The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death.
Autophagic cell death is morphologically characterized by an accumulation of autophagic vacuoles. Here, we show that inactivation of LAMP2 by RNA interference or by homologous recombination leads to autophagic vacuolization in nutrient-depleted cells. Cells that lack LAMP2 expression showed an enhanced accumulation of vacuoles carrying the marker LC3, yet a decreased colocalization of LC3 and lysosomes, suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. While a fraction of mitochondria from starved LAMP2-expressing cells colocalized with lysosomal markers, within autophagolysosomes, no such colocalization was found on removal of LAMP2 from the experimental system. Of note, LAMP1 depletion had no such effects and did not aggravate the phenotype induced by LAMP2-specific small interfering RNA. Serum and amino acid-starved LAMP2-negative cells exhibited an accumulation of autophagic vacuoles and then succumbed to cell death with hallmarks of apoptosis such as loss of the mitochondrial transmembrane potential, caspase activation and chromatin condensation. While caspase inhibition retarded cell death, it had no protective effect on mitochondria. Stabilization of mitochondria by overexpression of Bcl-2 or the mitochondrion-targeted cytomegalovirus protein vMIA, however, blocked all signs of apoptosis. Neither caspase inhibition nor mitochondrial stabilization antagonized autophagic vacuolization in LAMP2-deficient cells. Altogether, these data indicate that accumulation of autophagic vacuoles can precede apoptotic cell death. These findings argue against the clear-cut distinction between type 1 (apoptotic) and type 2 (autophagic) cell death.
González-Polo
Rosa-Ana
RA
CNRS-UMR8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.
Boya
Patricia
P
Pauleau
Anne-Laure
AL
Jalil
Abdelali
A
Larochette
Nathanael
N
Souquère
Sylvie
S
Eskelinen
Eeva-Liisa
EL
Pierron
Gérard
G
Saftig
Paul
P
Kroemer
Guido
G
D017209:Apoptosis / Q000502:physiology*; D001343:Autophagy / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000096:biosynthesis / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron; D008859:Microscopy, Polarization; D034622:RNA Interference; D014162:Transfection; D014617:Vacuoles / Q000378:metabolism / Q000502:physiology*; D052118:Lysosomal-Associated Membrane Protein 1
D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C497811:LAMP2 protein, human