Figure 1.
ANKRD24 localization to hair cell stereocilia rootlets. (A and B) Mass spectrometry quantitation from cochlea (A) and utricle (B) using DIA. Pou4f3-GFP–positive hair cells (green) and –negative cells (black). Mean ± SEM (n = 4). (C) ANKRD24 staining in midcochlea on P7.5. Left, horizontal view through rootlets. Right, reslices of OHCs (top) or IHCs (bottom). (D) ANKRD24 staining in P7.5 utricle. Left, horizontal view through rootlets; inset, magnified view of the rootlets. Right, reslice images from type I (left) or type II (right) hair cells. (E) Developmental progression of ANKRD24 in midcochlea (OHCs, top; IHCs, bottom). Image intensities adjusted to compare labeling. (F) Triple labeling of OHCs and IHCs for actin (cyan; Act), ANKRD24 (magenta; A), and TRIOBP (yellow; T). Right panels, ANKRD24 and TRIOBP. (G) Development of the rootlet and associated ANKRD24 and TRIOBP labeling from P0.5 through P8.5. Brackets in actin panels highlight rootlet development. (H and I) ANKRD24 overlap with TRIOBP in IHCs and OHCs. Arrows indicate position of ANKRD24 at apical surface, and arrowheads indicate ANKRD24 and TRIOBP in the lower rootlet. (J) ANKRD24 rings surrounding the membrane insertion in IHCs. Lower-intensity staining at rootlet in actin-free channel (arrowheads). (K) ANKRD24 in OHC. ANKRD24 at membrane insertion and extension through rootlet (arrowheads). (L) Cross-section of stereocilia rootlets within cuticular plate, near apical surface. ANKRD24 fills the actin-free region, while TRIOBP is more tightly associated with rootlet F-actin. (M) Montage of ANKRD24 labeling (Alexa Fluor 488 secondary antibody) from stereocilia insertion (z = 0 nm) in single x–y images at 220-nm intervals through the rootlet. ANKRD24 rings are apparent not only at insertion, but also within the rootlet. (N–R) Averages of actin (O and Q) or antibody labeling with Alexa Fluor 488 secondary antibody in row 1 IHC stereocilia (N, P, and R). (N) ANKRD24 at stereocilia insertions (average of 40 images). (O) Actin from ANKRD24 at rootlets (n = 50). (P) ANKRD24 at rootlets (≥440 nm below insertions; n = 50). (Q) Actin from TRIOBP at rootlets (n = 35). (R) TRIOBP at rootlets (≥440 nm below insertions; n = 50). (S) Line scans (31.3 nm wide) through center of average images in N–R. Offsets are applied to N and O for clarity. Data (lighter colored points) are fitted with single (actin, TRIOBP) or double (ANKRD24) Gaussians (thick lines). (T) Cartoon showing arrangement of actin (truncated stereocilium with rootlet), TRIOBP, and ANKRD24 in rootlet area. Last panel shows all three proteins but with rootlet ANKRD24 and TRIOBP partially removed to show their concentric relationship relative to actin. Panel full widths: C left, 25 µm; C right, 4.5 µm; D left, 44 µm (inset, 5 µm); D right, 4.5 µm; E, 17 µm; F, 18 µm; G and K, 3 µm; H, 4 µm; J, 1.8 µm; L, 2.1 µm; M, 2 × 2 montage of 470-nm panels; N–R, 625 nm. Superresolution modality: A–I, Airyscan; J–R, lattice SIM. AU, arbitrary unit.

ANKRD24 localization to hair cell stereocilia rootlets. (A and B) Mass spectrometry quantitation from cochlea (A) and utricle (B) using DIA. Pou4f3-GFP–positive hair cells (green) and –negative cells (black). Mean ± SEM (n = 4). (C) ANKRD24 staining in midcochlea on P7.5. Left, horizontal view through rootlets. Right, reslices of OHCs (top) or IHCs (bottom). (D) ANKRD24 staining in P7.5 utricle. Left, horizontal view through rootlets; inset, magnified view of the rootlets. Right, reslice images from type I (left) or type II (right) hair cells. (E) Developmental progression of ANKRD24 in midcochlea (OHCs, top; IHCs, bottom). Image intensities adjusted to compare labeling. (F) Triple labeling of OHCs and IHCs for actin (cyan; Act), ANKRD24 (magenta; A), and TRIOBP (yellow; T). Right panels, ANKRD24 and TRIOBP. (G) Development of the rootlet and associated ANKRD24 and TRIOBP labeling from P0.5 through P8.5. Brackets in actin panels highlight rootlet development. (H and I) ANKRD24 overlap with TRIOBP in IHCs and OHCs. Arrows indicate position of ANKRD24 at apical surface, and arrowheads indicate ANKRD24 and TRIOBP in the lower rootlet. (J) ANKRD24 rings surrounding the membrane insertion in IHCs. Lower-intensity staining at rootlet in actin-free channel (arrowheads). (K) ANKRD24 in OHC. ANKRD24 at membrane insertion and extension through rootlet (arrowheads). (L) Cross-section of stereocilia rootlets within cuticular plate, near apical surface. ANKRD24 fills the actin-free region, while TRIOBP is more tightly associated with rootlet F-actin. (M) Montage of ANKRD24 labeling (Alexa Fluor 488 secondary antibody) from stereocilia insertion (z = 0 nm) in single x–y images at 220-nm intervals through the rootlet. ANKRD24 rings are apparent not only at insertion, but also within the rootlet. (N–R) Averages of actin (O and Q) or antibody labeling with Alexa Fluor 488 secondary antibody in row 1 IHC stereocilia (N, P, and R). (N) ANKRD24 at stereocilia insertions (average of 40 images). (O) Actin from ANKRD24 at rootlets (n = 50). (P) ANKRD24 at rootlets (≥440 nm below insertions; n = 50). (Q) Actin from TRIOBP at rootlets (n = 35). (R) TRIOBP at rootlets (≥440 nm below insertions; n = 50). (S) Line scans (31.3 nm wide) through center of average images in N–R. Offsets are applied to N and O for clarity. Data (lighter colored points) are fitted with single (actin, TRIOBP) or double (ANKRD24) Gaussians (thick lines). (T) Cartoon showing arrangement of actin (truncated stereocilium with rootlet), TRIOBP, and ANKRD24 in rootlet area. Last panel shows all three proteins but with rootlet ANKRD24 and TRIOBP partially removed to show their concentric relationship relative to actin. Panel full widths: C left, 25 µm; C right, 4.5 µm; D left, 44 µm (inset, 5 µm); D right, 4.5 µm; E, 17 µm; F, 18 µm; G and K, 3 µm; H, 4 µm; J, 1.8 µm; L, 2.1 µm; M, 2 × 2 montage of 470-nm panels; N–R, 625 nm. Superresolution modality: A–I, Airyscan; J–R, lattice SIM. AU, arbitrary unit.

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