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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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Comparison of the kinetics and onset of cyt-c-GFP and Smac/DIABLO-YFP release in MCF-7 cells. (A) Confocal image series of two typical cells transfected with cyt-c-GFP or Smac/DIABLO-YFP. Both cells show a redistribution of the fluorescence signal in response to 3 μM STS. The onset of release was set to time point zero. The individual traces of the standard deviation of the pixel intensities for the two cells are shown below. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bar, 5 μm. (B and C) Individual traces of Smac/DIABLO-YFP– or cyt-c-GFP–expressing MCF-7 cells treated with 3 μM STS. The release of Smac/DIABLO-YFP and cyt-c-GFP was detected as a reduction in the standard deviation of the YFP or GFP pixel intensity, respectively. Changes in mitochondrial TMRM uptake were calculated by determining the average pixel intensity in the TMRM-sensitive channel. Diamonds and squares indicate corresponding TMRM- and YFP-fluorescence changes of two individual cells. (D) Scatter plot showing the onset of cyt-c-GFP (black diamonds) or Smac/DIABLO-YFP (open squares) release in individual cells and their corresponding half-life time of the standard deviation decrease, a measure of the release duration (see Materials and methods). Cells were transfected with either cyt-c-GFP or Smac/DIABLO-YFP, treated with 3 μM STS and observed by confocal microscopy. Mean values are represented by a gray diamond or square, respectively (±SD in both dimensions). The duration of the Smac/DIABLO-YFP release was greater than that of cyt-c-GFP (t test; P < 0.05), whereas no significant difference was observed regarding the time point of release onset (t test). Data were collected from n = 18 and 25 cells in three and five experiments, respectively. Asterisk indicates significance (t test); P < 0.05; n.s., not significant. (E) Kinetics of Smac/DIABLO-YFP release in MCF-7 cells in response to TNF-α/CHX. Individual trace of a typical cell is shown. Similar traces were obtained from n = 8 cells in three separate experiments.
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fig5: Comparison of the kinetics and onset of cyt-c-GFP and Smac/DIABLO-YFP release in MCF-7 cells. (A) Confocal image series of two typical cells transfected with cyt-c-GFP or Smac/DIABLO-YFP. Both cells show a redistribution of the fluorescence signal in response to 3 μM STS. The onset of release was set to time point zero. The individual traces of the standard deviation of the pixel intensities for the two cells are shown below. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bar, 5 μm. (B and C) Individual traces of Smac/DIABLO-YFP– or cyt-c-GFP–expressing MCF-7 cells treated with 3 μM STS. The release of Smac/DIABLO-YFP and cyt-c-GFP was detected as a reduction in the standard deviation of the YFP or GFP pixel intensity, respectively. Changes in mitochondrial TMRM uptake were calculated by determining the average pixel intensity in the TMRM-sensitive channel. Diamonds and squares indicate corresponding TMRM- and YFP-fluorescence changes of two individual cells. (D) Scatter plot showing the onset of cyt-c-GFP (black diamonds) or Smac/DIABLO-YFP (open squares) release in individual cells and their corresponding half-life time of the standard deviation decrease, a measure of the release duration (see Materials and methods). Cells were transfected with either cyt-c-GFP or Smac/DIABLO-YFP, treated with 3 μM STS and observed by confocal microscopy. Mean values are represented by a gray diamond or square, respectively (±SD in both dimensions). The duration of the Smac/DIABLO-YFP release was greater than that of cyt-c-GFP (t test; P < 0.05), whereas no significant difference was observed regarding the time point of release onset (t test). Data were collected from n = 18 and 25 cells in three and five experiments, respectively. Asterisk indicates significance (t test); P < 0.05; n.s., not significant. (E) Kinetics of Smac/DIABLO-YFP release in MCF-7 cells in response to TNF-α/CHX. Individual trace of a typical cell is shown. Similar traces were obtained from n = 8 cells in three separate experiments.

Mentions: We monitored individual MCF-7 cells expressing either Smac/DIABLO-YFP or a cyt-c-GFP fusion protein (Luetjens et al., 2001; Dussmann et al., 2003a,b) by time lapse confocal microscopy during STS-induced apoptosis. As reported previously, individual cells released cyt-c-GFP at different time points after addition of the proapoptotic agent, and the majority of the cyt-c-GFP fusion protein was released in one step that was completed within 10 min (Fig. 5, A and B; Goldstein et al., 2000; Luetjens et al., 2001). The cyt-c-GFP redistribution was indicated by a decrease in the standard deviation of the average pixel. Confocal imaging of Smac/DIABLO-YFP redistribution revealed a comparable type of release. The majority of the fluorescence signal was redistributed in a single step, albeit the duration of the Smac/DIABLO-YFP release was significantly increased (Fig. 5, A and C). We simultaneously monitored changes in the mitochondrial uptake of the voltage-sensitive probe TMRM in order to determine the temporal relationship between Smac/DIABLO-YFP, cyt-c-GFP release, and mitochondrial dysfunction during apoptosis. In both cases, release of cyt-c-GFP and Smac/DIABLO-YFP coincided with ΔΨM depolarization indicated by a decrease in mitochondrial TMRM uptake (Fig. 5, B and C). This suggested that cyt-c-GFP and Smac/DIABLO-YFP are released at a similar time point during STS-induced apoptosis.


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

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

Comparison of the kinetics and onset of cyt-c-GFP and Smac/DIABLO-YFP release in MCF-7 cells. (A) Confocal image series of two typical cells transfected with cyt-c-GFP or Smac/DIABLO-YFP. Both cells show a redistribution of the fluorescence signal in response to 3 μM STS. The onset of release was set to time point zero. The individual traces of the standard deviation of the pixel intensities for the two cells are shown below. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bar, 5 μm. (B and C) Individual traces of Smac/DIABLO-YFP– or cyt-c-GFP–expressing MCF-7 cells treated with 3 μM STS. The release of Smac/DIABLO-YFP and cyt-c-GFP was detected as a reduction in the standard deviation of the YFP or GFP pixel intensity, respectively. Changes in mitochondrial TMRM uptake were calculated by determining the average pixel intensity in the TMRM-sensitive channel. Diamonds and squares indicate corresponding TMRM- and YFP-fluorescence changes of two individual cells. (D) Scatter plot showing the onset of cyt-c-GFP (black diamonds) or Smac/DIABLO-YFP (open squares) release in individual cells and their corresponding half-life time of the standard deviation decrease, a measure of the release duration (see Materials and methods). Cells were transfected with either cyt-c-GFP or Smac/DIABLO-YFP, treated with 3 μM STS and observed by confocal microscopy. Mean values are represented by a gray diamond or square, respectively (±SD in both dimensions). The duration of the Smac/DIABLO-YFP release was greater than that of cyt-c-GFP (t test; P < 0.05), whereas no significant difference was observed regarding the time point of release onset (t test). Data were collected from n = 18 and 25 cells in three and five experiments, respectively. Asterisk indicates significance (t test); P < 0.05; n.s., not significant. (E) Kinetics of Smac/DIABLO-YFP release in MCF-7 cells in response to TNF-α/CHX. Individual trace of a typical cell is shown. Similar traces were obtained from n = 8 cells in three separate experiments.
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fig5: Comparison of the kinetics and onset of cyt-c-GFP and Smac/DIABLO-YFP release in MCF-7 cells. (A) Confocal image series of two typical cells transfected with cyt-c-GFP or Smac/DIABLO-YFP. Both cells show a redistribution of the fluorescence signal in response to 3 μM STS. The onset of release was set to time point zero. The individual traces of the standard deviation of the pixel intensities for the two cells are shown below. For direct comparison of the release kinetics, traces were scaled from 100 (baseline before release) to 0 (baseline after completion of the release). Bar, 5 μm. (B and C) Individual traces of Smac/DIABLO-YFP– or cyt-c-GFP–expressing MCF-7 cells treated with 3 μM STS. The release of Smac/DIABLO-YFP and cyt-c-GFP was detected as a reduction in the standard deviation of the YFP or GFP pixel intensity, respectively. Changes in mitochondrial TMRM uptake were calculated by determining the average pixel intensity in the TMRM-sensitive channel. Diamonds and squares indicate corresponding TMRM- and YFP-fluorescence changes of two individual cells. (D) Scatter plot showing the onset of cyt-c-GFP (black diamonds) or Smac/DIABLO-YFP (open squares) release in individual cells and their corresponding half-life time of the standard deviation decrease, a measure of the release duration (see Materials and methods). Cells were transfected with either cyt-c-GFP or Smac/DIABLO-YFP, treated with 3 μM STS and observed by confocal microscopy. Mean values are represented by a gray diamond or square, respectively (±SD in both dimensions). The duration of the Smac/DIABLO-YFP release was greater than that of cyt-c-GFP (t test; P < 0.05), whereas no significant difference was observed regarding the time point of release onset (t test). Data were collected from n = 18 and 25 cells in three and five experiments, respectively. Asterisk indicates significance (t test); P < 0.05; n.s., not significant. (E) Kinetics of Smac/DIABLO-YFP release in MCF-7 cells in response to TNF-α/CHX. Individual trace of a typical cell is shown. Similar traces were obtained from n = 8 cells in three separate experiments.
Mentions: We monitored individual MCF-7 cells expressing either Smac/DIABLO-YFP or a cyt-c-GFP fusion protein (Luetjens et al., 2001; Dussmann et al., 2003a,b) by time lapse confocal microscopy during STS-induced apoptosis. As reported previously, individual cells released cyt-c-GFP at different time points after addition of the proapoptotic agent, and the majority of the cyt-c-GFP fusion protein was released in one step that was completed within 10 min (Fig. 5, A and B; Goldstein et al., 2000; Luetjens et al., 2001). The cyt-c-GFP redistribution was indicated by a decrease in the standard deviation of the average pixel. Confocal imaging of Smac/DIABLO-YFP redistribution revealed a comparable type of release. The majority of the fluorescence signal was redistributed in a single step, albeit the duration of the Smac/DIABLO-YFP release was significantly increased (Fig. 5, A and C). We simultaneously monitored changes in the mitochondrial uptake of the voltage-sensitive probe TMRM in order to determine the temporal relationship between Smac/DIABLO-YFP, cyt-c-GFP release, and mitochondrial dysfunction during apoptosis. In both cases, release of cyt-c-GFP and Smac/DIABLO-YFP coincided with ΔΨM depolarization indicated by a decrease in mitochondrial TMRM uptake (Fig. 5, B and C). This suggested that cyt-c-GFP and Smac/DIABLO-YFP are released at a similar time point during STS-induced apoptosis.

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