Fig. 6

TMEM63A distribution and function in stretch-activated Ca2+ signaling in OLs (A and B) Maximum intensity-projected confocal z stack (A) and single-plane STED (B) image of an OL in T63a-eYFP/+ Cx immunostained for CNP (magenta) and eYFP (green). Arrowheads: TMEM63A puncta in the soma and processes. Higher magnification of boxed area in (B) shows TMEM63A puncta on the OL somatic membrane. Note intracellular pool of TMEM63A. (C) Electron micrographs of optic nerves from T63a-eYFP/+ mice (top row). Arrowheads show anti-GFP (TMEM63A, 10 nm gold particles; red) and anti-carbonic anhydrase II (CAII, 15 nm gold particles; cyan) labeling. Primary antibody (anti-GFP, bottom left) and no primary antibody (bottom right) control sections from T63a+/+ mice. (D and E) Time-series projection of jGCaMP8s signals in cultured T63a+/+ (D) and T63a−/− (E) OLs at baseline and after mechanical stimulation. (F and G) Frequency of Ca2+ events in each CaM (F) and average amplitude (Z score) of Ca2+ (G) at baseline. (H–K) Intensity vs. time traces for soma and five CaMs (H and J) and duration and intensity (Z scores) of Ca2+ transients in all CaMs (I and K) in T63a+/+ (H and I) and T63a−/− (J and K) mice. Stim, time of stimulation. Note, no response to Stim in T63a−/−, but baseline Ca2+ transients remain intact. (L) Proportion of T63a+/+ vs. T63a−/− responder and non-responder OLs. (M and N) Normalized maximum Ca2+ amplitude at baseline (Ctrl) and after stimulation (Stim) in T63a+/+ (M) and T63a−/− (N) OLs. Green: responders, red: non-responders, and gray: response in Ca2+-free extracellular recording solution. (O and P) Frequency of Ca2+ events in each CaM before (Ctrl) or after bath application of TPC2-A1-N (TPC, 10 μM, O) and activation ratio (ratio between event frequencies before and after application (P). Data represent mean ± SEM. ns, non-significant, p > 0.05, ∗p < 0.05. See also Videos S2 and S3 and Table S3 (sample sizes and statistical tests).