Article Figures & Data
Figures
- Figure 1.
Septate junction-like features and localization of Caspr at type I hair cell calyceal synapses. a, Thin-section electron microscopy of mouse vestibular epithelia showing regular intercellular gap between the conspicuously electron-dense membranes of the calyx (Cx) and the type I hair cells (HC). b, Higher magnification shows the presence of periodic elements lining up the extracellular surface of the membranes (inset, arrows). Some of these periodic elements seem to be connected to form cross-bridges. c, Freeze-fracture image of the protoplasmic fracture face of a rat vestibular type I hair cell membrane shows parallel linear arrays of intramembrane particles (arrows). d, Confocal immunofluorescence image of type I hair cells of adult rat (red) shows localization of Caspr (arrows, red) at the internal part of the calyx (counterstained with anti-βIII tubulin; green). The first hemiparanode (arrow) shows Caspr immunoreactivity (green). e, Schematic diagram showing in red the regions that are positive for Caspr in the calyceal innervation (green) of the type I hair cell. f, Caspr immunogold-labeled thin section (of adult rat ampula) showing the contact between the Cx and the HC equivalent to the region shown by the rectangle in e. Majority of the gold particles line up with the nerve calyx membrane. Scale bars: a, 500 nm; b, c, 50 nm; d, 5 μm; e, 50 nm.
- Figure 2.
Ablation of Caspr leads to disruption of the calyceal synaptic contact. a, b, Low (a) and high (b) magnification thin-section micrographs showing the irregular and often enlarged gap between the membranes of the calyx (Cx) and the type I hair cell (HC) from a Caspr−/− mouse ampula (asterisk). The contacting membranes also lost their characteristic electron density as seen in Figure 1. c, Box plot of the distance between the calyx and the type I hair cell membranes measured in Caspr+/+ (average, 28 ± 4 nm; n = 279) and Caspr−/− (average, 64 ± 55 nm; n = 436) mice; filled square, mean; middle line of the box, median; top line of the box, 75th percentile; bottom line of the box, 25th percentile; top and bottom whiskers, maximum and minimum, respectively. Scale bars: a, 400 nm; b, 200 nm.
- Figure 3.
Localization of KCNQ4 at the calyceal membrane depends on Caspr. a, Confocal optical section grazing the calyx-type I hair cell (from adult rat ampulla); the contact region shows perfectly matched patterns of distribution of the immunofluorescence for Caspr (red) and KCNQ4 (green), indicating that these two proteins are colocalized. b, c, Caspr immunoreactivity in ampulla of Caspr+/+ and Caspr−/− at P18. b, Caspr and KCNQ4 colocalize at the calyceal synapses in Caspr+/+ mice. c, The labeling for KCNQ4 in the membranes of the Caspr−/− calyx is faint and sparsely distributed. d, e, Immunogold labeling of KCNQ4 confirms its localization at the internal membranes of the nerve calyx in Caspr+/+ and along both internal and external membranes of the calyx in Caspr−/−. f, g, Schematic diagram shows the colocalization of Caspr and KCNQ4 in Caspr+/+ (f) and the redistribution of KCNQ4 in Caspr−/− (g). A yet unidentified binding partner for the Caspr complex on the hair cell membrane is represented in gray (f, g). Scale bars: a, 2 μm; b, c, 10 μm; d, e, 200 nm.
- Figure 4.
Ablation of Caspr leads to decreases in KCNQ4 expression and its redistribution in the calyx membranes. a, Representative image showing the different distribution of fluorescence intensity between inner (arrowheads) and outer (arrows) faces of the calyceal region of two KCNQ4-labeled cells from Caspr+/+ (blue) and Caspr−/− (red). A line crossing hair cells and calyx indicates the sampling plane used in the profile plots shown in b. Maximum values for peak, corresponding to inner (i, i′) and outer (o, o′) faces of the calyx, were used to calculate the ratio of fluorescence distribution at the calyx membranes.
