Loss of CAVIN4 in mouse skeletal muscle causes ultrastructural defects and abnormal distribution of CAV3. (A) Confocal images of CAVIN4 (with Phalloidin-Alexa488 counterstain, inset) and CAV3 in WT and Cavin4^−/− 3-d-old mouse skeletal hindlimb muscle. Inverted images show the magnified area highlighted by an asterisk. Arrowhead highlights internal CAV3 labeling. Scale bars: 20 µm. (B) Ratio of sarcolemmal to internal CAV3 fluorescence intensity in WT and Cavin4^−/− skeletal hindlimb muscle (n = 2 each of WT and Cavin4^−/− samples, two-tailed t test). Quantitation was performed in coded (blinded) samples; colored circles represent measurements from individual images. *, P ≤ 0.05. (C–F) EM of hindlimb skeletal muscle from 3-d-old WT and Cavin4^−/− pups. In WT muscle (C and D), sparse vesicular elements were visible between the myofibers, generally as vesicular or short tubular structures. Cavin4^−/− muscle (E and F) exhibited numerous long tubular elements between the myofibers, often forming complex stacked arrays (E) not seen in WT muscle. Tubular structures highlighted in green in D and F. Scale bars: 1 µm. (G) Membrane/cytosolic volume (%) in WT and Cavin4^−/− muscle (n = 2 each of WT and Cavin4^−/− samples, two-tailed t test). Colored circles represent separate images. ****, P ≤ 0.0001. Error bars represent mean ± SD.