Real-Time Single Cell Analysis of Smac/DIABLO Release during Apoptosis

• Published in: The Journal of cell biology 2003-09, Vol.162 (6), p.1031-1043
• Main Authors: Rehm, Markus, Düßmann, Heiko, Jochen H. M. Prehn
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Bacterial Proteins; bcl-2-Associated X Protein; Carrier Proteins - drug effects; Carrier Proteins - metabolism; Caspase 3; Caspase Inhibitors; Caspases - deficiency; Caspases - genetics; Caspases - metabolism; Cell division; Cell Line; Cell lines; Cells; Cellular biology; Cytochrome c Group - drug effects; Cytochrome c Group - metabolism; Cytochromes; Cytosol; Enzyme Inhibitors - pharmacology; Eukaryotic Cells - cytology; Eukaryotic Cells - drug effects; Eukaryotic Cells - metabolism; Fluorescence; Green Fluorescent Proteins; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Luminescent Proteins; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mitochondria; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondrial DNA; Mitochondrial Proteins - drug effects; Mitochondrial Proteins - metabolism; Peptide Hydrolases - drug effects; Peptide Hydrolases - metabolism; Proto-Oncogene Proteins - deficiency; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins c-bcl-2; Standard deviation; Time Factors
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.200303123

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.