Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism\u27s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca(2+)-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery

Molecular remodeling of tip links underlies mechanosensory regeneration in auditory hair cells.

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism\u27s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca(2+)-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery

Formation of a transient PCDH15–PCDH15 tip link and its subsequent replacement with PCDH15–CDH23 link.

<p>The model assumes that BAPTA disrupts PCDH15–CDH23 bonds <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Sotomayor2" target="_blank">[39]</a> and MET channels are bound to or located near the lower end of the tip link <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Beurg1" target="_blank">[3]</a>. The transduction channel becomes nonfunctional after tip link disruption. Therefore, its location immediately after BAPTA treatment is unknown, although illustrated as present at the tip of stereocilium. Alternatively, MET channels may migrate away from the tip of a stereocilium as a complex with PCDH15 molecules. Harmonin-based complexes linking CDH23 to the cytoskeleton <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Grillet1" target="_blank">[41]</a> are shown as blue circles near the upper end of the tip link.</p

MET regenerates in two distinct steps.

<p>(A) Maximal MET current in control IHCs (black) and in IHCs at different times after BAPTA treatment (red). All records were obtained in “optimal” position of the probe (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio-1001583-g003" target="_blank">Figure 3</a>) at holding potential of −90 mV. n, number of cells. (B) Number of “tip” (blue) and “top” (black) links per stereocilium in control IHCs (dashed lines) and in IHCs recovering from BAPTA (solid lines). From 5 to 12 IHCs (154–502 stereocilia) were analyzed for each data point. (C) Representative SEM image of an IHC stereocilia bundle shows tip (blue dots) and top (black dots) links. See the text for exact definitions of “tip” and “top” links. (D) Current–displacement relationships of MET current at different stages of regeneration. Data were fitted to a second-order Boltzmann function and normalized to the maximum MET current obtained from the fit <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Stepanyan2" target="_blank">[50]</a>. (E) Overlapping MET currents evoked by a small bundle deflection (150 nm, bottom trace) that increases the open probability of MET channels (P/P_max) by ∼30%. To compare adaptation at different time points of tip link regeneration, MET responses were normalized to peak current and averaged. Standard errors were calculated for each point of the trace, but shown only at five time points for clarity of the figure. (F) Extent of adaptation calculated at 1.6 ms (1) and 7.6 ms (2) after the beginning of bundle deflection at different stages of MET recovery. Black dashed lines show the data from control IHCs untreated with BAPTA. The same MET records contribute to (A), (D), (E), and (F). All averaged data are shown as mean ± SE. Asterisks indicate statistical significance from the control values: * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001 (<i>t</i> test of independent variables). Age of the cells: P3+2–4 div (dissected at P3 and kept <i>in vitro</i> for 2–4 d).</p

Regenerating stereocilia links appear at the tips but not at the bottom of stereocilia.

<p>(A–B) Conventional (secondary electron) SEM images of IHC stereocilia before (A) and immediately after (B) link disruption. Dashed rectangles indicate the areas magnified in insets. Arrows point to the tip links. (C) Distribution of the links along the height of stereocilia (0%, bottom; 100%, top) in the third (shortest) row at different stages of link recovery. (D) Backscatter SEM image of IHC bundle immuno-labeled with anti-PDCH15 antibody, HL5614 (10 nm gold particles seen as white dots). (E) The same as in (D), but primary antibody was omitted. (F) Percentage of immuno-gold particles observed on links of second and third row stereocilia in IHCs at two different dilutions of HL5614. (G) Representative images of HL5614 labeling in third row stereocilia immediately and 20 min after BAPTA treatment. (H) Cumulative distribution of PCDH15 immuno-gold particles on third row stereocilium in control and during link recovery. For each time point, 50–70 stereocilia images were scaled to a common template (dashed line) and the location of every gold particle was shown by a semitransparent grey circle. (I) Distribution of PCDH15 immuno-gold particles along the height of third row stereocilia. Data in panels (C), (F), and (I) are shown as mean ± SE. Age of the cells: P3–4 plus 2–3 days <i>in vitro</i> (P3–4+2–3 div).</p

Redistribution of PCDH15 and CDH23 over the surface of stereocilia during tip link regeneration.

<p>(A) Backscattered SEM image of an IHC stereocilia bundle, labeled with anti-CDH23 antibody, C2367 (10 nm gold particles seen as white dots). (B) The same as in (A), but primary antibody was omitted. Insets in (A) and (B) show tip links at higher magnification. (C) Average number of all visible anti-PCDH15 (red) and anti-CDH23 (blue) immuno-gold particles on the surface of the first and second row stereocilia (per stereocilia pair) in control IHCs (open symbols) and in IHCs at different time points after BAPTA treatment (solid symbols). (D–E) Distribution of anti-PCDH15 (D, red) and anti-CDH23 (E, blue) immuno-gold particles on the first and second row stereocilia near the tip link region (different stereocilia row than in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio-1001583-g001" target="_blank">Figure 1H</a> and different antibody dilution than in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio-1001583-g005" target="_blank">Figure 5C</a>). 50 (PCDH15) or 80–90 (CDH23) images of stereocilia pairs were aligned and scaled to a template (leftmost cartoon in D) and the location of each gold particle was marked with a semi-transparent circle. (F) Template showing the regions of interest: tip link area (green), side link area (yellow), and the remaining surface of the stereocilia pair (grey). (G–I) Changes of the number of anti-PCDH15 (red) and anti-CDH23 (blue) immuno-gold particles in the indicated regions of interest. Arrows indicate time of BAPTA treatment. Black circles in (G) show number of tip links per stereocilia pair counted in the immuno-SEM images. The same cells contributed to (D–I). All averaged data are shown as mean ± SE. Asterisks indicate statistical significance from the control values: * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001 (<i>t</i> test of independent variables). Antibody dilutions: C2367, 1∶100; HL5614, 1∶1000 (C), 1∶100 (D–I). Age of the cells: P3+2–3 div.</p

Vertical positioning of the probe is crucial for meaningful MET recordings.

<p>(A) Three different probe positions drawn to scale: “very high” (I), “high” (II), and “optimal” (III). Dashed cyan lines indicate the boundary layer of fluid entrained by a fast moving probe. The main panels show a “back view” of the probe deflecting stereocilia bundle, while insets represent side views. In positions (I) and (II), the liquid boundary layer may entrain the outermost stereocilia of the bundle during forward movement of the probe. Therefore, these outermost stereocilia are free to “spring back” when the probe stops. In contrast to bullfrog sacculus stereocilia, which are tightly coupled and move in unison <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Kozlov1" target="_blank">[48]</a>, stereocilia of mammalian IHCs are coupled more loosely and are unlikely to move in unison <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Stauffer1" target="_blank">[20]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001583#pbio.1001583-Smith1" target="_blank">[49]</a>. (B) MET currents (top traces) evoked by the graded deflections of stereocilia (bottom traces) in <i>the same cell</i> at different probe positions (I, II, III) at positive (+90 mV) and negative (−90 mV) holding potentials. Positive holding potential prevents Ca²⁺ influx into the cell and, therefore, eliminates Ca²⁺-dependent adaptation. Black traces highlight MET current evoked by the smallest deflection of the probe. (C) The current–displacement relationships of MET current (left); the extent of adaptation, i.e. percent changes of the MET current at the end of bundle deflection of 10 ms duration (middle); and the time constant of MET adaptation, τ_ad, determined by a single exponential fit (right) at different probe positions (I, blue; II, green; III, black). Data in panel (C) are calculated from traces in panel (B) at holding potential of −90 mV. Age of the cell: P3+4 div.</p