Fig. 2.

V-ATPase activity controls lysosomal degradation of endocytic cargoes. (A-B′) Dextran uptake in live mosaic Vha68-2^R6 Drosophila eye discs. Discs were counterstained to detect the intracellular domain of Notch (NICD) to assess colocalization (B′). Mutant tissue is outlined. Compared with WT tissue, mutant tissue internalizes less Dextran after 10 minutes (A), whereas after 40 minutes the amount of Dextran internalized is similar (B). Note that some of the internalized Dextran at 40 minutes colocalizes with the accumulated Notch (B′). (C,C′) Notch internalization assay in live imaginal discs. In WT tissue (C), surface-bound anti-Notch extracellular domain (NECD) is internalized into endosomes and degraded after 60 minutes. In Vha68-2^R6 tissue (C′), anti-NECD is internalized but is not degraded, accumulating intracellularly (actin staining is used to demarcate cell boundaries). (D) Vha68-2^R6 mosaic eye imaginal disc stained to detect ubiquitin. No ubiquitin accumulation is observed in the mutant tissue. (E-F′) Vha68-2^R6 mosaic eye imaginal discs stained with anti-NICD and anti-Avl (E) or anti-Hrs (F); separate channels are shown in E′,E′,F′,F′. No colocalization of Notch is observed with either endosomal marker. (G-G′) Vha68-2^R6 mosaic eye imaginal discs expressing GFP-Lamp-1 and stained to detect NICD (G); separate channels are shown in G′,G′. Partial colocalization in the mutant tissue is observed. Scale bars: 10 μm.