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First published online 19 April 2005
doi: 10.1242/jcs.02324

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The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies

¹ Department of Cellular and Physiological Sciences, University of British Columbia, 2177 Wesbrook Mall, Vancouver V6T 1Z3, British Columbia, Canada
² Département de Pathologie et Biologie Cellulaire, Université de Montréal, CP6128 Succursale Centre-Ville, Montréal H3C 3J7, Québec, Canada
³ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto M5G 1X5, Ontario, Canada

View larger version (124K): [in a new window]   Fig. 1. U18666A treatment induces the accumulation of swollen cholesterol-rich, LAMP-2-positive vacuoles. Untransfected Mv1Lu cells (A-D,I-L) and Mgat5-transfected M9 clones (E-H,M-T) were cultured in regular medium for 6 days (A-H,Q-T) and selected cultures incubated with U18666A for the final 24 hours (I-P). The cells were then fixed and triple labelled with anti-LAMP-2 antibody (A,E,I,M,Q), Nile Red (B,F,J,N,R) and filipin (C,G,K,O,S). Merged images (D,H,L,P,T) show LAMP-2 in green, Nile Red in red, and filipin in blue. In untreated M9 cells, filipin labelling is concentrated at the limiting membrane of the vacuoles (Q-T). In the presence of U18666A, the LAMP-2/Nile Red-positive vacuoles (arrowheads) accumulate cholesterol and are all positive for filipin after 24 hours. Bar, 5 µm.

View larger version (216K): [in a new window]   Fig. 2. U18666A induces MLB expression. Mv1Lu (A,C) and Mgat5-transfected M9 (B,D) cells were incubated with U18666A for 24 hours (C,D) and processed for electron microscopy. Cytoplasmic MLBs are present in M9 cells as well as in both untransfected Mv1Lu and M9 clones following treatment with U18666A. Bar, 2.5 µm.

View larger version (223K): [in a new window]   Fig. 3. U18666A induces MLB expression despite leupeptin treatment. Mv1Lu (A,C) and Mgat5-transfected M9 (B,D) cells were incubated with leupeptin (2 µg/ml) for 96 hours and U18666A was added to selected cultures (C,D) for the final 24 hours before processing for electron microscopy. In the absence of U18666A, leupeptin induces the accumulation of dense autophagic vacuoles however U18666A still induces MLB production in leupeptin-treated cells. Bar, 2.5 µm.

View larger version (154K): [in a new window]   Fig. 4. U18666A treatment induces accumulation of swollen LAMP-2-positive vacuoles in the presence of the autophagy inhibitor 3-MA. Untransfected Mv1Lu (A-D,I-L) and Mgat5-transfected M9 (E-H,M-P) cells were grown in medium supplemented with 10 mM 3-MA for 3 days and selected cultures were incubated with U18666A for the final 24 hours (I-P). The cells were fixed and then triple labelled with anti-LAMP-2 (A,E,I,M), Nile Red (B,F,J,N) and filipin (C,G,K,O). Merged images (D,H,L,P) show LAMP-2 in green, Nile Red in red and filipin in blue. Large phospholipid-rich, LAMP-2-positive vacuoles are not observed following 3-MA treatment but U18666A is still able to induce the formation of swollen, filipin-positive lysosomal vacuoles. Bar, 5 µm.

View larger version (146K): [in a new window]   Fig. 5. Swollen vacuoles induced by U18666A in the presence of 3-MA lack concentric lamella. Untransfected Mv1Lu (A-C) and Mgat5-transfected M9 (D-F) cells were incubated with 10 mM 3-MA for 3 days and selected cultures treated with U18666A for 24 hours (B,C,E,F) prior to processing for electron microscopy. 3-MA treatment results in the disappearance of MLBs in M9 cells and expression of membrane-bound, non-lamellar inclusion bodies that lack concentric lamella. Bar, 0.5 µm (A,C,E,F); 2 µm (B,D).

View larger version (61K): [in a new window]   Fig. 6. Treatment with 3-MA reduces MDC labelling of swollen, lysosomal vacuoles. M9 cells were grown in regular medium (A,C) or in medium supplemented with 10 mM 3-MA for 3 days (B,D) and selected cultures were incubated with U18666A for the final 24 hours (C,D). Cells were incubated with MDC and Nile Red for 10 minutes and images of MDC labelling of unfixed cells are shown. (E) Mean MDC labelling intensity of lysosomal vacuoles positive for Nile Red was quantified in untreated M9 cells as well as in U18666A-treated M9 cells in the presence or absence of 3-MA (mean±s.d. of three independent experiments). MLBs present in untreated M9 cells and cells treated with U18666A are strongly labelled for MDC, however MDC labelling of swollen, lysosomal vacuoles formed in the presence of 3-MA treatment is significantly reduced. *P<0.005 when compared to intensity in control M9 cells. Bar, 10 µm.

View larger version (64K): [in a new window]   Fig. 7. Serum starvation stimulates of cholesterol-rich MLBs. (A) Untransfected Mv1Lu cells were grown for 6 days and U18666A was added for the final 24 hours. Selected cultures were then washed and incubated for 6 hours in serum-containing (B) or serum-free (C) media and labelled with filipin. The expression of filipin-labelled MLBs was quantified from both untransfected Mv1Lu (D) and Mgat5-transfected M9 cells (E) incubated in serum-containing (empty bars) or serum-free (filled bars) media for 0.5, 1, 3 and 6 hours labelled with filipin and the nuclear dye Sytox Green. The average area of filipin-labelled vacuoles and the total area covered by these vacuoles per cell were quantified (mean±s.e.m. of three independent experiments). *P<0.005, **P<0.01, ***P<0.05 compared to areas in control cells at the same time point. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 8. 3-MA treatment does not affect accessibility of LAMP-2-positive vacuoles to fluid-phase endocytosis. M9 cells either untreated (Ctl, A-F), treated with U18666A for 24 hours (U24; G-L) or grown for 3 days in the presence of 10 mM 3-MA with U18666A added for the final 24 hours (U24 + 3-MA; M-R) were incubated for 30 minutes (A-C,G-I,M-O) or 4 hours (D-F,J-L,P-R) in the presence of 5 mg/ml FITC-dextran prior to labelling for LAMP-2. LAMP-2 labelling (A,D,G,J,M,P) is shown in red and FITC-dextran labelling (B,E,H,K,N,Q) in green and merged images are presented in C,F,I,L,O,R. The percentage of LAMP-2/Nile Red-positive swollen vacuoles containing FITC-dextran at 0.5, 1, 2 and 4 hours of FITC-dextran endocytosis was quantified from Mv1Lu and M9 cells treated or not with 3-MA for 3 days and/or U18666A for the final 24 hours, as indicated (S). For all treatment conditions, FITC-dextran does not accumulate in swollen LAMP-2-positive vacuoles at 30 minutes and the majority are accessible after only 4 hours of endocytosis even in the presence of 3-MA. Bar, 5 µm.

View larger version (51K): [in a new window]   Fig. 9. 3-MA treatment does not affect fluid-phase endocytosis to lysosomes. M9 cells were grown in the presence of 3-MA for 3 days and then incubated with FITC-dextran for 30 minutes (A-C) or 4 hours (D-F) prior to labelling for LAMP-2 (A,D). FITC-dextran labelling (B,E) is shown in green and LAMP-2 in red in the merged images (C,F). FITC-dextran does not accumulate in lysosomal structures at 30 minutes but reaches them after 4 hours of endocytosis. Bar, 5 µm.

View larger version (59K): [in a new window]   Fig. 10. Non-lamellar, cholesterol-rich LAMP-2-positive vacuoles are acidic, lysosomal organelles. M9 cells were grown in regular medium (A-C,G-I) or in media supplemented with 10 mM 3-MA for 3 days (D-F,J-L) and selected cultures were incubated with U18666A for the final 24 hours (G-L). Viable cells were labelled with Lysotracker Red (A,D,G,J) and after fixation, with filipin (B,E,H,K). Merged images (C,F,I,L) show Lysotracker Red in red and filipin in blue. The large cholesterol-rich swollen lysosomal vacuoles induced by U18666A are still acidic in the presence of 3-MA. Bar, 5 µm.

View larger version (28K): [in a new window]   Fig. 11. An autophagic contribution is required for the formation of phospholipid and cholesterol-rich MLBs. (A) Extensive interaction occurs between the endocytic (black) and autophagic (red) pathways. Maturation of autolysosomes into MLBs requires lysosomal degradation and is inhibited by leupeptin. U18666A-mediated lysosomal cholesterol accumulation (+U) induces the formation of cholesterol-rich MLBs from autolysosomes or pre-existing phospholipid-rich MLBs independently of lysosomal degradation (blue). (B) Upon inhibition of autophagy with 3-MA, the formation of phospholipid-rich MLBs is inhibited and U18666A-mediated lysosomal cholesterol accumulation results in the formation, not of MLBs, but of non-lamellar lysosomal vacuoles.