Figure 3

Figure 3 Lysosomal acidification is impaired in vma21 mutants.

Representative images of (A) wild-type (WT)/heterozygous sibling control and (B) vma21^{Δ14ins21/Δ14ins21} zebrafish larvae at 5 days post fertilization (dpf) stained with LysoTracker Red, a marker of lysosomal acidification. Scale bars: 45 mm. (C) Bar graph showing quantification of LysoTracker Red staining. Each dot represents the average of gray values (technical replicate) and three biological replicates were performed. Data are shown as mean ± SEM. Significance at *P < 0.05 (unpaired two-tailed t test). (D) Z-stack and (E) single Z plane confocal micrographs of whole-mount zebrafish embryos at 5 dpf labeled with Lamp1 (green), Dystrophin (magenta), and DAPI (blue). Lamp1 (left panels in (D, E)) shows a punctate staining pattern and localizes in the myofibers (yellow arrowheads) and at the myotendinous junctions (MTJs) (yellow arrows). Lamp1 puncta are less bright in vma21 mutants compared to WT. Scale bars: 20 mm (D) and 10 mm (E). (F) Single Z plane confocal micrographs of myofibers isolated from zebrafish embryos at 5 dpf and labeled with Lamp1 (green), Dystrophin (magenta), and DAPI (blue). Lamp1 puncta (left panels) are present throughout the myofibers, at the MTJs (yellow arrows), and in increased numbers around the nuclei (white arrowheads). Lamp1 puncta are less bright in vma21 mutant myofibers compared to WT myofibers. Scale bar: 10 mm. (G) Quantification of Lamp1 fluorescence intensity at the MTJs shows significantly reduced levels of Lamp1 in vma21 mutants. n = 16 WT and n = 8 vma21 mutants. Data represented as mean ± SEM. Significance at ***P < 0.001 (unpaired two-tailed t test). (H) Quantification of Lamp1 fluorescence intensity in the entire myofiber shows significantly reduced levels of Lamp1 in vma21 mutants. n = 11 WT and n = 15 vma21 mutants. Data represented as mean ± SEM. Significance at **P < 0.01 (unpaired two-tailed t test). Source data are available online for this figure.