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The Journal of Neuroscience, June 13, 2007, 27(24):6478-6488; doi:10.1523/JNEUROSCI.0342-07.2007
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Cellular/Molecular
Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning
Nicolas Michalski,1 * Vincent Michel,1 * Amel Bahloul,1 Gaëlle Lefèvre,1 Jérémie Barral,3 Hideshi Yagi,2 Sébastien Chardenoux,1 Dominique Weil,1 Pascal Martin,3 Jean-Pierre Hardelin,1 Makoto Sato,2 andChristine Petit1 1Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche en Santé 587, Collège de France, Institut Pasteur, 75724 Paris cedex 15, France, 2Division of Cell Biology and Neuroscience, Department of Morphological and Physiological Sciences, Research and Education Program for Life Science, University of Fukui, Eiheiji, Fukui 910-1193, Japan, and 3Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Institut Curie, 75248 Paris cedex 05, France
Abstract
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Abstract
Introduction
Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1–/– mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1–/– mice. In postnatal day 7 Vlgr1–/– mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-link-mediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1–/– mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1–/– mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.
Key words: cochlea; hair bundle; ankle link; Vlgr1; adenylyl cyclase 6; Usher syndrome
Introduction
The two types of cochlear sensory cells, the inner hair cells (IHCs), the genuine sensory cells, and the outer hair cells (OHCs), the cochlear amplifiers, transduce sound-evoked mechanical stimuli into electrical signals in the hair bundle. The hair bundle is composed of an array of modified microvilli, the stereocilia, organized in three rows of graded length. During development, it also harbors a genuine cilium, the kinocilium. The architecture, orientation, and stiffness of the hair bundle are critically involved in the mechanoelectrical transduction (MET) process. Strong evidence supports the role of the tip link, a single link extending upward the tip of each stereocilium to the side of the adjacent taller stereocilium, in this process via mechanical coupling to the MET apparatus (Pickles et al., 1984; Assad et al., 1991
The study of the proteins encoded by genes defective in Usher syndrome type 1 (USH1), characterized by congenital profound deafness, has shown that cadherin 23 (USH1D) and probably protocadherin 15 (USH1F) make up lateral links that interconnect the stereocilia as well as the stereocilia to the kinocilium during hair bundle differentiation (Ahmed et al., 2001
The ankle links are present in all vertebrate species, both in the auditory and vestibular hair bundles, although they are only transient in the mammalian cochlear hair cells. Recently usherin and very large G-protein-coupled receptor 1 (Vlgr1), two transmembrane proteins encoded by genes defective in Usher syndrome type 2 (USH2A and USH2C, respectively), characterized by a less severe hearing impairment than in USH1, were suggested to be components of the ankle links because they are both located at the base of stereocilia (Adato et al., 2005b
120 aa Calx-ß domains. The disappearance of the ankle links in Vlgr1del7TM mutant mice further suggests that Vlgr1 is an ankle-link component (McGee et al., 2006
Materials and Methods
Animals and antibodies. Animal experiments were performed in accordance with the Institut de la Santé et de la Recherche Médicale animal welfare guidelines, approved by the French Ministère de l'Agriculture, de l'Alimentation, de la Pêche et de la Ruralité. The Vlgr1–/– mice have been reported previously (Yagi et al., 2005Whole-mount fluorescence. Animals at different developmental stages were killed by exposure to CO2 followed by decapitation, and cochleas were dissected from temporal bones. The cochlear shells were removed and placed in PBS. The organ of Corti was exposed by removing the cochlear lateral wall, and the tissues were then fixed in 4% paraformaldehyde PBS for 1 h at room temperature. The tissues were washed three times in PBS (10 min for each wash), followed by permeabilization and blocking in PBS containing 1% bovine serum albumin (BSA), 20% normal goat serum, and 0.3% Triton X-100 for 1 h at room temperature. After PBS washes, primary antibodies, diluted in PBS containing 1% BSA and 0.25% Triton X-100, were incubated overnight at 4°C. Omission of the primary antibody was used as a negative control. After three 10 min washes in PBS, the tissues were incubated with secondary antibodies diluted in 1% BSA-PBS containing TRITC-conjugated or Alexa 488-conjugated phalloidin (1:2000 dilution) for 1 h at room temperature. After three washes in PBS, the tectorial membrane was carefully dissected away and the organs of Corti were mounted in Fluorsave (Calbiochem, La Jolla, CA), under glass coverslips with the hair bundles directed toward the coverslip. Whole-mount preparations were viewed with a 63x Plan Apochromat oil-immersion objective (numerical aperture, 1.2) using a Zeiss (Oberkochen, Germany) LSM-510 confocal microscope.
For double-labeling experiments using two rabbit primary antibodies (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), the following modifications were done to the above protocol. The fixed tissue samples were sequentially incubated with the primary antibody (anti-Vlgr1) and a Cy5-conjugated secondary antibody (1:500 dilution) for 1.5 and 1 h at room temperature, respectively. Tissue samples were fixed again in 4% paraformaldehyde PBS for 15 min at room temperature. They were then sequentially incubated with the second primary antibody (anti-usherin or anti-vezatin) overnight at 4°C and an Alexa 488-conjugated secondary antibody (1:800 dilution) for 15 min at room temperature, followed by a 15 min fixation in 4% paraformaldehyde PBS. Finally, tissue samples were incubated with Cy3-conjugated phalloidin (1:2000 dilution). Omission of the second primary antibody (anti-usherin or anti-vezatin) was used as a negative control.
BAPTA and subtilisin treatments. After dissection (see above), organs of Corti were placed in microwells and incubated with either PBS, PBS containing 5 mM BAPTA, or PBS containing 50 µg/ml subtilisin [Sigma (St. Louis, MO) Protease type XXIV] for 15 min at room temperature. After treatment with PBS, BAPTA, or subtilisin, the solutions were removed, and fixative (4% paraformaldehyde in PBS) was added. After fixation for 1 h at room temperature, the organs of Corti were immunolabeled as described above.
Scanning electron microscopy. Whole inner ears from postnatal day 6 (P6) Vlgr1+/+, Vlgr1+/–, and Vlgr1–/– mice were removed, pierced at the apex of the cochlea and in the round and oval windows, and fixed by immersion in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 2 h at room temperature. After three washes with the buffer alone, the inner ears were dissected to give direct access to the organ of Corti. The samples were then dehydrated by successive washes in ethanol (50, 70, 80, 90, and 100%), critical point dried, mounted on a stub, sputter coated with gold-palladium, and examined under a JEOL (Peabody, MA) JSM 6700F scanning microscope.
In vitro binding experiments. The cDNA fragments encoding the cytoplasmic regions of the mouse usherin (NCBI accession number NM_021408.2, amino acids 5055–5193) and Vlgr1 (NCBI accession number NM_054053, amino acids 6150–6298) and the following truncated fragments of whirlin (GenBank accession number AY739114.1, amino acids 1–260, amino acids 240–550, and amino acids 420–907) were reverse transcription (RT)-PCR amplified from mouse inner ear mRNA and cloned into pGEX-4T-1 vector for protein production in Escherichia coli BL21 cells. Glutathione S-transferase (GST) fusion proteins were purified from bacterial extracts by using glutathione–Sepharose 4B beads. The cDNA encoding the long isoform of vezatin (GenBank accession number AY753561) was subcloned in pCMV-tag 3b and expressed in HEK293 cells. The Myc-tagged vezatin was purified from cell extracts on an anti-Myc affinity column. Finally, the His-tagged myosin VIIa tail fragment corresponding to the C-terminal MyTH4-FERM repeat (NCBI accession number NM_000260, amino acids 1747–2215) was produced in a baculovirus system and purified on a Ni2+-agarose column.
To test for myosin VIIa tail interactions, the same amount (4 µg) of GST-tagged usherin, GST-tagged Vlgr1, or GST alone was bound to glutathione–Sepharose beads (25 µl) for 90 min at 4°C and then incubated with the purified myosin VIIa C-terminal fragment (3 µg,
To test for vezatin interactions, the anti-Myc affinity beads bound to Myc-tagged vezatin (25 µl) were washed intensively with cell lysis buffer (PBS complemented with 50 mM NaCl, 0.5% Triton X-100, 0.1% SDS, 5% glycerol, 0.5 mM DTT, 1 mM orthovanadate, and complete inhibitor protease mixture) and incubated with 2 µg of each GST-fusion protein [whirlin fragments (
Single-cell RT-PCR. To obtain single hair cells, P6 isolated organs of Corti were collected in 100 µl of PBS with 5% trypsin at room temperature and were slowly disassembled mechanically for 4 min. A total of 100 µl of fetal calf serum was added for neutralization of trypsin. Isolated hair cells were then harvested with a patch pipette filled with PBS under a Nikon inverted microscope. Each hair cell was individually placed directly in 10 µl of the RT mix using the SuperScript II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, except that the RT reaction was incubated overnight at 37°C. For each cell, we used the extracellular fluid as a negative control. A multiplex nested PCR was then performed on each hair cell or control tube using the Expand High Fidelity PCR System kit (Roche) according to the manufacturer's instructions. The first PCR was done in a total volume of 100 µl (including the 10 µl of the RT reaction), using specific primers (at a concentration of 1 µM each) for the nine adenylyl cyclase (AC) transcripts that were tested and for two control transcripts, namely myosin VIIa, which is expressed in both OHCs and IHCs, and prestin, which is only expressed in OHCs. The PCR consisted of 20 amplification cycles (40 s at 95°C, 45 s at 58°C, and 50 s at 72°C). Primer sets were chosen on different exons to be able to discriminate between genomic DNA and cDNA (for primer sequences, see supplemental Table, available at www.jneurosci.org as supplemental material). Amplified DNA fragments had a molecular weight ranging between 300 and 500 bp. The nested PCR was performed in a 25 µl mix containing 2 µl of the first PCR mix and only one set of inner primers at a time through 35 cycles (40 s at 95°C, 45 s at 58°C, and 50 s at 72°C). To test whether the various cDNA fragments could be selectively amplified by the primer sets, we first performed a PCR amplification assay on entire cochlea cDNA in the same conditions. All adenylyl cyclase transcripts and control transcripts could be detected in this way.
Thirty cells were systematically collected for each experiment. For result analysis, only cells that showed the proper myosin VIIa and prestin expression profiles were computed. The number of positive cells for each adenylyl cyclase was then counted and compared statistically to the number of extracellular fluid samples showing cDNA amplification (n = 10) by using the
2 method (with Yates correction for small samples) to check for significance (p < 0.05).
Electrophysiological recordings. Electrophysiological recordings from cochlear explants were performed on P7 Vlgr1+/+, Vlgr1+/–, and Vlgr1–/– mice as reported previously in the rat (Kennedy et al., 2003
Hair cells were whole-cell voltage clamped at room temperature (20–25°C) using an EPC-9 patch-clamp amplifier and the Patchmaster software (HEKA, Lambrecht, Germany). No correction was made for liquid junction potentials. Borosilicate patch pipettes (2–3 M
) were approached parallel to the hair cell rows through a hole in the reticular lamina. During this approach, extracellular solution was abundantly perfused to avoid contact between EGTA and the MET apparatus, which is sensitive to calcium chelators. Patch-clamped OHCs and IHCs were located at a distance of
Data were analyzed using the IgorPro software (WaveMetrics, Lake Oswego, OR). Po-displacement curves were fitted with a second order Boltzmann function (Kros et al., 2002
Results
Vlgr1 is a component of the ankle links of the hair bundle
The specificity of our affinity-purified antibody directed against the N-terminal extracellular region of the mouse Vlgr1 was tested by immunofluorescence imaging on whole-mount cochlear preparations from Vlgr1+/+, Vlgr1+/–, and Vlgr1–/– mice (Fig. 1D,E and data not shown). At embryonic day 17 (E17), an intense Vlgr1 staining was present at the base of the emerging hair bundle and on a peripheral subpopulation of microvilli located at the neural edge of the hair cell apical surface in Vlgr1+/+ cochlear hair cells (Fig. 1A,A'). At P0–P1, the staining extended from the very base of the stereocilia, i.e., where they anchor in the apical cell surface, to their basal first quarter (Fig. 1B,B'). From P4 to P9, the labeling became restricted, forming a basal band-like pattern at the ankle-link level, above the now well distinguishable stereocilia tapering or sharpened region (Fig. 1C,C',D). From P12 onward, Vlgr1 was no longer detected in the cochlear hair cells (Fig. 1F). This spatiotemporal expression profile suggested that Vlgr1 is an ankle-link component. Indeed, the ankle links could not be detected in P6 Vlgr1–/– mice by scanning electron microscopy, whereas other lateral links were still present (Fig. 2A–B'). This result confirms recently published results by McGee et al. (2006)
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Figure 1. Vlgr1 in normal hair bundle development. Whole-mount preparations of wild-type mouse cochlear middle-coil double labeled for actin (red) and Vlgr1 (green). A, A', At E17, the Vlgr1 staining of hair cells is located at the base of the emerging hair bundles and extends as a fine peripheral band to the neural edge short microvilli (A, arrowheads). B, B', At P1, Vlgr1 labeling is restricted to the basal end of the hair bundles both in OHCs (B) and IHCs (B'). C–E, At P4 and P8, in both hair cell types, the Vlgr1 labeling forms a tight band at the base of stereocilia (C–D), which is not detected in Vlgr1–/– mice (E). F, Vlgr1 is no longer detected in P12 Vlgr1+/+ hair bundles. G, The Vlgr1 labeling disappears after BAPTA or subtilisin treatment of P5 cochlear explants. Scale bars, 4 µm.
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Figure 2. Morphological defects of Vlgr1–/– hair bundles. A–B', Scanning electron microphotographs of OHC and IHC hair bundles from P6 Vlgr1+/+ and Vlgr1–/– mice. Insets, Enlarged views of stereocilia bases. A, B, Arrowheads point to the ankle links in Vlgr1+/+ hair bundles. A', B', Ankle links are absent in Vlgr1–/– hair bundles. C–E', Scanning electron microphotographs of the surface of the organ of Corti at the apical, middle, and basal coils of cochleas from P6 Vlgr1+/+ and Vlgr1–/– mice. C', D', Arrowheads point to neural side supernumerary microvilli in Vlgr1–/– IHCs. Scale bars, 4 µm.
Hair bundle abnormalities and persistence of peripheral microvilli in Vlgr1–/– hair cells
We studied the structure of the apical region of Vlgr1–/– hair cells by scanning electron microscopy. In agreement with the anomalies reported recently in Vlgr1/del7TM mice (McGee et al., 2006Lack of Vlgr1 results in the absence of usherin, vezatin, and whirlin at the stereocilia base
We asked whether usherin, a protein with a large extracellular region that is present at the base of the stereocilia (Adato et al., 2005b
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Figure 3. Hair bundle distribution of usherin, vezatin, and whirlin in P6 Vlgr1+/+ and Vlgr1–/– cochlear hair cells. A–B', In Vlgr1+/+ mice, both usherin (A) and vezatin (B) are detected at the base of stereocilia and along the kinocilium. In Vlgr1–/– mice, usherin (A') and vezatin (B') labelings are no longer present at the base of the hair bundle but remain in the kinocilium (A', B',arrows) both in OHCs and IHCs. C, C', In Vlgr1+/+ mice, whirlin is detected both at the base and apical end of stereocilia. In Vlgr1–/– mice, only the apical labeling can be seen (C', arrowheads), both in stereocilia and supernumerary neural edge microvilli. Scale bar, 4 µm.
Two additional proteins, namely vezatin, a putative transmembrane protein (Küssel-Andermann et al., 2000bAbnormal distributions of Vlgr1, usherin, and vezatin in shaker-1 and whirler mouse mutants
Based on the absence of harmonin, a hair bundle PDZ domain-containing protein, from the stereocilia of myosin VIIa defective mice, we have proposed that myosin VIIa conveys harmonin to the hair bundle (Boëda et al., 2002
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Figure 4. Distribution of Vlgr1, usherin, and vezatin in the apical region of inner hair cells from P6 shaker-1 and whirler mice. In the absence of myosin VIIa (shaker-1 mutant) or whirlin (whirler mutant), the proper hair bundle distributions of Vlgr1, usherin, and vezatin are no longer detected. A–A", In shaker-1 hair cells, the Vlgr1 labeling forms clumps beneath the apical cell surface (A'), whereas in whirler hair cells, it is detected at the very base of stereocilia, i.e., below the ankle links (A"). B–B", A faint usherin staining can be seen beneath the apical cell surface in shaker-1 hair cells (B', arrowheads), whereas usherin cannot be detected in whirler hair cells (B"). C–C", The vezatin staining forms clumps beneath the apical cell surface in shaker-1 hair cells (C') and is not detected in whirler hair cells (C"). Scale bar, 4 µm.
Myosin VIIa–Vlgr1, myosin VIIa–usherin, and vezatin–usherin in vitro interactions
In vitro direct interactions between whirlin and the cytoplasmic regions of Vlgr1 (van Wijk et al., 2006
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Figure 5. Myosin VIIa–Vlgr1, myosinVIIa–usherin, and vezatin–usherin in vitro interactions. A, Agarose beads carrying the Myc-tagged vezatin bind to a GST-fusion protein containing the cytoplasmic region of usherin (GST-CytoUsherin) but not to GST alone. B, The myosin VIIa C-terminal MyTH4-FERM fragment (Myosin VIIa Cter) binds to GST-fusion proteins containing the cytoplasmic domain of either Vlgr1 (GST-CytoVlgr1) or usherin (GST-CytoUsherin) but not to GST alone. The positions of the molecular mass markers are indicated on the right.
Lack of Vlgr1 affects the stereociliar distribution of adenylyl cyclase 6
Vlgr1 is a class B orphan GPCR (Foord et al., 2005s subunits that stimulate ACs, hence cAMP production. AC1, AC4, AC6, AC7, and AC9 transcripts have been found in the mouse and rat organs of Corti by RT-PCR analysis (Drescher et al., 1997
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Figure 6. Adenylate cyclase 6 in Vlgr1+/+ and Vlgr1–/– hair bundles. A, Detection of AC transcripts in P6 OHCs by using single-cell RT-PCR. Only AC6 and AC9 transcripts could be detected. B–F, AC6 distribution in the hair bundles of middle-coil cochlear hair cells at different postnatal stages. B, In P1 wild-type mice, AC6 is detected all along OHC stereocilia but not in IHC stereocilia. C, E, In P2 and P7 wild-type OHCs, the AC6 labeling is restricted to the stereocilia base. D, F, In wild-type IHCs, the AC6 labeling can be detected at the stereocilia base from P3. G, H, H', In P7 Vlgr1–/– OHCs and IHCs, the AC6 labeling is more intense and located all along stereocilia. Scale bars, 4 µm.
We then analyzed the stereociliar distribution of AC6 during development. Between E16 and P1, AC6 labeling was present all along the stereocilia of OHCs but was almost undetectable in IHC hair bundles (Fig. 6B,C). From P3 onward, the labeling of OHC hair bundles was restricted to their base, and AC6 became detectable at the base of IHC stereocilia (Fig. 6D–F). In mature OHCs and IHCs, AC6 was still detected at the stereocilia base. In P7 Vlgr1–/– mice, a dramatic increase in the AC6 labeling was found in OHC and IHC stereocilia (Fig. 6G–H'). In addition, instead of being restricted to the stereocilia base, it extended from stereocilia base to apex in both hair cell types. In contrast, the distribution of AC9 was not modified in Vlgr1–/– mice (data not shown).MET currents are abnormal in Vlgr1–/– outer hair cells
To determine whether the ankle links are involved in the MET process, we performed a comparative analysis of the response of Vlgr1+/+ and Vlgr1–/– hair bundles to mechanical stimulation. In P7 Vlgr1+/+ mice, IHCs exhibited large transducer currents in response to a fast mechanical stimulus applied to the hair bundle in the excitatory (positive) direction, i.e., along the tip-link axis in the direction of the tallest stereocilia. This is the first time that MET currents are recorded in mouse IHCs using a stiff probe to stimulate the hair bundle. These currents had magnitudes comparable with those reported recently in the rat IHCs (Beurg et al., 2006
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Figure 7. Mechanoelectrical transduction current recordings in the excitatory direction. A, Examples of MET current recordings in IHCs (red) and OHCs (blue) from Vlgr1+/+ and Vlgr1–/– P7 mice while applying different displacement steps in the excitatory direction (shown on the left) and one 150 nm step in the opposite direction. Cells were voltage clamped at –80 mV. B, Relative Po-displacement curves plotted for Vlgr1+/+ (n = 5) and Vlgr1–/– (n = 8) IHCs (left) and for Vlgr1+/+ (n = 5) and Vlgr1–/– (n = 11) OHCs (right). Relative Po-displacement curves were fit with a normalized second-order Boltzmann function: I = 1/[1 + {exp(a(X0 – X))}{1 + exp(b(X1 – X))}], where a = 0.044 nm–1, b = –0.037 nm–1, X0 = 78 nm, X1 = 60.3 nm for Vlgr1+/+ IHCs, and a = 0.024 nm–1, b = –0.017 nm–1, X0 = 123.6 nm, X1 = 86.5 nm for Vlgr1–/– IHCs, and a = 0.049 nm–1, b = –0.036 nm–1, X0 = 80.4 nm, X1 = 46.9 nm for Vlgr1+/+ OHCs, and a = 0.027 nm–1, b = –0.020 nm–1, X0 = 166 nm, X1 = 120.4 nm for Vlgr1–/– OHCs. B', X0.5 denotes average displacements required to open 50% of the MET channels and average sensitivity is the slope mean value calculated for displacements corresponding to Po values between 0.2 and 0.8 in Vlgr1+/+ and Vlgr1–/– hair cells. Statistically significant differences (Student's t test) are denoted by asterisks.
In contrast, Vlgr1–/– OHCs showed greatly impaired MET currents at P7. In Vlgr1+/+ mice at the apical turn (20–30%) of the cochlea, transducer currents had a mean saturating amplitude of 853 ± 200 pA (n = 5) and a fast adaptation time constant of 363 ± 70 µs (n = 5) (Fig. 7A). In Vlgr1–/– mice, all recorded OHCs exhibited MET currents. However, there were both a significant decrease in transducer current mean saturating amplitude and an important heterogeneity between hair cells: 220 ± 152 pA (n = 12) in Vlgr1–/– OHCs at the apical turn (20–30%; Student's t test vs wild-type, p < 10–3). Comparing the normalized relative Po-displacement curves in Vlgr1+/+ and Vlgr1–/– OHC hair bundles showed that the displacement required to open 50% of the MET channels and the slope mean value calculated for displacements corresponding to Po values between 0.2 and 0.8 were significantly higher (Student's t test, p < 10–2) and smaller (n = 11; Student's t test, p = 0.03) in Vlgr1–/– OHCs, respectively (Fig. 7B'). Fast adaptation was systematically present in Vlgr1–/– OHCs, and its time constant (437 ± 133 µs; n = 8) was similar to that in wild-type OHCs (Student's t test vs wild-type, p = 0.19).Paradoxical response of the Vlgr1–/– hair bundles to displacements in the inhibitory direction
Strikingly, for all Vlgr1–/– hair bundles tested at P7, we recorded a current when they were pushed in the inhibitory or negative direction. In Vlgr1–/– IHCs (Fig. 8A), the threshold for MET activation in the negative direction was 182 ± 82 nm (n = 12), and for a 1 µm negative displacement, the mean recorded current was 212 ± 162 pA (n = 13). In Vlgr1–/– OHCs (Fig. 8C), the mean MET current threshold was 104 ± 70 nm (n = 9), and the mean current was 103 ± 87 pA (n = 9) for a 1 µm negative displacement. In contrast, in Vlgr1+/+ hair cells (Fig. 8A), only 2 of 24 Vlgr1+/+ IHCs displayed MET currents for displacements < 250 nm in the negative direction and the IHC mean threshold for MET activation was 856 ± 444 nm (Student's t test vs mutant, p < 10–3). For a 1 µm negative displacement, the mean current value was very small: 32 ± 52 pA (n = 26) (Student's t test vs mutant, p < 10–2). Similar results were obtained for Vlgr1+/+ OHC hair bundles (Fig. 8C), with a mean activation threshold of 993 ± 434 nm (Student's t test vs mutant, p < 10–4) and a mean current amplitude of 7 ± 9 pA (n = 11) for a 1 µm negative displacement (Student's t test versus mutant, p = 0.01). Both in Vlgr1–/– IHCs and OHCs, these currents elicited by stimulation in the negative direction were reversibly abolished by the perfusion of hair bundles with dihydrostreptomycin, an MET channel blocker (Fig. 8B,D).
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Figure 8. Mechanoelectrical transduction current recordings in the inhibitory direction. A, C, Examples of MET current recordings in IHCs (red) and OHCs (blue) from Vlgr1+/+ and Vlgr1–/– P7 mice while applying different displacement steps in the inhibitory direction. Cells were voltage clamped at –80 mV. B, D, Dihydrostreptomycin (DHS) application suppresses the MET current in P7 Vlgr1–/– IHCs and OHCs.
Cadherin 23 class A isoforms are absent from Vlgr1–/– hair bundles
Because loss of cohesion of the hair bundle could account for the abnormal MET current recordings in Vlgr1–/– hair cells, we asked whether other interstereociliary links could be missing in Vlgr1–/– mice that lack the ankle links. We focused on cadherin 23 because it has been shown to form transient lateral links during hair bundle development (Lagziel et al., 2005
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Figure 9. Hair bundle distribution of cadherin 23 in Vlgr1+/+ and Vlgr1–/– inner hair cells. A, B, C, In P1 (A) and P6 (B, C) wild-type hair cells from cochlear middle coil, the N1 (extracellular) and Ela3 (both intracellular and extracellular) epitopes of cadherin 23 are both detected in the apical part of the stereocilia. A', B', C', In Vlgr1–/– hair cells, the N1 epitope is detected at P0 (A') but not at P6 (B'). In contrast, the distribution of Ela3 is preserved (C'). Scale bar, 4 µm.
Discussion
Based on the molecular identification of the avian ALA and immunoelectron microscopy analysis of the developing hair bundle in wild-type and Vlgr1/del7TM mutant mice, McGee et al. (2006)
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Figure 10. Schematic representation of proteins involved in the ankle-link complex. In vitro direct interactions between proteins are indicated by arrows.
According to the GPCR subclass of Vlgr1, it is expected to activate the G-proteinBecause electrophysiological recordings in Vlgr1–/– mice showed the presence of MET currents in response to positive deflections of the hair bundle in both IHCs and OHCs, we conclude that ankle links are not necessary for the setting up of the MET apparatus and its mechanical stimulation. This result does not corroborate recent data obtained in Vlgr1/del7TM mice by McGee et al. (2006)
Both USH2C patients, who carry mutations in VLGR1, and Vlgr1–/– mice suffer from moderate to severe hearing impairment and have severely impaired otoacoustic emissions (Weston et al., 2004
Footnotes
Received Jan. 25, 2007; revised May 2, 2007; accepted May 10, 2007.*N.M. and V.M. contributed equally to this work.
This work was supported by the Fondation Raymonde et Guy Strittmatter, the European Commission FP6 Integrated Project EuroHear LSHG-CT-2004-512063, Ernst-Jung Stiftung für Medizin Preis, and A & M Suchert-Retina Kontra Blindheit. N.M. was supported by a fellowship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie. We are deeply indebted to R. Fettiplace for teaching hair-cell patch clamping to N.M. and for his suggestions for improving this manuscript. We thank M. Beurg and L. Prado de Carvalho for numerous pieces of advice. We also thank E. Bizard and S. Nouaille for technical help; Y. Yamasaki and G. P. Richardson for providing us with the anti-whirlin and anti-Ptprq antibodies, respectively; J. Levilliers for her help in the preparation of this manuscript; and J. Ashmore for careful reading of this manuscript.
Correspondence should be addressed to Christine Petit, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 587, Unité de Génétique des Déficits Sensoriels, Collège de France, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France. Email: cpetit{at}pasteur.fr
Copyright © 2007 Society for Neuroscience 0270-6474/07/276478-11$15.00/0
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