Real-time single cell analysis of Smac/DIABLO release during apoptosis.
We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)-deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk-sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.
Rehm
Markus
M
Interdisciplinary Center for Clinical Research, University Münster Clinics, Münster, Germany.
Düssmann
Heiko
H
Prehn
Jochen H M
JH
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000187:drug effects / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D013997:Time Factors; D051028:bcl-2-Associated X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D001426:Bacterial Proteins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C108711:DEVDase