). We co-expressed the six mutants with human full length TMC1 and analyzed the effect of CIB2 mutations on association with TMC1 using FRET assays. Three out of six mutants (p.E64D, p.F91S, and p.C99W) affected interactions of CIB2 with TMC1 (Fig. 8b, bottom, Supplementary Fig. 10), suggesting that these mutations disrupt the putative TMC1-CIB2 complex in hair cells and thus cause deafness in humans.