Figure 10.
Abnormalities of stereocilia rootlets and taper regions evoked by TPRN deficiency. (A) Ultrastructural features of stereocilia tapers and rootlets in control (left), TprnN259/N259 (middle), and Tprn−/− (right) IHCs. Images represent maximum intensity projections of the stacks of individual FIB-SEM sections with ∼2 × 2 × 20-nm resolution through different overall thicknesses: 1.6 µm (top) and 400 nm (bottom). Arrows point to three typical abnormalities of the rootlets in TPRN-deficient IHCs: (1) “kinks” in the lower portion of the rootlet within cuticular plate, (2) branching at the lower end of the rootlet, and (3) F-actin breaks at stereocilia pivot points. All scale bars are 500 nm. The age of IHCs (P17, P32, or P34) is indicated. (B) Percentages of stereocilia with F-actin breaks at the pivot points (left), abnormal kinked rootlets (middle), and branched rootlets (right) in WT (n = 8/3, cells/mice, black open circles), TprnN259/N259 (n = 3/2, cells/mice, red closed circles), and Tprn−/− (n = 2/1, cells/mice, magenta closed circles) IHCs. In all three categories (F-actin breaks, kinked and branched rootlets), the difference between genotypes was highly significant (P < 0.001, one-way ANOVA). Asterisks show significance of the differences from the WT (post hoc Bonferroni test: **P < 0.01; ***P < 0.001; n.s., not significant). (C) Diameters of the lower portion of rootlets were quantified by measuring at the level of 100-nm down from IHC stereocilia pivot points (schematic in the inset of C) in the same WT, TprnN259/N259, and Tprn−/− IHCs as in B. Each point is one rootlet. The decrease in diameters of rootlets from in rows 1 and 2 of TprnN259/N259 stereocilia was significantly different from WT (P < 0.0001, one-way ANOVA, post hoc Bonferroni test: ***P < 0.001; n.s., not significant). P17 TprnN259/N259 and P32 Tprn−/− IHCs were processed and analyzed in parallel with corresponding WT IHCs (n = 4 for each group). Since no statistically significant differences were found between P17 and P34 for the WT, the data for WT were combined. (D) Drawings illustrate adult WT (top) and adult TPRN-deficient (bottom) stereocilia with magnified views of their rootlets and tapers and localization of stereocilia base proteins discussed in this study. The enlarged view of a WT stereocilia taper region is shown in a circle of top panel. In the bottom panel, an enlarged image shows details of structural disruptions at the taper region in the absence of TPRN.

Abnormalities of stereocilia rootlets and taper regions evoked by TPRN deficiency. (A) Ultrastructural features of stereocilia tapers and rootlets in control (left), TprnN259/N259 (middle), and Tprn−/− (right) IHCs. Images represent maximum intensity projections of the stacks of individual FIB-SEM sections with ∼2 × 2 × 20-nm resolution through different overall thicknesses: 1.6 µm (top) and 400 nm (bottom). Arrows point to three typical abnormalities of the rootlets in TPRN-deficient IHCs: (1) “kinks” in the lower portion of the rootlet within cuticular plate, (2) branching at the lower end of the rootlet, and (3) F-actin breaks at stereocilia pivot points. All scale bars are 500 nm. The age of IHCs (P17, P32, or P34) is indicated. (B) Percentages of stereocilia with F-actin breaks at the pivot points (left), abnormal kinked rootlets (middle), and branched rootlets (right) in WT (n = 8/3, cells/mice, black open circles), TprnN259/N259 (n = 3/2, cells/mice, red closed circles), and Tprn−/− (n = 2/1, cells/mice, magenta closed circles) IHCs. In all three categories (F-actin breaks, kinked and branched rootlets), the difference between genotypes was highly significant (P < 0.001, one-way ANOVA). Asterisks show significance of the differences from the WT (post hoc Bonferroni test: **P < 0.01; ***P < 0.001; n.s., not significant). (C) Diameters of the lower portion of rootlets were quantified by measuring at the level of 100-nm down from IHC stereocilia pivot points (schematic in the inset of C) in the same WT, TprnN259/N259, and Tprn−/− IHCs as in B. Each point is one rootlet. The decrease in diameters of rootlets from in rows 1 and 2 of TprnN259/N259 stereocilia was significantly different from WT (P < 0.0001, one-way ANOVA, post hoc Bonferroni test: ***P < 0.001; n.s., not significant). P17 TprnN259/N259 and P32 Tprn−/− IHCs were processed and analyzed in parallel with corresponding WT IHCs (n = 4 for each group). Since no statistically significant differences were found between P17 and P34 for the WT, the data for WT were combined. (D) Drawings illustrate adult WT (top) and adult TPRN-deficient (bottom) stereocilia with magnified views of their rootlets and tapers and localization of stereocilia base proteins discussed in this study. The enlarged view of a WT stereocilia taper region is shown in a circle of top panel. In the bottom panel, an enlarged image shows details of structural disruptions at the taper region in the absence of TPRN.

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