ACF7 distribution in mouse vestibular and cochlear hair cells. A–D, F–M, Images acquired using confocal microscopy. A–D, F, G, Adult mouse vestibular hair cells labeled with ACF7 antibody (green) and phalloidin (red). A, ACF7 is localized toward the base of the CP (bracket). B, ACF7 encircles the CP and forms a ring within the fonticulus. C, ACF7 coats the lumen of the fonticulus (arrowhead). Bracket denotes CP, above which are stereocilia with normal morphology and a few bent stereocilia (arrows). D, Cell colabeled with tubulin antibody (blue). E, Fluorescence intensity profile from hair cell in D, using the yellow-line region of interest (ROI), demonstrates that ACF7-associated signal (green) rests between phalloidin-associated (red) and tubulin-associated (blue) signals. Intensity scale is linear, but the units are arbitrary. X-axis represents the length of the ROI, from top (0 μm) to bottom (4 μm). F, In 3D reconstruction, yellow indicates volume of red and green overlap. Bracket indicates position of CP. G, The volume of overlap from F with non-overlapping signals removed. No significant differences in ACF7 localization were noted between type I and type II hair cells. H–M, 3D reconstructions of P2 mouse cochlear hair cells labeled with ACF7 antibody (green) and phalloidin (red). H, I, Images of a basal turn of a cochlea demonstrate that ACF7 forms a ring around each fonticulus and labeling is appreciable in and around each CP. Each arrow points out a typical fonticulus. Moreover, increased ACF7 levels are observed closer to the fonticulus. Labeling of cells in the bottom row is out of the plane of focus. J–M, Two inner hair cells. L, Image of two hair cells, from a lateral perspective, demonstrates that ACF7 resides basal to, and within, the CP. Yellow indicates overlapping signals. M, Yellow signal from L demonstrates that ACF7 overlaps with actin at the CP and at a restricted region near the cortical surface of the hair cell (arrowhead). Scale bar, 1 μm.