Figure 3.

Premature autolysosomal fusion resulting from Atp6v0ca deficiency, as well as Spns1 deficiency. (A) Appearance of EGFP-Lc3B- and mCherry-Lamp1-positive autolysosomal fusion with Atp6v0ca deficiency, as well as Spns1 deficiency, was detectable. Embryos of EGFP-Lc3B- and mCherry-Lamp1-double transgenic spns1-mutant (Tg[CMV:EGFP-Lc3B;mCherry-Lamp1];spns1^hi891/hi891) fish and EGFP-Lc3B- and mCherry-Lamp1-double transgenic atp6v0ca-mutant (Tg[CMV:EGFP-lc3b;eef1a1l1:mCherry-lamp1];atp6v0ca^{hi1207/hi1207}) fish were examined to confirm autophagosome-lysosome fusion at 76 hpf. Scale bar: 10 µm. Quantification of the EGF (green), mCherry (red) and merged (yellow) fluorescence-positive particle numbers is shown in the right graph (n = 6); the number (n) of animals is for each genotype and phenotype. Error bars represent the mean ± SD, *P < 0.005, **P < 0.001. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. Western blot analysis using anti-Lc3A/B antibody shows endogenous Lc3A/B protein levels, which can confirm an increase of the total amount of Lc3A/B in wt, atp6v0ca^{hi1207/hi1207}- and spns1^hi891/hi891-mutant fish. Increased Lc3A/B-II conversion or accumulation was detected slightly in atp6v0ca^{hi1207/hi1207} and more significantly in spns1 mutants. (B) Most of the EGFP-Lc3B-positive autolysosomes were not LysoTracker Red-positive compartments. LysoTracker Red DND-99 staining of EGFP-Lc3B-transgenic spns1-mutant (Tg[CMV:EGFP-lc3b];spns1^hi891/hi891) or atp6v0ca-mutant (Tg[CMV:EGFP-lc3b];atp6v0ca^{hi1207/hi1207}) embryos was performed at 76 hpf. Scale bar: 10 µm. Quantification of the EGFP (green) and LysoTracker (red) fluorescence-positive particle numbers is shown in the right graph (the number of animals for each genotype and phenotype = 6). Error bars represent the mean ± SD, **P < 0.001, ***P < 0.0005; ns, not significant. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals.