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. 2008 Sep 5;283(36):24584–24593. doi: 10.1074/jbc.M803715200

FIGURE 2.

FIGURE 2.

Amine-induced vacuoles are created through a heterotypic fusion of lysosomes with late endosomes. A, immunofluorescence images of NPC1+/+ cells showing the localization of NPC1 (without amine treatment). NPC1 (green) does not significantly colocalize with the late endosome-specific mannose 6-phosphate receptor (MPR, red) and does colocalize with the lysosome-associated membrane protein-1 (LAMP-1, red). B, immunofluorescence and phase contrast images of NPC1+/+ cells incubated with NR to induce vacuolization. NPC1, LAMP-1, and MPR are all localized to the membrane of the newly formed vacuoles. C, fluorescence-labeled dextran macromolecules specifically colocalize with amine-induced vacuoles in normal fibroblasts following a pulse-chase protocol (see “Experimental Procedures”). D, Western blot analysis of organelle-specific proteins contained in the purification of terminal endocytic compartments (see “Experimental Procedures”). Aliquots of the post nuclear supernatant (PNS), the material flowing through the magnetic column (flow through, F.T.), and the fraction retained on the column (retained) were all analyzed for the Golgi-specific protein Golgin-84, the lysosome-specific protein LAMP-1, and the early endosome antigen 1 (EEA1). E, Western blot analysis of MPR in purified terminal endocytic compartments before and after amine (NR) treatments. NPC1+/+ fibroblasts become enriched in MPR after amine treatment, which does not occur with NPC1-/- fibroblasts. F, fluorescence images of NPC1+/+ fibroblasts transfected with NPC1-GFP (green) and with the late endosome-specific Rab9-YFP (red). An increase NPC1/Rab9 colocalization is observed following treatment with the amine CQ. G, vacuolization of lysosomes with sucrose occurs regardless of NPC1 functional status and does not involve fusion with late endosomes. Phase contrast images show that the addition of 0.1% sucrose induced vacuolization (sucrosomes formation) in both NPC1+/+ and NPC1-/- fibroblasts. Immunofluorescence analysis of LAMP-1 (green) and MPR (red) reveals that they do not colocalize with or without sucrose. Scale bars represent 10 μm.