Published 15 September 2003. doi:10.1083/jcb.200303123
© The Rockefeller University Press,
0021-9525/2003/9/1031 $5.00
The Journal of Cell Biology, Volume 162, Number 6, 1031-1043
Real-time single cell analysis of Smac/DIABLO release during apoptosis
Markus Rehm1,2,
Heiko Düßmann1 and
Jochen H.M. Prehn1,2
1 Interdisciplinary Center for Clinical Research, Research Group "Apoptosis and Cell Death", University Münster Clinics, D-48149 Münster, Germany
2 Center for Neurology and Neurosurgery, Experimental Neurosurgery, Johann Wolfgang Goethe-University Clinics, D-60590 Frankfurt, Germany
Address correspondence to Jochen H.M. Prehn, Experimental Neurosurgery Center for Biological Chemistry (ZBC), HS 25 B, 4. OG, Johann Wolfgang Goethe-University Clinics, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Tel.: 49-69-6301-6930. Fax: 49-69-6301-5575. email: prehn{at}rcsi.ie
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.
Key Words: mitochondria; cytochrome c; caspases; confocal microscopy; fluorescence resonance energy transfer
Abbreviations used in this paper: Casp-3, caspase-3; CHX, cycloheximide; cyt-c, cytochrome c; 
M, inner mitochondrial membrane potential; Eto, etoposide; FRET, fluorescence resonance energy transfer; IAP, inhibitor of apoptosis protein; STS, staurosporine; TMRM, tetramethyl rhodamine methyl ester; z-VAD-fmk, z-Val-Ala-Asp(O-methyl)-fluoromethylketone.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
-
Huber, H. J., Rehm, M., Plchut, M., Dussmann, H., Prehn, J. H. M.
(2007). APOPTO-CELL a simulation tool and interactive database for analyzing cellular susceptibility to apoptosis. Bioinformatics
23: 648-650
[Abstract]
[Full Text]
-
Munoz-Pinedo, C., Guio-Carrion, A., Goldstein, J. C., Fitzgerald, P., Newmeyer, D. D., Green, D. R.
(2006). Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration. Proc. Natl. Acad. Sci. USA
103: 11573-11578
[Abstract]
[Full Text]
-
Tan, C., Dlugosz, P. J., Peng, J., Zhang, Z., Lapolla, S. M., Plafker, S. M., Andrews, D. W., Lin, J.
(2006). Auto-activation of the Apoptosis Protein Bax Increases Mitochondrial Membrane Permeability and Is Inhibited by Bcl-2. J. Biol. Chem.
281: 14764-14775
[Abstract]
[Full Text]
-
Ward, M. W., Rehm, M., Duessmann, H., Kacmar, S., Concannon, C. G., Prehn, J. H. M.
(2006). Real Time Single Cell Analysis of Bid Cleavage and Bid Translocation during Caspase-dependent and Neuronal Caspase-independent Apoptosis. J. Biol. Chem.
281: 5837-5844
[Abstract]
[Full Text]
-
Valentijn, A. J., Gilmore, A. P.
(2004). Translocation of Full-length Bid to Mitochondria during Anoikis. J. Biol. Chem.
279: 32848-32857
[Abstract]
[Full Text]
-
Twiddy, D., Brown, D. G., Adrain, C., Jukes, R., Martin, S. J., Cohen, G. M., MacFarlane, M., Cain, K.
(2004). Pro-apoptotic Proteins Released from the Mitochondria Regulate the Protein Composition and Caspase-processing Activity of the Native Apaf-1/Caspase-9 Apoptosome Complex. J. Biol. Chem.
279: 19665-19682
[Abstract]
[Full Text]
-
Karbowski, M., Arnoult, D., Chen, H., Chan, D. C., Smith, C. L., Youle, R. J.
(2004). Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J. Cell Biol.
164: 493-499
[Abstract]
[Full Text]
