Two phases of signalling between mitochondria during apoptosis leading to early depolarisation and delayed cytochrome c release.
We investigated the mode of signalling between mitochondria during apoptosis by monitoring the behaviour of non-irradiated mitochondria following microscopic photosensitisation of half the mitochondria in single human osteosarcoma cells loaded with CMXRos. Following partial irradiation of cells, non-irradiated mitochondria underwent a rapid depolarisation (within 10 minutes). The depolarisation was not inhibited by the caspase inhibitor zVAD-fmk but was suppressed by the intracellular Ca(2+) chelator BAPTA and overexpression of Bcl-2. Significantly, such depolarisation occurred even after prior conversion of extended filamentous mitochondria into individual punctate structures, indicating that lumenal continuity is not required for communication between the irradiated and non-irradiated mitochondria. Partial irradiation of cells expressing cytochrome c-GFP revealed cytochrome c-GFP release from non-irradiated mitochondria at a delayed but unpredictable time interval (between 30 minutes and more than 2.5 hours) following irradiation, which was unaffected by zVAD-fmk. Once activated, cytochrome c-GFP release occurred within a 10 minute period. Immunocytochemistry failed to reveal the recruitment of Bax to non-irradiated mitochondria, which suggests that Bax does not mediate the release of cytochrome c from mitochondria. We conclude that signals (mediated by Ca(2+)) emanating from irradiated mitochondria are processed by their non-irradiated counterparts and comprise two temporally distinct phases, both independent of caspase-mediated amplification, which generate an initial rapid depolarisation and subsequent delayed release of cytochrome c.
Lum
May-Ghee
MG
Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
Nagley
Phillip
P
D000111:Acetylcysteine / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D020013:Calcium Signaling; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D016572:Cyclosporine; D045304:Cytochromes c; D020169:Caspases; D000111:Acetylcysteine