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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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Casp-3 and z-VAD-fmk–sensitive caspases do not accelerate the kinetics of Smac/DIABLO-YFP release in response to STS. (A) Quantification of Smac/DIABLO-YFP release and mitochondrial TMRM uptake in cells expressing Smac/DIABLO-YFP. MCF-7/Casp-3 cells, Casp-3–deficient MCF-7 cells and 200 μM z-VAD-fmk–treated MCF-7 cells were equilibrated with 30 nM TMRM and treated with 3 μM STS. Mean traces were calculated from single cell kinetics synchronized to the time of Smac/DIABLO-YFP release. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bars, ±SEM. (B) Comparison of Smac/DIABLO-YFP release kinetics. Single cell release kinetics were fitted with an exponential decay function and the corresponding half-life times were calculated. Asterisk indicates significance (ANOVA and Tukey test). Error bars, ±SEM. (C) Comparison of the mean standard deviation baseline value reached after completion of the Smac/DIABLO-YFP redistribution. Data in A–C were collected from 9 to 18 cells in three to seven independent experiments per treatment. n.s. , not significant. (D and E) Release of Smac/DIABLO-YFP in HeLa D98 cells. Cells were treated with 3 μM STS in the absence (D) or presence (E) of 200 μM z-VAD-fmk. Individual traces of typical cells are shown. Similar traces were obtained from n = 29 and 15 cells in two separate experiments per treatment.
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fig7: Casp-3 and z-VAD-fmk–sensitive caspases do not accelerate the kinetics of Smac/DIABLO-YFP release in response to STS. (A) Quantification of Smac/DIABLO-YFP release and mitochondrial TMRM uptake in cells expressing Smac/DIABLO-YFP. MCF-7/Casp-3 cells, Casp-3–deficient MCF-7 cells and 200 μM z-VAD-fmk–treated MCF-7 cells were equilibrated with 30 nM TMRM and treated with 3 μM STS. Mean traces were calculated from single cell kinetics synchronized to the time of Smac/DIABLO-YFP release. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bars, ±SEM. (B) Comparison of Smac/DIABLO-YFP release kinetics. Single cell release kinetics were fitted with an exponential decay function and the corresponding half-life times were calculated. Asterisk indicates significance (ANOVA and Tukey test). Error bars, ±SEM. (C) Comparison of the mean standard deviation baseline value reached after completion of the Smac/DIABLO-YFP redistribution. Data in A–C were collected from 9 to 18 cells in three to seven independent experiments per treatment. n.s. , not significant. (D and E) Release of Smac/DIABLO-YFP in HeLa D98 cells. Cells were treated with 3 μM STS in the absence (D) or presence (E) of 200 μM z-VAD-fmk. Individual traces of typical cells are shown. Similar traces were obtained from n = 29 and 15 cells in two separate experiments per treatment.

Mentions: Our digitonization/immunoblotting and immunofluorescence experiments suggested that the release of Smac/DIABLO from mitochondria during STS-induced apoptosis was not influenced by Casp-3 or z-VAD-fmk–sensitive caspases. However, the possibility remained that significant differences existed at the single cell level, which could not be resolved using the above techniques. Therefore, we analyzed the kinetics of Smac/DIABLO-YFP release in response to STS by time-lapse confocal microscopy in MCF-7/Casp-3 cells, MCF-7 cells, and z-VAD-fmk–treated MCF-7 cells. In parallel, we monitored changes in mitochondrial TMRM uptake, indicative of ΔΨM depolarization. Confocal imaging of Smac/DIABLO-YFP fluorescence redistribution revealed that the majority of Smac/DIABLO-YFP was released in one single step, regardless of the level of caspase activation (Fig. 7 A). Moreover, the release was always associated with a decrease in mitochondrial TMRM uptake. To calculate the average kinetics of Smac/DIABLO-YFP release, cells were synchronized to the time of Smac/DIABLO-YFP release (Fig. 7 A). Analysis of the half-life time of the release indicated no significant differences besides a modest difference between MCF-7/Casp-3 cells and z-VAD-fmk–treated MCF-7 cells, with faster release kinetics in the z-VAD-fmk–treated MCF-7 cells (Fig. 7 B). This difference was not detected in z-VAD-fmk plus lactacystin cotreated cells (Fig. 7 B). Analysis of the final standard deviation after the Smac/DIABLO-YFP-release indicated no significant differences in the extent of Smac/DIABLO-YFP redistribution, suggesting no differences in the magnitude of release (Fig. 7 C). As shown previously in MCF-7/Casp-3 cells, ΔΨM depolarized during STS-induced apoptosis until a new steady-state level was reached (ΔΨM−cyt-c; Dussmann et al., 2003b). z-VAD-fmk–treated MCF-7 cells showed an increased TMRM uptake when ΔΨM−cyt-c was reached, an effect largely attributable to caspase-dependent plasma membrane potential depolarization (Dussmann et al., 2003b).


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

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

Casp-3 and z-VAD-fmk–sensitive caspases do not accelerate the kinetics of Smac/DIABLO-YFP release in response to STS. (A) Quantification of Smac/DIABLO-YFP release and mitochondrial TMRM uptake in cells expressing Smac/DIABLO-YFP. MCF-7/Casp-3 cells, Casp-3–deficient MCF-7 cells and 200 μM z-VAD-fmk–treated MCF-7 cells were equilibrated with 30 nM TMRM and treated with 3 μM STS. Mean traces were calculated from single cell kinetics synchronized to the time of Smac/DIABLO-YFP release. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bars, ±SEM. (B) Comparison of Smac/DIABLO-YFP release kinetics. Single cell release kinetics were fitted with an exponential decay function and the corresponding half-life times were calculated. Asterisk indicates significance (ANOVA and Tukey test). Error bars, ±SEM. (C) Comparison of the mean standard deviation baseline value reached after completion of the Smac/DIABLO-YFP redistribution. Data in A–C were collected from 9 to 18 cells in three to seven independent experiments per treatment. n.s. , not significant. (D and E) Release of Smac/DIABLO-YFP in HeLa D98 cells. Cells were treated with 3 μM STS in the absence (D) or presence (E) of 200 μM z-VAD-fmk. Individual traces of typical cells are shown. Similar traces were obtained from n = 29 and 15 cells in two separate experiments per treatment.
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Related In: Results  -  Collection

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fig7: Casp-3 and z-VAD-fmk–sensitive caspases do not accelerate the kinetics of Smac/DIABLO-YFP release in response to STS. (A) Quantification of Smac/DIABLO-YFP release and mitochondrial TMRM uptake in cells expressing Smac/DIABLO-YFP. MCF-7/Casp-3 cells, Casp-3–deficient MCF-7 cells and 200 μM z-VAD-fmk–treated MCF-7 cells were equilibrated with 30 nM TMRM and treated with 3 μM STS. Mean traces were calculated from single cell kinetics synchronized to the time of Smac/DIABLO-YFP release. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bars, ±SEM. (B) Comparison of Smac/DIABLO-YFP release kinetics. Single cell release kinetics were fitted with an exponential decay function and the corresponding half-life times were calculated. Asterisk indicates significance (ANOVA and Tukey test). Error bars, ±SEM. (C) Comparison of the mean standard deviation baseline value reached after completion of the Smac/DIABLO-YFP redistribution. Data in A–C were collected from 9 to 18 cells in three to seven independent experiments per treatment. n.s. , not significant. (D and E) Release of Smac/DIABLO-YFP in HeLa D98 cells. Cells were treated with 3 μM STS in the absence (D) or presence (E) of 200 μM z-VAD-fmk. Individual traces of typical cells are shown. Similar traces were obtained from n = 29 and 15 cells in two separate experiments per treatment.
Mentions: Our digitonization/immunoblotting and immunofluorescence experiments suggested that the release of Smac/DIABLO from mitochondria during STS-induced apoptosis was not influenced by Casp-3 or z-VAD-fmk–sensitive caspases. However, the possibility remained that significant differences existed at the single cell level, which could not be resolved using the above techniques. Therefore, we analyzed the kinetics of Smac/DIABLO-YFP release in response to STS by time-lapse confocal microscopy in MCF-7/Casp-3 cells, MCF-7 cells, and z-VAD-fmk–treated MCF-7 cells. In parallel, we monitored changes in mitochondrial TMRM uptake, indicative of ΔΨM depolarization. Confocal imaging of Smac/DIABLO-YFP fluorescence redistribution revealed that the majority of Smac/DIABLO-YFP was released in one single step, regardless of the level of caspase activation (Fig. 7 A). Moreover, the release was always associated with a decrease in mitochondrial TMRM uptake. To calculate the average kinetics of Smac/DIABLO-YFP release, cells were synchronized to the time of Smac/DIABLO-YFP release (Fig. 7 A). Analysis of the half-life time of the release indicated no significant differences besides a modest difference between MCF-7/Casp-3 cells and z-VAD-fmk–treated MCF-7 cells, with faster release kinetics in the z-VAD-fmk–treated MCF-7 cells (Fig. 7 B). This difference was not detected in z-VAD-fmk plus lactacystin cotreated cells (Fig. 7 B). Analysis of the final standard deviation after the Smac/DIABLO-YFP-release indicated no significant differences in the extent of Smac/DIABLO-YFP redistribution, suggesting no differences in the magnitude of release (Fig. 7 C). As shown previously in MCF-7/Casp-3 cells, ΔΨM depolarized during STS-induced apoptosis until a new steady-state level was reached (ΔΨM−cyt-c; Dussmann et al., 2003b). z-VAD-fmk–treated MCF-7 cells showed an increased TMRM uptake when ΔΨM−cyt-c was reached, an effect largely attributable to caspase-dependent plasma membrane potential depolarization (Dussmann et al., 2003b).

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