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Control of stereocilia length during development of hair bundles [1]

['Jocelyn F. Krey', 'Oregon Hearing Research Center', 'Vollum Institute', 'Oregon Health', 'Science University', 'Portland', 'Oregon', 'United States Of America', 'Paroma Chatterjee', 'Julia Halford']

Date: 2023-04

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later—in early stage IV—and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23 v2J or Pcdh15 av3J ), transduction channels (Tmie KO ), or the row 1 tip complex (Myo15a sh2 ). Cdh23 v2J/v2J and Pcdh15 av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of Tmie KO/KO row 1 stereocilia, they accumulated normally in Cdh23 v2J/v2J and Pcdh15 av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all Tmie KO/KO , Cdh23 v2J/v2J , and Pcdh15 av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction’s role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

Funding: This work was supported by the National Institute on Deafness and Other Communication Disorders (R01DC002368 to PGBG, R21DC019195 to CLC and R01DC015495 to BJP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Krey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Thus, at least 5 key multimolecular assemblies control stereocilia dimensions: (1) tip links; (2) transduction channels; (3) the row 1 complex; (4) row 2 cappers; and (5) actin-severing proteins. Each of these assemblies is activated at different times during development and is responsible for 1 or more steps of hair-bundle development. Here, we defined with higher precision the changes in stereocilia dimensions during postnatal development of apical IHCs from postnatal day 0.5 (P0.5) to P21.5. Moreover, we used mutant mice to define more specifically the role of tip links on bundle development and the localization of tip-protein complexes. We examined stereocilia dimensions and row protein localization in Pcdh15 av3J and Cdh23 v2J null hair cells, as well as in Tmie KO and Myo15a sh2 null hair cells. Besides gating transduction channels, we found that the tip-link proteins play an essential role in regulating stereocilia lengths; they also have a significant impact on accumulation of row-specific proteins at stereocilia tips, which in turn modulate stereocilia length. These observations complement previous results and provide us with a more comprehensive molecular understanding of bundle development in this hair-cell type.

F-actin can be depolymerized at stereocilia tips due to the action of the ADF/CFL family members DSTN and CFL1 [ 42 , 43 ]; these proteins sever actin filaments, which can lead to dissolution of actin structures [ 44 ]. Proteins in the ADF/CFL family typically associate with WDR1 (AIP1), which is located in stereocilia [ 45 ] and participates in length regulation [ 42 ]. DSTN and CFL1 are specifically located at row 2 stereocilia tips during early postnatal development, and this localization is disrupted in mutants lacking mechanotransduction [ 43 ]. Actin dynamics controlled by DSTN and CFL1 may underlie pruning of the shortest rows of stereocilia that occurs during hair-bundle maturation [ 46 ].

When tip links are under tension, the stereocilia membrane lifts off of the underlying F-actin core, a phenomenon called membrane tenting [ 38 , 39 ]; once the membrane is pulled away, actin polymerization is enhanced, especially on the side of stereocilium where the tip link anchors [ 17 , 40 ]. This phenomenon gives row 2 stereocilia a pronounced beveled shape [ 17 , 40 ]. These results suggest the possibility that tension in tip links promotes actin polymerization in the shorter stereocilia rows, independent of channel gating [ 41 ]. Because tip links gate transduction channels [ 38 ], Cdh23 and Pcdh15 mutants lose both transduction and membrane tenting [ 37 ], and shorter stereocilia in homozygous Cdh23 and Pcdh15 mutants have rounded tips instead of beveled ones [ 37 ].

Tip links are made up of dimers of CDH23, which project from the side of a taller stereocilium, and dimers of PCDH15, projecting from tips of short stereocilia [ 24 – 26 ]. The molecular motor MYO7A positions both molecules and tensions tip links [ 27 – 29 ]. All 3 of these proteins are members of the Usher I protein complex [ 30 ], and all are necessary for formation of tip links and activation of transduction. CDH23 and PCDH15 contribute to transient lateral links [ 24 , 26 , 31 , 32 ], which interconnect stereocilia along their shafts during development, as well as kinocilial links [ 33 , 34 ], which connect several central row 1 stereocilium to the axonemal kinocilium. Mutant mouse lines lacking functional genes for Cdh23 or Pcdh15 develop ragged staircases, with stereocilia of irregular lengths but relatively uniform diameters [ 35 – 37 ].

Tension in tip links, extracellular filaments that interconnect stereocilia rows, elicits transduction currents by opening cation-conducting mechanotransduction channels [ 2 , 16 ]. Transduction regulates the final dimensions of stereocilia. Blockade of transduction channels elicits stereocilia shortening, suggesting a dynamic balance between actin polymerization and depolymerization [ 17 ]. Establishment of transduction currents requires the small membrane protein TMIE [ 18 ] and either TMC1 or TMC2, 2 channel-like proteins [ 19 , 20 ]. An ordered stereocilia staircase still forms in mouse mutants lacking either Tmie or both Tmc1 and Tmc2 [ 18 , 19 ], but row-specific distinctions of stereocilia width and length become more muted [ 6 , 21 ]. The presence of transduction-channel proteins also regulates the localization of proteins specific for row 1 and row 2 [ 6 ]. Although row 1 lacks transduction [ 22 ], in transduction mutants, GNAI3 and GPSM2 do not accumulate substantially at row 1 tips over postnatal development [ 6 ]. Significantly, transduction-channel proteins do localize to row 1 during early postnatal development [ 23 ].

Lengths of the shorter rows of stereocilia are regulated differently. Several other proteins, including TWF2, EPS8L2, and CAPZB, are found predominantly at row 2 tips [ 13 – 15 ]; these proteins have actin-filament capping activity, so their presence may slow lengthening. Stereocilia lacking Capzb are narrow, then shorten and disappear [ 14 ], suggesting that CAPZB (and its binding partner TWF2) contribute both to stereocilia widening but also length stability.

During development, stereocilia in each row differentially widen and lengthen in specific stages [ 1 , 6 ]. MYO15A and EPS8 are found together at all stereocilia tips during early postnatal development and likely control the initial lengthening of stereocilia [ 7 ]. A complex of GPSM2 and GNAI3, coupled to MYO15A and EPS8 by WHRN, catalyzes lengthening of stereocilia beyond about a micrometer in postnatal stereocilia development [ 7 – 9 ]. This complex concentrates exclusively in row 1 after P7 and hence is referred to here as the row 1 tip complex. The 5 proteins together undergo phase separation, and the presence of GPSM2 both enhances phase separation and promotes F-actin bundling by EPS8 [ 10 , 11 ], activity that presumably underlies stereocilia lengthening. Mutations in any of the genes encoding these 5 proteins leads to hair bundles that have short stereocilia and a minimal staircase, i.e., only very small changes of stereocilia length in successive rows [ 12 ].

Sensory hair cells of the inner ear are distinguished by the staircase arrangement of the approximately 100 stereocilia in their hair bundles, the sensory organelle that converts mechanical stimuli into electrical signals [ 1 , 2 ]. The staircase architecture enables directional sensitivity of mechanotransduction [ 3 ] and increases transduction sensitivity [ 4 ]. In the mature mammalian cochlea, stereocilia of inner hair cells (IHCs) and outer hair cells (OHCs) are arranged in 3 rows, albeit with distinct stereocilia lengths and widths [ 5 ].

Results

Quantitation of stereocilia actin-core dimensions using lattice structured illumination microscopy We determined dimensions of stereocilia F-actin cores, labeled with fluorescent phalloidin, which were imaged in IHCs of C57BL/6 mice at precise times during early postnatal development. We used IHCs from the basal half of the apical turn of the cochlea, corresponding to 17% to 33% of cochlear length. Our measurements should reflect native lengths and widths; dimensions of mildly fixed, phalloidin-labeled stereocilia are not significantly different from dimensions of live stereocilia labeled with membrane dyes [47]. We improved resolution over conventional confocal microscopy by using lattice structured illumination microscopy (lattice SIM) [48–50], which has a point-spread function (PSF) of approximately 150 nm under our conditions [50]; typical confocal microscopy PSFs are approximately 230 nm [51]. We rendered image surfaces from each phalloidin-stained hair bundle (Fig 1A–1F); rendered surfaces are 3D models of specific structures computed from stacks of images by preprocessing, segmentation, and connected-component labeling steps. We saved separate surfaces for each row 1, 2, or 3 stereocilium, as well as for some row 4 stereocilia and apical-surface microvilli (referred together as row 4+ stereocilia). Because of their relatively large sizes, rows 1 and 2 stereocilia could be measured reliably; dimensions of rows 3 and 4+ stereocilia and microvilli could also be measured, but required careful choices of rendering parameters and editing of surfaces after rendering. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Stereocilia dimensions during development of C57BL/6J apical IHCs. (A–F) Imaris reconstruction of phalloidin-labeled IHC stereocilia from indicated ages. Stereocilia surfaces are color-coded according to row; each box is 14 × 35 μm. Imaris reconstructions show overlapping hair bundles of adjacent cells, presumably an artefact of sample preparation. (G–N) IHC stereocilia dimension measurements using reconstructed stereocilia surfaces. (G) Rows 1 and 2 stereocilia length during development; center 10 stereocilia in each row. Row 1 data were fit with a line with slope of zero from P0.5–P7.5 followed by an exponential climb from P7.5–P21.5; row 2 data were fit with an exponential decline. (H) Rows 1 and 2 stereocilia width (center 10). In the panel, data for row 1 and for row 2 from P0.5–P7.5 were fit with an exponential climb; data for 2 from P7.5–P21.5 were fit with an exponential climb. The data could also be fit linearly; between P0.5 and P7.5, the slope for row 1 was 0.026 μm • day-1 (95% confidence interval of 0.025–0.026), while the row 2 slope was 0.034 μm • day-1 (95% confidence interval of 0.032–0.036). (I) Volume per individual row 1 or row 2 stereocilium (center 10). Data were fit with a linear increase from P0.5–P7.5, following by an exponential climb (row 1) or decline (row 2) from P7.5–P21.5. (J) Rows 1 and 2 stereocilia cross-sectional area (center 10). Data were fit with a linear increase from P0.5–P7.5, followed by an exponential climb (row 1) or decline (row 2) from P7.5–P21.5. (K) Total stereocilia volume per cell using rows 1, 2, and 3 stereocilia. Data were fit with an exponential climb. (L) Number of stereocilia per cell. Data were fit with an exponential decline. (M) Length difference between row 1 and row 2 stereocilia in the same column. Data were fit with a line with slope of zero from P0.5–P7.5, followed by an exponential climb from P7.5–P21.5. (N) Length difference between adjacent (side-by-side) stereocilia in the same row. Data were fit by exponential declines. For each plot, dimension measurements of stereocilia from each bundle were averaged to give an individual cell mean; then all means for individual cells (5–6 cells from each of 3–4 cochleas) were averaged and plotted ± SEM. (O) Diagrams of IHC bundle structure at each Tilney stages in mouse cochlea. Tip links are indicated as blue lines connecting adjacent stereocilia; they appear during stage II. Transducing stereocilia are shaded red and are the stereocilia that contain active transduction channels. The principal stereocilia-building function in each stage is indicated. The data underlying all the graphs shown in the figure can be found in figshare (https://doi.org/10.6084/m9.figshare.21632636.v2). IHC, inner hair cell. https://doi.org/10.1371/journal.pbio.3001964.g001 For row 1 and row 2, the variability in stereocilia length within each IHC hair bundle (represented by the coefficient of variation, CV) was high at P0.5 and declined by P7.5 (S1A Fig). The majority of length variability at intermediate ages during development was because the peripheral stereocilia in a row were systematically shorter than the central stereocilia of that row [6], a phenomenon that disappears by P11 [47] (depicted schematically in S1E–S1G Fig). We therefore restricted most of our measurements to the center 10 stereocilia of each row (panel F of S1 Fig), which could be reliably carried out only with rows 1 and 2. By focusing on these center stereocilia, length variability was reduced significantly in row 1 and row 2 at all ages except P0.5 and was constant across development (S1B Fig). The width CV for each bundle was also significantly lower for row 1 and especially row 2 for the center 10 stereocilia at most ages (S1C and S1D Fig).

Stereocilia dimensions during stages III and IV of early postnatal development Tilney [1] suggested that hair-bundle development in chickens could be divided into 4 stages, where stage I hair cells have apical surfaces with equal-length microvilli, stereocilia in stage II cells begin to lengthen and form the staircase, stage III stereocilia widen without lengthening, and stage IV stereocilia undergo final lengthening (Fig 1O). While these stages are present in apical IHCs of C57BL/6 mice [6], the temporal and spatial resolution of previous measurements was insufficient to define transition points between the stages. We improved on those measurements by (1) using the objective measurement approach described above; (2) including a larger number of samples for each time point; and (3) expanding the number of time points examined (Fig 1G–1N). For length and width measurements, as well as individual stereocilium volumes, we calculated the mean and CV for the center 10 stereocilia of each row of a single bundle; each symbol (each time point) in Fig 1G–1N corresponds to data from 22 to 24 single hair cells from 4 different cochleas. Total stereocilia volume and number measurements were made for all stereocilia in a bundle, also with a total of 22 to 24 single-cell measurements from 4 cochleas. As previously reported [52], the number of IHC stereocilia decreased between P0.5 and P21.5, eventually reaching approximately 17 each of row 1 and row 2 stereocilia (Fig 1L). Row 1 stereocilia length remained constant between P0.5 and P8.5, and then increased exponentially through P21.5, nearly doubling in length over that time period (Fig 1G). The transition between the flat and exponential regimes was sharp. By contrast, row 2 lengths were greatest at P0.5; the length then decreased linearly until P8.5, when the length stabilized (Fig 1G). Row 2 stereocilia widened about 30% faster than row 1 stereocilia (Fig 1H). The difference in width between the rows at each time point was somewhat less than we previously estimated using a different quantitation method [6]. The widening of row 1 over the entire developmental period was fit by an exponential function, while row 2 started with exponential growth and switched to narrowing with an exponential time course. The more sustained widening in row 1 stereocilia meant that they eventually became about 20% wider than those in row 2 (Fig 1H). If new actin filaments were added to a stereocilium at a constant rate, stereocilium cross-sectional area (but not width) would grow linearly. Indeed, stereocilia cross-sectional area for IHCs increased linearly between P0.5 and P8.5 (row 1) or P7.5 (row 2); after that time, row 1 area increased exponentially in row 1 and decreased exponentially in row 2 (Fig 1J). Again, the transition between the linear and exponential regimes occurred sharply around P8 (Fig 1J). The volume of each center 10 row 1 or 2 IHC stereocilium increased linearly until P8.5 (row 1) or P7.5 (row 2); row 1 volume increased exponentially afterwards, while row 2 volume decreased exponentially (Fig 1I). To measure total stereocilia volume in a hair bundle, we included all stereocilia from rows 1 to 3; volume increased exponentially between P0.5 and P21.5 (Fig 1K), with no apparent transitions. We also measured the difference in length between adjacent IHC stereocilia. The difference in length between adjacent rows 1 and 2 stereocilia pairs—those that are in a single column along the tip-link axis—was constant at approximately 1 μm between P0.5 and P7.5, then increased exponentially to over 4 μm by P21.5 (Fig 1M). Unsurprisingly, this growth profile resembled that of row 1 lengthening and was enhanced by the minor row 2 shortening over the same period. Immediately adjacent stereocilia (side-by-side) in a row were very similar in length (Fig 1N). Using the central 10 stereocilia of each row, we measured the absolute value of the difference in length between each pair of adjacent stereocilia, then plotted the mean and CV for each hair bundle. Row 1 and row 2 stereocilia pairs showed a narrow distribution of length differences within a bundle, averaging approximately 0.3 μm (Fig 1N).

Total hair-bundle volume does not change in mutants Total stereocilia volume per hair cell was not significantly different between Cdh23v2J/+, Pcdh15av3J/+, TmieKO/+ heterozygotes and C57BL/6 wild-type mice at P7.5 or at P21.5 (Fig 8). Despite the variability in stereocilia number, length, and width, total stereocilia volumes in Cdh23v2J/v2J, Pcdh15av3J/av3J, and TmieKO/KO IHC hair bundles were statistically indistinguishable at P7.5 (Fig 8A). At P21.5, differences between Cdh23v2J and TmieKO controls and mutants were modest, albeit statistically significant (Fig 8B). The similarity in stereocilia volume is broadly consistent with Tilney’s proposal that each hair cell has a constant amount of F-actin in its bundle, but cells deploy it differently depending on other factors [60]. Although Myo15a was an outlier at P21.5 (Fig 8B), actin and actin crosslinkers intended for stereocilia may have been directed instead to cytocauds in the cell body [61]. PPT PowerPoint slide

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TIFF original image Download: Fig 8. Total stereocilia volume in mutant IHCs. (A, B) Total stereocilia volume per hair bundle and volume CV for Cdh23v2J, Pcdh15av3J, TmieKO, and Myo15ash2 IHCs. Volume measurements used reconstructed stereocilia surfaces. Plotting was as in Fig 3; statistical tests used one-way ANOVA analysis with the Šidák correction. (A) P7.5. No significant differences in volume between any of the genotypes. (B) P21.5. Modest differences in total stereocilia volume were seen for Cdh23v2J/+ vs. Cdh23v2J/v2J and TmieKO/+ vs. TmieKO/KO; a large difference in volume was seen for Myo15ash2 IHCs. The data underlying all the graphs shown in the figure can be found in figshare (https://doi.org/10.6084/m9.figshare.21632636.v2). CV, coefficient of variation; IHC, inner hair cell. https://doi.org/10.1371/journal.pbio.3001964.g008

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