Figure 3. Normal expression and subcellular distribution of otoferlin in Otof ^C2C/C2C IHCs.

(A) Confocal microscopy images of IHCs from whole-mount preparations of the organ of Corti from P15 Otof ^C2C/+ and Otof ^C2C/C2C mice triple-labeled for otoferlin (blue), ribeye (red), and postsynaptic GluA2 receptors (green). Insets: detailed views of the boxed synaptic areas. n: cell nucleus. Scale bar: 5 µm. Inset scale bar: 1 µm. (B) Confocal microscopy image of IHCs from a whole-mount preparation of an organ of Corti from a P15 Otof ^-/- mouse triple-labeled for otoferlin (green), the hair cell marker myosin VI (red), and the cell nucleus marker DAPI (blue). Note that the luminosity of the green channel (otoferlin) has been enhanced to show the absence of otoferlin expression in Otof ^-/- mice. Scale bar: 5 µm. (C) Top: Summed projected z-stack confocal microscopy images of IHCs from whole-mount preparations of organs of Corti from P15 Otof ^C2C/+ and Otof ^C2C/C2C mice labeled for otoferlin (green). Scale bar: 5 µm. Bottom: Quantification of otoferlin fluorescence in Otof ^C2C/+ (n = 51 cells in 7 mice) and Otof ^C2C/C2C IHCs (n = 51 cells from 7 mice) at the apex, middle, and the base of IHCs. Data information: in (C), data are presented as the mean ± SEM. ns, not significant (Student's t-test with Welch correction).

Figure 3—figure supplement 1. Normal number of ribbon synapses in Otof ^C2C/C2C IHCs.

Confocal microscopy images of IHCs from whole-mount preparations of the P15 organ of Corti, triple-labeled for the synaptic ribbon protein ribeye (red), presynaptic Ca_v1.3 L-type Ca²⁺ channels (blue), and postsynaptic GluA2 glutamate receptors (green), in Otof ^C2C/+ and Otof ^C2C/C2C mice. The Ca_v1.3 labeling overlaps with the ribeye labeling. The cell contours are indicated by dotted lines. n: cell nucleus. Scale bars: 5 µm. Insets: detailed views of the circled synaptic areas. Inset scale bars: 1 µm.

Figure 3—figure supplement 2. Normal colocalization of otoferlin and Vglut3 in Otof ^C2C/C2C IHCs.

Analysis of two representative STED images of individual IHCs from whole-mount preparations of organs of Corti from a P15 Otof ^C2C/+ mouse (left panels) and a P15 Otof ^C2C/C2C mouse (right panels) double-labeled for otoferlin (green) and Vglut3 (red). For each example, the STED image (top left), the STED image superimposed on the resolution-limited spots representing structures stained for otoferlin and Vglut3 (top right), and the corresponding distributions of nearest neighbor distances from otoferlin spots to the closest Vglut3 spots and from Vglut3 spots to the closest Vglut3 spots (bottom) are shown. Data information: Images are shown after demixing of the Atto565- and the DY485-channel images. The numbers of Vglut3 and otoferlin spots were similar in each example IHC, ***p<0.001 (Kolmogorov-Smirnov test).