V-ATPase activity is required for biosynthetic delivery of apical membrane proteins. (A) p75-GFP localization in the gut of aa24.2pd1209 mutants at 3–5 dpf. Arrows point to intracellular accumulation. n ≥ 5 larvae per genotype per stage. Scale bars are 10 µm. (B) Experimental strategy for a 1- or 4-h TGN release in the presence of 750 nM bafilomycin or DMSO. (C) p75-GFP surface delivery (arrows) is blocked by bafilomycin, leading to subapical accumulation (arrowheads). n = 6 for bafilomycin and 4 for DMSO. Scale bars are 10 µm. (D) Line profiles of p75-GFP from the cyan arrows in C. Phalloidin marks the BB. Rel., relative. (E) p75-GFP accumulates in apical vacuoles within 4 h of TGN release with bafilomycin. Arrows point to the apical membrane; arrowheads to subapical accumulation; asterisks mark vacuoles. n = 6 for bafilomycin and 3 for controls. Scale bars are 10 µm and 2 µm (white box). (F) Line profiles of p75-GFP from the cyan arrows in E. (G) Experimental design. p75-GFP was allowed to reach steady-state accumulation at the apical membrane by a 24-h TGN release. Then, 750 nM bafilomycin or DMSO was applied for 1 or 2 h, and larvae were processed for imaging. (H) No apical membrane localization (arrows) or subapical accumulation were observed after 1 h of bafilomycin treatment. Only sparse subapical accumulations (arrowheads) were seen after 2 h of bafilomycin treatment. At least five bafilomycin-treated larvae and at least four controls were imaged per time point. Scale bars are 10 µm.
