Relative timing of redistribution of cytochrome c and Smac/DIABLO from mitochondria during apoptosis assessed by double immunocytochemistry on mammalian cells.
Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (< or = 15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.
Lim
Maria L R
ML
Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Victoria, Australia.
Chen
Baohong
B
Beart
Philip M
PM
Nagley
Phillip
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C412403:DIABLO protein, human; C499605:Diablo protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D045304:Cytochromes c; D020169:Caspases; D019311:Staurosporine