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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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Smac/DIABLO-YFP release precedes DEVDase activation. (A and B) Individual traces of a MCF-7/Casp-3 cell and a Casp-3–deficient MCF-7 cell treated with 3 μM STS. The release of Smac/DIABLO-YFP was detected as a reduction in the standard deviation of the YFP pixel intensity. DEVDase activation was measured by proteolytical FRET-disruption of a CFP-DEVD-YFP fusion protein, indicated by an increase in the CFP/YFP emission ratio (see Materials and methods). (C) Statistical analysis of the lag-time between the onset of Smac/DIABLO-YFP release and DEVDase activation (Materials and methods). Data were collected from 9 and 10 cells in four and five individual experiments per treatment, respectively. DEVDase activation was significantly delayed in MCF-7 cells (t test, P < 0.05). Error bars, ±SEM. Asterisk indicates significance (t test); P < 0.05.
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fig8: Smac/DIABLO-YFP release precedes DEVDase activation. (A and B) Individual traces of a MCF-7/Casp-3 cell and a Casp-3–deficient MCF-7 cell treated with 3 μM STS. The release of Smac/DIABLO-YFP was detected as a reduction in the standard deviation of the YFP pixel intensity. DEVDase activation was measured by proteolytical FRET-disruption of a CFP-DEVD-YFP fusion protein, indicated by an increase in the CFP/YFP emission ratio (see Materials and methods). (C) Statistical analysis of the lag-time between the onset of Smac/DIABLO-YFP release and DEVDase activation (Materials and methods). Data were collected from 9 and 10 cells in four and five individual experiments per treatment, respectively. DEVDase activation was significantly delayed in MCF-7 cells (t test, P < 0.05). Error bars, ±SEM. Asterisk indicates significance (t test); P < 0.05.

Mentions: Release of cyt-c triggers the formation of the caspase-activating apoptosome, a process which in many cell types may be sensitive to IAPs (Holcik and Korneluk, 2001). From a mechanistic point of view, release of Smac/DIABLO during apoptosis could, therefore, represent the rate-limiting step in apoptosome formation. However, it is currently unknown how much time is required for apoptosome formation and subsequent DEVDase activation after the release of Smac/DIABLO. To address this question, we used time-lapse imaging experiments in MCF-7/Casp-3 and MCF-7 cells cotransfected with plasmids coding for Smac/DIABLO-YFP and a recombinant FRET probe. The probe was comprised of CFP, a linker peptide containing the optimal effector caspase-cleavage site (DEVD), and YFP. The DEVD linker peptide of the FRET-fusion protein is cleaved upon activation of DEVDases, resulting in a loss of resonance energy transfer and an increase in the CFP/YFP emission ratio (Tyas et al., 2000; Rehm et al., 2002). We have shown previously that the cleavage of the probe during apoptosis correlated well with the cleavage of endogenous cytosolic or nuclear caspase substrates and the activation of executioner Casp-3 and caspase-7 (Rehm et al., 2002). Fig. 8 A demonstrates CFP/YFP ratio changes and changes in the YFP redistribution in a MCF-7/Casp-3 cell in response to 3 μM STS. The cell initially showed a stable baseline CFP/YFP emission ratio, followed by a rapid cleavage of the FRET probe in <10 min. Of note, the majority of Smac/DIABLO-YFP was released before the onset of the FRET probe cleavage. As reported previously, Casp-3–deficient MCF-7 cells demonstrated significantly slower FRET probe cleavage in response to STS, indicating decreased DEVDase activity (Rehm et al., 2002; Fig. 8 B). Interestingly, in these cells, the entire Smac/DIABLO-YFP release was completed before DEVDase activity could be detected. A quantitative analysis of the time span between release of Smac/DIABLO-YFP and onset of DEVDase activity showed that MCF-7/Casp-3 cells required 5.8 ± 1.5 min for the activation of DEVDases. MCF-7 cells required a significantly longer time period, but surprisingly also achieved efficient DEVDase activity within 10 min (mean value, 9.5 ± 0.8 min; Fig. 8 C).


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

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

Smac/DIABLO-YFP release precedes DEVDase activation. (A and B) Individual traces of a MCF-7/Casp-3 cell and a Casp-3–deficient MCF-7 cell treated with 3 μM STS. The release of Smac/DIABLO-YFP was detected as a reduction in the standard deviation of the YFP pixel intensity. DEVDase activation was measured by proteolytical FRET-disruption of a CFP-DEVD-YFP fusion protein, indicated by an increase in the CFP/YFP emission ratio (see Materials and methods). (C) Statistical analysis of the lag-time between the onset of Smac/DIABLO-YFP release and DEVDase activation (Materials and methods). Data were collected from 9 and 10 cells in four and five individual experiments per treatment, respectively. DEVDase activation was significantly delayed in MCF-7 cells (t test, P < 0.05). Error bars, ±SEM. Asterisk indicates significance (t test); P < 0.05.
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fig8: Smac/DIABLO-YFP release precedes DEVDase activation. (A and B) Individual traces of a MCF-7/Casp-3 cell and a Casp-3–deficient MCF-7 cell treated with 3 μM STS. The release of Smac/DIABLO-YFP was detected as a reduction in the standard deviation of the YFP pixel intensity. DEVDase activation was measured by proteolytical FRET-disruption of a CFP-DEVD-YFP fusion protein, indicated by an increase in the CFP/YFP emission ratio (see Materials and methods). (C) Statistical analysis of the lag-time between the onset of Smac/DIABLO-YFP release and DEVDase activation (Materials and methods). Data were collected from 9 and 10 cells in four and five individual experiments per treatment, respectively. DEVDase activation was significantly delayed in MCF-7 cells (t test, P < 0.05). Error bars, ±SEM. Asterisk indicates significance (t test); P < 0.05.
Mentions: Release of cyt-c triggers the formation of the caspase-activating apoptosome, a process which in many cell types may be sensitive to IAPs (Holcik and Korneluk, 2001). From a mechanistic point of view, release of Smac/DIABLO during apoptosis could, therefore, represent the rate-limiting step in apoptosome formation. However, it is currently unknown how much time is required for apoptosome formation and subsequent DEVDase activation after the release of Smac/DIABLO. To address this question, we used time-lapse imaging experiments in MCF-7/Casp-3 and MCF-7 cells cotransfected with plasmids coding for Smac/DIABLO-YFP and a recombinant FRET probe. The probe was comprised of CFP, a linker peptide containing the optimal effector caspase-cleavage site (DEVD), and YFP. The DEVD linker peptide of the FRET-fusion protein is cleaved upon activation of DEVDases, resulting in a loss of resonance energy transfer and an increase in the CFP/YFP emission ratio (Tyas et al., 2000; Rehm et al., 2002). We have shown previously that the cleavage of the probe during apoptosis correlated well with the cleavage of endogenous cytosolic or nuclear caspase substrates and the activation of executioner Casp-3 and caspase-7 (Rehm et al., 2002). Fig. 8 A demonstrates CFP/YFP ratio changes and changes in the YFP redistribution in a MCF-7/Casp-3 cell in response to 3 μM STS. The cell initially showed a stable baseline CFP/YFP emission ratio, followed by a rapid cleavage of the FRET probe in <10 min. Of note, the majority of Smac/DIABLO-YFP was released before the onset of the FRET probe cleavage. As reported previously, Casp-3–deficient MCF-7 cells demonstrated significantly slower FRET probe cleavage in response to STS, indicating decreased DEVDase activity (Rehm et al., 2002; Fig. 8 B). Interestingly, in these cells, the entire Smac/DIABLO-YFP release was completed before DEVDase activity could be detected. A quantitative analysis of the time span between release of Smac/DIABLO-YFP and onset of DEVDase activity showed that MCF-7/Casp-3 cells required 5.8 ± 1.5 min for the activation of DEVDases. MCF-7 cells required a significantly longer time period, but surprisingly also achieved efficient DEVDase activity within 10 min (mean value, 9.5 ± 0.8 min; Fig. 8 C).

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