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.

Lysosomotropic amines can stimulate the function of NPC1 in the clearance of lysosomal contents and late endosome/lysosome hybrid organelle formation. A, cumulative lysosomal-dextran secreted into the cell culture medium after 24 h in both NPC1^+/+ and NPC1^-/- fibroblasts pretreated with indicated molecules. Bars represent average ± S.D. for the percent deviation in dextran amount released relative to untreated NPC1^+/+ fibroblasts (value set to zero). Positive differences in dextran release were observed for the lysosomotropic amines neutral red (70 μm, NR, n = 12), morpholine (10 mm, MOR, n = 8), and imidazole (10 mm, IMDZ, n = 7) in NPC1^+/+ fibroblasts relative to the untreated control (^*, p < 0.001 by unpaired t test). Statistical differences were not observed in NPC1^-/- cells treated with the same compounds. Amine-containing molecules that are known not to accumulate in the lysosome, such as rhodamine-123 (R123, 70 μm, n = 7), had no effect on dextran release. U18666A (10 μm, n = 7) inhibited NPC1 function in dextran secretion in normal cells and had no influence on NPC cells. The formation of sucrosomes with sucrose (0.1% w/v, n = 4) had no statistically significant influence of dextran secretion. B, the degree of late endosome/lysosome hybrid organelle formation in NPC1^+/+ and NPC1^-/- fibroblasts pretreated with indicated molecules. Bars represent average ± S.D. for the percent deviation in FRET signal relative to untreated NPC1^+/+ fibroblasts (value set to zero). Positive deviations in hybrid organelle formation (relative to untreated normal fibroblasts) were observed for MOR (5 mm, n = 3) and IMDZ (10 mm, n = 3) with NPC1^+/+ fibroblasts. Treatments with U18666A (10 μm, n = 3) and nocodazole (50 μm, n = 3) decreased late endosome/lysosome fusion relative to control using NPC1^+/+ fibroblasts (^*, p < 0.01 by unpaired t test). All treatments using NPC1^-/- fibroblasts (n = 3 for each) showed no differences in FRET signal in comparison to untreated NPC1^-/- fibroblasts.

Structures of weakly basic amine-containing compounds shown to either stimulate or inhibit the NPC1-dependent trafficking of lysosomal cargo. All molecules shown are lysosomotropic with respect to their physicochemical properties. U18666A and imipramine are amphiphilic and have been shown to inhibit NPC1 function. NR, MOR, and IMDZ are not considered to be as amphiphilic and were all found to stimulate NPC1 function.

Amine-induced vacuolization requires functional NPC1 and an intact microtubule network. A, NPC1^+/+ and NPC1^-/- fibroblasts incubated with NR and imaged using confocal microscopy. NR causes vacuolization in NPC1^+/+ fibroblasts but not in NPC1^-/- cells. Nocodazole (NOC) pretreatment disrupts the NPC1-dependent vacuolization process. Scale bars represent 5 μm. B, quantitative analysis of average amine-induced vacuole diameters. NR-containing compartments in normal cells are approximate twice the size of NR compartments in NPC1^-/- fibroblasts or from fibroblasts pretreated with NOC (^*, p < 0.01 by unpaired t test). C, combined phase contrast and NPC1 immunofluorescence (green) images of NPC1^+/+ fibroblasts pretreated with NPC1 siRNA and subsequently incubated with 100 μm chloroquine (CQ) to induce vacuolization. Cells that did not contain NPC1 (i.e. efficiently transfected, T) did not form visible vacuoles. Conversely, cells inefficiently transfected (NT) contained NPC1 and were able to form amine-induced vacuoles. Scale bars represent 10 μm. D, the amine-induced vacuolization phenotype is rescued in NPC1^-/- cells by transfection with a functional NPC1-GFP construct. Cells were transfected with a NPC1-GFP plasmid and subsequently exposed to 100 μm CQ to form vacuoles. Scale bars represent 10 μm.

NPC1 is required for the efficient clearance of amines that accumulate in lysosomes. A, Western blot analysis of NPC1 expression in normal fibroblasts before and after transfection with NPC1 siRNA. Actin expression was used as a sample loading control. B, quantitative evaluation of NR release shows NPC1^+/+ fibroblasts (○) clear NR more efficiently than NPC1^-/- fibroblasts (•) or NPC1^+/+ fibroblasts pretreated with NPC1 siRNA (▾). Each time point represents the mean ± S.D. of 3–10 individual measurements. C, fluorescence micrographs of the lysosomal vital stain LTR showing the enhanced clearance in NPC1^+/+ fibroblasts relative to the NPC1^-/- fibroblasts. D, lysosomal clearance of LTR is not impaired in lysosomal storage disorder fibroblasts mucolipidosis type IV (MLIV) and Sandhoff disease. All LTR images are representative of data collected from at least three independent experiments.

NPC1-deficient cells are more susceptible to the toxic effects of the lysosomotropic aldehyde and polyamine metabolite 3-AP. A, NPC1^+/+ fibroblasts exposed to 3-AP were used in comparative cytotoxicity evaluations upon knockdown of NPC1 expression using siRNA. NPC1 depleted cells (•) exposed to 3-AP were found to have an IC₅₀ of 9.8 ± 1.3 μm, whereas cells transfected with a nonspecific scrambled control siRNA (•) had an IC₅₀ value of 27.3 ± 2.7 μm. Each point represents average ± S.D. from three independent experiments. B, ibuprofen (a non-lysosomotropic drug) had no preferential toxicity toward NPC1-deficient fibroblasts relative to normal fibroblasts. Data points represent averages ± S.D. from three independent experiments. C, polyamine oxidase, the enzyme responsible for the metabolism of spermine to spermidine and 3-AP, and spermidine to putrescine and 3-AP, is differentially expressed in fibroblasts with dysfunctional NPC1 as shown by Western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was included for protein loading comparisons. D, β-hexosaminidase activity, a lysosomal enzyme, is greater in isolated cytosol from 3-AP-treated NPC cells (gray bar) relative to that from 3-AP-treated normal fibroblasts (black bar). Cells were treated with 100 μm 3-AP for 12 h prior to analysis. Bars represent average ± S.D. from five independent experiments (^*, p < 0.01 by unpaired t test).