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Real-time single cell analysis of Smac/DIABLO release during apoptosis.

Rehm M, Düssmann H, Prehn JH - J. Cell Biol. (2003)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Clinical Research, University Münster Clinics, Münster, Germany.

ABSTRACT
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.

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Release of Smac/DIABLO during apoptosis can occur independent of Bax. Bax-deficient DU145 cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results.
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fig3: Release of Smac/DIABLO during apoptosis can occur independent of Bax. Bax-deficient DU145 cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results.

Mentions: To investigate whether Bax expression is necessary for the release of Smac/DIABLO from mitochondria, Bax-deficient human DU-145 prostate cancer cells (Honda et al., 2001) were treated with 3 μM STS. The cytosolic accumulation of Smac/DIABLO and cyt-c detected after digitonization and immunoblotting. Interestingly, both Smac/DIABLO and cyt-c were released from Bax-deficient mitochondria in a similar time course (Fig. 3).


Real-time single cell analysis of Smac/DIABLO release during apoptosis.

Rehm M, Düssmann H, Prehn JH - J. Cell Biol. (2003)

Release of Smac/DIABLO during apoptosis can occur independent of Bax. Bax-deficient DU145 cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172837&req=5

fig3: Release of Smac/DIABLO during apoptosis can occur independent of Bax. Bax-deficient DU145 cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results.
Mentions: To investigate whether Bax expression is necessary for the release of Smac/DIABLO from mitochondria, Bax-deficient human DU-145 prostate cancer cells (Honda et al., 2001) were treated with 3 μM STS. The cytosolic accumulation of Smac/DIABLO and cyt-c detected after digitonization and immunoblotting. Interestingly, both Smac/DIABLO and cyt-c were released from Bax-deficient mitochondria in a similar time course (Fig. 3).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Clinical Research, University Münster Clinics, Münster, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus