Heterodimeric capping protein is required for stereocilia length and width regulation

Abstract

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.

Figure 1.

Mass spectrometry identification and quantitation of hair-bundle actin cappers in chick and mouse inner ear. (A) Data-dependent acquisition (DDA) mass spectrometry of E20 chick hair bundle proteins detected in three out of three chick datasets. Actin-associated proteins enriched twofold or more in bundles are indicated by red callouts; bold red callouts indicate actin cappers. Bars for actin cross-linkers, actin-membrane connectors, and actin filaments indicate the approximate number of each per stereocilium. (B) DDA analysis of P23 mouse bundle proteins detected in four out of four biological replicates. (C) Capper levels in chick and mouse stereocilia estimated by DDA mass spectrometry. Mean ± SD, n = 4 for all. (D) DIA mass spectrometry of isolated cells at different developmental ages. Utricle and cochlea cells were separately isolated by FACS from Pou4f3-GFP mice; hair cells are GFP positive (GFP+), and all other cells are GFP negative (GFP−). Dashed lines in the CAPZB panels indicate the sum of the CAPZA1 and CAPZA2 mean peptide intensities. Note y axis expansion for GSN in utricle. Mean ± SD, n = 3 for all.

Figure 2.

Actin cappers during postnatal mouse cochlea and utricle development. (A–L) Localization of actin cappers during development. Primary antibodies are indicated on the left, and developmental age and organ on the top. (A) CAPZB2 in cochlea. (B) CAPZB2 in utricle. Arrow indicates medium-sized hair bundle with strong CAPZB2 labeling. (C) CAPZA1 in cochlea. (D) CAPZA1 in utricle. (E) EPS8 in cochlea. Arrow indicates strong labeling at tips of row 1 (tallest) inner hair cell (IHC) stereocilia. (F) EPS8 in utricle. Arrow indicates labeling at tips of tallest stereocilia. (G) EPS8L2 in cochlea. Arrow indicates strong labeling at tips of row 2 inner hair cell stereocilia, although row 1 stereocilia are also labeled. (H) EPS8L2 in utricle. Arrow indicates labeling of tips of intermediate-length stereocilia. (I) GSN in cochlea. (J) GSN in utricle. Asterisk indicates occasional tip labeling in utricle bundles. (K) TWF2 in cochlea. Labeling in inner hair cells is similar to that of ankle links (arrow) rather than stereocilia tips. (L) TWF2 in utricle. Labeling at stereocilia tips is strong at P8; labeling near ankle links is also apparent (asterisks). (M) CAPZB2 labeling in P22 apical cochlea inner hair cells. Arrow indicates strong labeling at row 2 tips. (N) TWF2 labeling in P22 apical cochlea inner hair cells. Arrow indicates strong labeling at row 2 tips. (O) Double labeling of CAPZB2 (green) and TWF2 (magenta). Green and magenta channels were subjected to two-pixel Gaussian filtering. Arrowheads indicate several coincident green and magenta spots. (P) High-magnification view (projected x-z reslices) of increased CAPZB2 labeling density in short utricle bundles (arrows). Asterisks indicate labeling at supporting cell apical surfaces. (Q) CAPZB2 density in bundles during development. Confocal images (averaged z-planes) showing no labeling in small bundles (asterisks), strong labeling in medium-sized bundles (arrows), and reduced labeling in tall bundles. (R) Relation between bundle height (in tranches of 1 µm) and CAPZB2/phalloidin ratio. Gray line is exponential rise for the first three points, exponential fall for remaining points. Mean ± SEM is plotted; number of bundles measured (n) for each height tranche is indicated. Panel full widths are as follows: In A, C, E, G, and I, low-power outer hair cell (OHC) and inner hair cell panels are 25 µm wide, and the high-magnification inner hair cell image is 8 µm wide. In B, D, F, H, and J, the left three panels are maximum projections of 10-µm-deep x-z reslices, taken from z-series, and are 25 µm wide; high-magnification panels on the right are 8 µm wide. In K and L, panels are 20 µm wide. In M, panels are 7 µm wide. In N, the panel is 14.5 µm wide. In O, panels are 25 µm wide. All high-magnification panels used maximum projection of several z-sections (not x-z reslices) from images acquired with Airyscan detector.

Figure 3.

Immunoprecipitation of capping protein from mouse crude stereocilia extracts. Crude mouse stereocilia were solubilized with RIPA buffer and then subject to sequential control and CAPZ subunit immunoprecipitations. (A–C) Immunoblot analysis. Total and flow-through (F/T) samples are loaded at 33%; immunoprecipitates are loaded at 100%. (D and E) Quantitative mass spectrometry analysis comparing each protein's relative abundance (riBAQ) in the total sample (x axis) and the specific immunoprecipitate (y axis). Capping protein subunits, contaminating immunoglobulins (IgG), and actin are indicated in each plot. In CAPZA immunoprecipitates, large amounts of multiple subunits of the PDH complex were observed (D). In CAPZB2 immunoprecipitates, twinfilins TWF1 and TWF2 were each substantially enriched (indicated by vertical distance from dashed gray unity line). Only proteins detected in two out of three total replicates or two out of two immunoprecipitation replicates are plotted. ND, not detected. (F) Immunoprecipitate/total riBAQ ratio for the 25 most-enriched proteins in the CAPZB2 immunoprecipitations; note log scale. riBAQ ratios for CAPZA immunoprecipitates are also indicated. Mean ± SD is plotted, n = 2. Asterisks indicate not detected in CAPZA immunoprecipitations.

Figure 4.

Effects of Capzb deletion on auditory and vestibular function. (A and B) Auditory brainstem response (ABR). (A) Waveform examples at indicated decibel sound pressure level at 8 kHz for wild-type, heterozygous, and Capzb^CKO mice are at top. (B) Summarized data (mean ± SEM). (C and D) Distortion-product otoacoustic emissions (DPOAE). (C) Waveform examples for indicated f1 and f2 primaries and the 2f1-f2 distortion product (asterisks) for wild-type, heterozygous, and Capzb^CKO mice. (D) Summarized data (mean ± SEM). (E and F) Vestibular evoked potentials (VsEPs). (E) Waveform examples in response to linear jerk pulses at indicated stimulus level for wild-type, heterozygous, and Capzb^CKO mice. (F) Summarized data (mean ± SEM). Calibration bars represent amplitudes in microvolts for ABR, decibel signal-to-noise ratio (dB SNR) for DPOAE, and microvolts for VsEP. Time is represented in milliseconds for ABR and VsEP waveforms. Frequency for DPOAE waveforms is represented in kilohertz. ***, P < 0.001.

Figure 5.

Consequences of Capzb deletion in cochlea. (A and B) Bundle and cell morphology in Capzb^CKO;Ai14 cochlea. Occasional hair cells (<1%) were Ai14 negative (*). Several phenotypes of Capzb^CKO hair cells are indicated in B: cells with no stereocilia (#), ruptures in cuticular plates (arrows), increased actin density in adherens junction region (arrowheads), and expanded cross-sectional area of hair cell (dashed box). (C and D) Reslice of z-stack through outer hair cells showing that CAPZB2 immunolabeling is diminished in hair cell somas of Capzb^CKO mice (asterisks), but some immunoreactivity remains in stereocilia (arrows). Arrowheads indicate increased actin density in adherens junction region. (E and F) High-magnification views of anti-CAPZB2 staining of control (E) and Capzb^CKO (F) inner-hair cell bundles; panels are 18 µm wide. z-projection of 1.5 µm completely through the bundles displayed. In F, some residual CAPZB2 immunoreactivity is apparent in tall stereocilia (arrow). Note too that the bundle on the left has a region where stereocilia are near normal length (*) and another region where stereocilia have shortened and thinned (***). (G–I) Scanning electron microscopy of control (G) and Capzb^CKO (H and I) cochleas. G and H are 45 µm wide, whereas panel I is 60 µm wide; all insets are 8 µm wide.

Figure 6.

Consequences of Capzb deletion in utricle. (A and B) Cell morphology in control;Ai14 and Capzb^CKO;Ai14 utricular macula at P21 using phalloidin and tdTomato. Note the reduced number of hair bundles and partially extruded cells in Capzb^CKO utricle. (C–H) Phalloidin staining of bundles (left) and cell apex (right) at indicated developmental times and genotypes. Disruption of cuticular plates in Capzb^CKO utricles is apparent even at P2. At P9, cytocauds are visible in Capzb^CKO utricles (arrows). At P15, only occasion normal-appearing bundles are seen in Capzb^CKO utricles (asterisk). (I and J) Utricle at ∼P100. Occasional normal-appearing bundles are seen in Capzb^CKO utricles (asterisk), but most bundles are very small (arrows), even if they have long kinocilia. (K and L) Scanning electron microscopy of control and Capzb^CKO utricles. Insets show representative bundles; note that the Capzb^CKO bundle has very short stereocilia except for the tallest one or two rows. (M and N) CAPZB2 staining of control and Capzb^CKO utricles. Left panels are CAPZB2 channel alone, right panels have merged phalloidin and CAPZB2 channels. The arrow in N indicates short stereocilia that stained with phalloidin but not anti-CAPZB2; asterisks indicate reduced CAPZB2 staining specifically in hair cell somas. (O and P) Pan-ESPN staining of control and Capzb^CKO utricles. The left panel is ESPN channel alone, and the right panel has both phalloidin and ESPN channels. Note the prominent cytocauds in the Capzb^CKO hair cells; they stain with anti-ESPN, albeit less strongly than do bundles. (Q and R) Isolated stereocilia from P23 heterozygous (Q) or Capzb^CKO (R) utricles. At this age, short and medium-length stereocilia that are relatively thick in diameter have strong CAPZB2 staining at tips (arrows). Panel full widths: (A and B) 300 µm; (C–H) 70 µm (left) and 18 µm (right); (I and J) 150 µm; (K and L) 50 µm (left) and 7.5 µm (right); (M–P) 20 µm; (Q and R) 40 µm.

Figure 7.

Capzb^CKO utricle hair bundles are short and have narrow stereocilia. (A–D) Confocal slices through hair bundles (A and C) or cell apex (B and D) of heterozygous control (A and B) or Capzb^CKO utricle. Insets in A and C show representative bundles; the lower intensity of the bundle in C indicates fewer actin filaments and hence a narrower diameter. (E–M) Measurements of hair cell properties from P21 heterozygous and Capzb^CKO utricles using image-scanning microscopy. (E) Stereocilia per bundle. (F) Cross-sectional area of bundle at base. (G) Cross-sectional area of cell apex. (H) Stereocilia diameter. (I) Mean tdTomato intensity at cell apex. (J) Relationship between stereocilia diameter and tdTomato mean intensity. (K) Length of tallest stereocilia. (L) Length of kinocilium. (M) Profile of bundles; for each height step in a confocal z-stack, the number of stereocilia still present was counted. (N–Q) Isolated stereocilia from P8 and P25 utricles. (R and S) Length distributions for P8 (R) and P25 (S) stereocilia. (T and U) Diameter distributions for P8 (T) and P25 (U) stereocilia; diameter estimated from full width at half-maximum of a Gaussian profile across the stereocilium. Panel full widths: (A–D) 45 µm; (N–Q) 30 µm.

Figure 8.

Heterologous MYC-CAPZB2 disrupts normal utricle stereocilia formation. (A) Stereocilia morphology in a nontransfected region (no ZsGreen signal) using z-stack reslice. (B) Stereocilia morphology in a transfected region (ZsGreen cell fill) using z-stack reslice. (C) Distribution of tallest stereocilia length of hair bundles from control (untransfected ears plus nontransfected regions) and MYC-CAPZB2–transfected hair cells. Two-Gaussian fits. (D and E) Single z-sections with phalloidin labeling of nontransfected (G) and transfected (H) regions; panels in Di and Ei are magnifications of boxed regions in D and E. Panel full-widths: (A and B) 30 µm; (D and E) 40 µm; (Di and Ei) 7.5 µm. (F) Purification of MYC-CAPZB2 from HEK cells. Left, Coomassie-stained SDS-PAGE gel with purified MYC-CAPZB2 and capping protein (CAPZA1:CAPZB2 heterodimer expressed in E. coli). Right, immunoblot analysis of purified MYC-CAPZB2 and capping protein. Detection antibodies and their targets are indicated below each immunoblot. (G) Inhibition of pyrene-actin polymerization by capping protein (left) or MYC-CAPZB2 (right). (H) Summed data. MYC-CAPZB2 data includes those with proteins purified by only 9E10 chromatography or with 9E10 and gel filtration; results were similar. Data were fit with Hill plots. For capping protein, IC50 = 2.1 ± 0.2 nM and Hill coefficient = 1.3 ± 0.2; for MYC-CAPZB2, IC50 = 190 ± 25 nM and Hill coefficient = 1.1 ± 0.1. (D–H) Transfection of utricle hair cells with MYC-CAPZB2.

Figure 9.

PLEKHO1 is up-regulated in Capzb^CKO inner ears. (A) Immunocytochemistry of wild-type mouse cochlea at P8 using antibody against PLEKHO1. The top panel shows inner hair cells, the middle panel shows outer hair cells, and the bottom panel shows outer hair cell somas. (B) PLEKHO1 immunoreactivity in Capzb^CKO cochlea. Asterisk indicates degenerating hair bundle with reduced PLEKHO1 immunoreactivity. (C) Quantitation of PLEKHO1 fluorescence in 2-µm z-projections using regions of interest encompassing outer hair cell (OHC) bundles or somas. (D) PLEKHO1 immunoreactivity in wild-type utricle. (E) PLEKHO1 immunoreactivity in Capzb^CKO utricle. Arrow indicates lack of staining of short stereocilia. (F) Colocalization of PLEKHO1 and CAPZB2 in P8 utricle stereocilia. Arrowheads show colocalized PLEKHO1 and CAPZB2 punctae. Panel full widths: (A and B) 50 µm; (D and E) 25 µm; (F) 12 µm (inset is 2 µm).

Figure 10.

CAPZB action in hair cells. (A) Model for capping protein interaction with actin. The β-tentacle of CAPZB (dark green) wraps around the first actin subunit (of three shown; magenta). CAPZA also binds to actin with its tentacle. Panel A was modified from Kim et al. (2010) with permission. (B) Model for action of capping protein in widening; the four stereocilia cartoons indicate sequential steps in the process. New actin filaments polymerize from the stereocilia base to the tip, alongside the preexisting original actin paracrystal. Capping protein transiently binds to elongating filaments, preventing their depolymerization when actin monomers are not available. (C) Development of hair cells with or without CAPZB. The middle trajectory indicates how stereocilia lengthening and widening proceed in a wild-type hair cell, with capping protein indicated using the same color scheme as in A and B. CAPZB enters stereocilia after the initial lengthening step and is present during widening. CAPZB accumulates at stereocilia tips, at least in shorter stereocilia. The lower trajectory diagrams the narrowing and shortening of stereocilia that occurs after CAPZB is lost. CAPZB derived from hair-cell progenitors is present in Capzb^CKO even after levels drop in the soma, presumably slowing the shortening and narrowing process. The top trajectory shows the cessation of stereocilia elongation that occurs when MYC-CAPZB2 is expressed in hair cells, as well as the abnormal stereocilia widening. MYC-CAPZB2 is represented with blue subunits, both as monomers and as heterodimers with CAPZA subunits. The placement of the MYC-CAPZB2 and CAPZA molecules in the MYC-CAPZB2 scenario is based on their presumed localization. (D) Diagram of interactions between capping protein (CP), TWF2, and PLEKHO1. The N terminus of CAPZB, which is not directly involved in binding actin filaments, binds to either TWF2 (shown with bound actin monomers) or PLEKHO1.