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First published online 28 June 2005
doi: 10.1242/jcs.02447

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The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death

Rosa-Ana González-Polo¹, Patricia Boya^1,*, Anne-Laure Pauleau¹, Abdelali Jalil², Nathanael Larochette¹, Sylvie Souquère³, Eeva-Liisa Eskelinen⁴, Gérard Pierron³, Paul Saftig⁴ and Guido Kroemer^1,

¹ CNRS-UMR8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France
² INSERM U487, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France
³ Institut André Lwoff, INSERM U504, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France
⁴ Institute of Biochemistry, University of Kiel, 24098 Kiel, Germany

View larger version (63K): [in a new window]   Fig. 1. LAMP2 knockdown sensitizes to vacuolization and cell death by nutrient depletion. (A,B) Immunoblot confirmation of the efficacy of LAMP1-(A) or LAMP2-specific (B) small interfering RNA (siRNA). HeLa cells were transfected with the indicated constructs specific for emerin (E), LAMP1 (A,B), and LAMP2 (64, 69), and the expression level of LAMP1 or LAMP2 was determined by immunoblot, after 24 or 48 hours. GAPDH expression was determined as a loading control. (C) Vacuolization induced by LAMP2 depletion. Twenty-four hours after transfection, the cells were cultured for 16 hours in nutrient-free (NF) medium – that is, in the absence of serum and amino acids, and then stained with CMFDA. Cytoplasmic vacuoles are visualized as holes. (D,E) Vesicular LC3 accumulation induced by LAMP2 depletion. Cells were transfected with siRNAs, then 24 hours later with an LC3-GFP fusion construct, and were nutrient deprived for 16 hours. Representative fluorescence microphotographs are shown in D, and the frequency of cells exhibiting the accumulation of LC3-GFP in vacuoles and apoptotic chromatin condensation (as determined by counterstaining with Hoechst 33342) was quantified (X±s.d., n=3) in E. Data in C and D are shown for LAMP1-A and LAMP2-64 siRNAs, and similar results were obtained for the LAMP1-B and LAMP2-69 siRNAs. (F) Electron microscopy of representative cells 6 hours after nutrient and serum depletion after siRNA specific for emerin (right) or siRNA specific for LAMP2 (64)+LAMP1 (A). Bars in C and D, 10 µm.

View larger version (43K): [in a new window]   Fig. 2. Effect of siRNA specific for LAMP1 and/or LAMP2 on cell death induced by serum and amino acid depletion. Cells transfected with the indicated siRNAs (24 hours) were cultured in complete medium (CM) or nutrient-free (NF) medium for 24 hours, followed by staining with propidium iodine (PI) and DiOC6(3) for determination of viability and the mitochondrial transmembrane potential, respectively. Representative FACS pictograms are shown in A. Numbers refer to the percentage of cells in each quadrant. Note that PI+ cells tend to be DiOC6(3)low, meaning that the former can be represented as a subpopulation of the latter. Results are means (±s.d.) of three independent experiments. The percentage of DiOC6(3)low cells and PI+ cells obtained in response to a variety of different siRNAs are shown in B and C, respectively. An electron microscopic picture of a representative cell subjected to LAMP2 siRNA (64) and nutrient depletion (20 hours) is shown in D. N, nucleus; AV, antophagic vacuole; M, mitochondrion.

View larger version (44K): [in a new window]   Fig. 3. Knockout of LAMP1 and LAMP2 sensitizes to nutrient depletion-induced cell death. (A) Cytoplasmic vacuolization induced by nutrient depletion in mouse embryonic fibroblasts (MEF) with a normal genotype (WT) or lacking both LAMP1 and LAMP2 (double knockout, DKO). Cytoplasmic vacuoles are visualized as CMFDA-negative holes. (B, C) LC3 accumulation observable in DKO cells. WT or DKO cells were first transfected with LC3-GFP (24 hours) and then nutrient-depleted for 15 hours (B) or for the indicated period (C), followed by quantitation of the frequency of cells with LC3 accumulation in cytoplasmic vacuoles and that of cells with manifest chromatin condensation, as detectable by counterstaining with Hoechst 33342. (D) WT or DKO cells were cultured in complete medium (CM) or nutrient-free (NF) medium for the indicated time, stained with PI and DiOC6(3) and subjected to cytofluorometric analysis (X±s.d., n=3). (E) Activation of caspase-3 WT and DKO cells, 24 hours after culture in the presence or absence of nutrients. Cells were stained with an antibody recognizing mature, cleaved caspase-3 and the percentage of cells containing active caspase-3 was quantified by immunofluorescence. Bar in A and B, 10 µm.

View larger version (60K): [in a new window]   Fig. 4. Colocalization of the autophagic marker LC3-GFP and LTR on nutrient depletion in controls and LAMP1 or LAMP2 knockdown cells. HeLa cells were transfected with siRNA specific for the indicated gene products (24 hours), transfected with LC3-GFP (24 hours), and cultured in complete medium (CM) of nutrient-free medium (NF) for 4 hours, followed by staining with lysotracker (LTR, 30 minutes) red and confocal scanning fluorescence microscopy. Representative cells are shown in A. Bar, 10 µm. The graphs (right panels) represent the fluorescence distribution determined for section of the cells, as indicated by the orientation of the arrow. The degree of colocalization (X±s.d.) was determined by image analysis for 50 cells each, as indicated in Materials and Methods.

View larger version (60K): [in a new window]   Fig. 5. Colocalization of mitochondrial and lyosomal markers on nutrient depletion in controls and LAMP1 (LA1) or LAMP2 (LA2) knockdown cells. HeLa cells were treated with siRNA specific for emerin, LAMP1 and/or LAMP2 (24 hours), then transfected with mt-DsRed (which localizes to mitochondria) and SytVII-GFP (which localizes to lysosomes), cultured for 24 hours and then incubated in CM or NF medium for 4 hours, followed by confocal microscopy as in Fig. 3. Representative cells are shown in A and the degree of colocalization (X±s.d., n=50) between mt-DsRed and SytVII-GFP is shown in B. The graphs (right panels) represent the fluorescence distribution determined for section of the cells, as indicated by the orientation of the arrow. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 6. Colocalization of mt-DsRed and SytVII-GFP in wild-type (WT) and LAMP1–/– LAMP2–/– (DKO) MEF. The degree of colocalization was measured similarly as in Fig. 4, before and after overnight starvation of amino acids and serum. Examples of the normal separation of mitochondria and lysosomes are shown in the upper part of A, whereas examples of how these organelles colocalize on starvation in WT but not DKO cells are shown in the middle and lower parts of A, respectively. Bar, 10 µm. The graphs (right panels) represent the fluorescence distribution determined for section of the cells, as indicated by the orientation of the arrow. Quantitative data are shown in B.

View larger version (48K): [in a new window]   Fig. 7. Contribution of caspases to vacuolization-associated death. (A,B) Activation of caspase-3 induced by a combination of LAMP-specific siRNA and starvation. Cells were treated as indicated and stained with an antibody recognizing activated caspase-3. Representative fluorescence microphotographs are shown in A and quantitative data (X±s.d., n=3) are shown in B. Bar, 10 µm. (C,D) Effect of the pan-caspase inhibitor Z-VAD-fmk. Cells were subjected to the indicated LAMP1 and/or LAMP2 knockdown and then cultured in the absence or presence of nutrients and/or Z-VAD-fmk for 24 hours. Then, the loss of the m (C) and viability (D) was measured with the indicated dyes, as in Fig. 2.

View larger version (48K): [in a new window]   Fig. 8. Effect of mitochondrial apoptosis inhibitors on vacuolization-associated death. (A,B) Effect of Bcl-2 and vMIA on cell death. HeLa cells stably transfected with Bcl-2 or vMIA were subjected to the indicated LAMP-specific siRNAs, and the frequence of mlow (A) or dead (B) cells was measured by staining with DiOC6(3) or PI, respectively. (C) Accumulation of the autophagic vacuole marker LC3-GFP in vesicles from Neo controls, Bcl-2-transfected and vMIA-transfected HeLa cells or Neo control vector-only transfected cells treated with Z-VAD-fmk. Note that none of the apoptosis inhibitors influenced the vacuolar accumulation of LC3-GFP. Bar, 10 µm.

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