Figure 7.

Lysosomes and lysosomal tubules show V-ATPase subdomains. (A) RAW264.7 cells transfected with ORP1L-GFP were subjected to lysosome enlargement by overnight treatment with 30 mM sucrose. Cells were stained with SidK-AL568 (magenta) and the distribution of V-ATPase in sucrosomes labeled with ORP1L (cyan) visualized. Side panels show individual SidK-AL568, ORP1L, and colocalization channels (left to right). Colocalization and Mander’s coefficient between ORP1L and SidK-AL568 (M) shown in orange. (B) RAW264.7 cells transfected with Arl8b-GFP were subjected to lysosome enlargement by treatment with 30 mM sucrose. Cells were stained with SidK-AL568 (magenta) and the distribution of V-ATPase in sucrosomes labeled with Arl8b (cyan) visualized. Side panels: Individual SidK-AL568, Arl8b, and colocalization channels (left to right). Colocalization and Mander’s coefficients between Arl8b and SidK-AL568 (M) shown in orange. (C–E) Localization of V-ATPases in tubular lysosomal structures in RAW264.7 cells (C), human macrophages (D), and HeLa cells (E). Cells were stained using SidK-AL568 (magenta) and immunostained for endogenous LAMP1 (cyan). Solid white arrowheads mark lysosomal regions that are SidK⁺ LAMP1^–; open arrowheads mark regions that are SidK^– LAMP1⁺. Side panels show individual SidK-AL568, LAMP1, and colocalization channels (top to bottom). Colocalization and Mander’s coefficients between LAMP1 and SidK-AL568 (M) shown in orange. (A–E) xy optical slices representative of ≥30 fields from three or more experiments of each type. Scale bars: 5 µm.