PMC

Shown are single confocal sections. SNX9 immunoreactivity visualized with FITC-conjugated secondary antibody was distinctly associated with the cuticular plate, which was visualized with rhodamine-conjugated phalloidin. (A) SNX9 immunoreactivity in the cuticular plate of 2-week old mouse cochlear hair cells. (B) SNX9 immunoreactivity in the cuticular plate of 3-week old mouse outer hair cells. (C) and (D) SNX9 immunoreactivity colocalized with FCHSD1 immunoreactivity, which was visualized with Cy5-conjugated secondary antibody, in the cuticular plate of 3 week-old mouse cochlear hair cells. The Novus FCHSD1 antibody was used in (C) and (D). Scale bars: 10 µm in (A–C), 5 µm in (D).

Immunogold labeling for spectrin, β-actin, and γ-actin. A–E, Rat OHCs. F, G, Guinea pig OHCs. A, Spectrin is distributed throughout the cuticular plate and is evident beneath the apical membrane and along the lateral margins (arrowheads). Labeling also occurs over the rootlets (arrow). B, Spectrin labeling is evident not only in the subapical layer (arrowheads) and rootlets (arrow) but also in the ankle region (asterisk) and stereocilia (s). C, β-Actin labeling is denser over the stereocilia and rootlets than the main matrix of the cuticular plate. A labeled rootlet extends below the cuticular plate (arrow). D, As well as prominent in the rootlets (r), β-actin labeling is concentrated over the dark meshwork regions of the plate (arrows). E, The labeling density for γ-actin is similar in the stereocilia and cuticular plate. Rootlets are also labeled, one extending deeper than the cuticular plate (arrow). F, γ-Actin labeling (small particles) is also strongest over the dense patches associated with the meshwork (arrows). Large particles represent β-actin. G, γ-Actin labeling is continuous from the cortical lattice (arrowheads) into the cuticular plate (cp). Scale bars: A, C, E, 400 nm; B, D, 200 nm; F, G, 100 nm.

Shown are single confocal sections. FCHSD1 or FCHSD2 immunoreactivity visualized with Cy5 or FITC-conjugated secondary antibody was distinctly associated with stereocilia or cuticular plate, which were visualized with rhodamine-conjugated phalloidin. (A) FCHSD1 immunoreactivity in the cuticular plate of 3-week old mouse cochlear hair cells. (B) FCHSD1 immunoreactivity in the cuticular plate of 3-week old mouse outer hair cells. (C) FCHSD2 immunoreactivity in the hair bundles of 6-week old mouse cochlear hair cells. (D) FCHSD2 immunoreactivity in the hair bundles of 7-month old mouse inner hair cells. (E) FCHSD2 immunoreactivity in the hair bundles of 2-week old mouse outer hair cells. The Novus FCHSD1 antibody was used in (A) and (B). Scale bars: 10 µm in(A-C), 5 µm in (D) and (E).

(A) TRIOBP-5–specific antibody signal (red) observed predominantly in the rootlet segment inside the cuticular plate. See single channel images in . (B) Anti-GFP antibody recognized EGFP-TRIOBP-4 in the stereocilia rootlet segment that resides above the cuticular plate within the stereocilia core (green). A diffuse EGFP signal is present in the cuticular plate (green). (C) In Deiters’ cells located between OHCs, both TRIOBP-4 and TRIOBP-5 are present in F-actin bundles of obliquely ascending processes (arrows). Outer pillar cells show predominantly a TRIOBP-5 signal (red, arrowheads) close to junctions with OHCs. (D) P32 wild-type mouse (C57BL/6J) organ of Corti stained with TRIOBP-4/5 antibody. The inset shows representative IHC, in which TRIOBP-4/5 immunoreactivity (green) is prominent in stereocilia above the cuticular plate and a weaker signal is present in the rootlet segment within the cuticular plate. TRIOBP-4/5 immunoreactivity is also present in stereocilia of OHCs (inset and arrowheads) and in Deiters’ cells (arrows). (E and F) Helios gene gun–mediated transfections of P3 wild-type organ of Corti explants with plasmids expressing either AcGFP1-TRIOBP-5 (E) or AcGFP1-TRIOBP-4 cDNA (F) reveal targeting of AcGFP1-TRIOBP-5 (E) to stereocilia rootlets (green) within the cuticular plate. (F) AcGFP1-TRIOBP-4 predominantly targets the upper half of the rootlets within the stereocilia core, recapitulating the endogenous differential localization of TRIOBP-4 and TRIOBP-5. A barely detectable signal of AcGFP1-TRIOBP-4 was found in the lower half of rootlets within the cuticular plate. F-actin is visualized by blue phalloidin-405 in A–C and rhodamine-phalloidin (red) staining in D–F. All images are maximum intensity projections of confocal Z-stacks or their subsets (inserts). Scale bars: 5 μm (all panels).