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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-DsRed is not released from mitochondria. MCF-7 cells cotransfected with cyt-c-GFP and Smac/DIABLO-DsRed before (A–C) and 40 min after (D–F) an exposure to 3 μM STS. The cell indicated by arrows released cyt-c-GFP, but retained its mitochondrial Smac/DIABLO-DsRed signal. Similar results were obtained in 16 cells from three separate time-lapse experiments. Bar, 10 μm. High resolution image of an MCF-7 control cell (G–I) and an MCF-7 cell treated for 6 h with 3 μM STS (J–L). Note the redistribution of cyt-c-GFP into the cytosol and nucleus after STS treatment, whereas Smac/ DIABLO-DsRed fluorescence retained in mitochondria. Bar, 10 μm.
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fig6: Smac/DIABLO-DsRed is not released from mitochondria. MCF-7 cells cotransfected with cyt-c-GFP and Smac/DIABLO-DsRed before (A–C) and 40 min after (D–F) an exposure to 3 μM STS. The cell indicated by arrows released cyt-c-GFP, but retained its mitochondrial Smac/DIABLO-DsRed signal. Similar results were obtained in 16 cells from three separate time-lapse experiments. Bar, 10 μm. High resolution image of an MCF-7 control cell (G–I) and an MCF-7 cell treated for 6 h with 3 μM STS (J–L). Note the redistribution of cyt-c-GFP into the cytosol and nucleus after STS treatment, whereas Smac/ DIABLO-DsRed fluorescence retained in mitochondria. Bar, 10 μm.

Mentions: The difference in release kinetics of cyt-c-GFP and Smac/DIABLO-YFP could be size dependent. To test this hypothesis further, we generated a Smac/DIABLO-DsRed fusion protein that was readily imported into mitochondria of MCF-7 cells judged by the colocalization with cyt-c-GFP (Fig. 6, A–C and G–I). As DsRed is only fluorescent as a tetramer (Baird et al., 2000), the size of the red emitting Smac/DIABLO–DsRed complex is ∼188 kD. Time-lapse confocal imaging experiments of MCF-7 cells cotransfected with Smac/DIABLO-DsRed and cyt-c-GFP revealed that treatment with 3 μM STS triggered the release of cyt-c-GFP, whereas Smac/DIABLO-DsRed retained in mitochondria (Fig. 6, D–F and J–L). Absence of Smac/DIABLO-DsRed release was observed in 16 out of 16 cyt-c-GFP release-positive cells in three separate time-lapse experiments.


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

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

Smac/DIABLO-DsRed is not released from mitochondria. MCF-7 cells cotransfected with cyt-c-GFP and Smac/DIABLO-DsRed before (A–C) and 40 min after (D–F) an exposure to 3 μM STS. The cell indicated by arrows released cyt-c-GFP, but retained its mitochondrial Smac/DIABLO-DsRed signal. Similar results were obtained in 16 cells from three separate time-lapse experiments. Bar, 10 μm. High resolution image of an MCF-7 control cell (G–I) and an MCF-7 cell treated for 6 h with 3 μM STS (J–L). Note the redistribution of cyt-c-GFP into the cytosol and nucleus after STS treatment, whereas Smac/ DIABLO-DsRed fluorescence retained in mitochondria. Bar, 10 μm.
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Related In: Results  -  Collection

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fig6: Smac/DIABLO-DsRed is not released from mitochondria. MCF-7 cells cotransfected with cyt-c-GFP and Smac/DIABLO-DsRed before (A–C) and 40 min after (D–F) an exposure to 3 μM STS. The cell indicated by arrows released cyt-c-GFP, but retained its mitochondrial Smac/DIABLO-DsRed signal. Similar results were obtained in 16 cells from three separate time-lapse experiments. Bar, 10 μm. High resolution image of an MCF-7 control cell (G–I) and an MCF-7 cell treated for 6 h with 3 μM STS (J–L). Note the redistribution of cyt-c-GFP into the cytosol and nucleus after STS treatment, whereas Smac/ DIABLO-DsRed fluorescence retained in mitochondria. Bar, 10 μm.
Mentions: The difference in release kinetics of cyt-c-GFP and Smac/DIABLO-YFP could be size dependent. To test this hypothesis further, we generated a Smac/DIABLO-DsRed fusion protein that was readily imported into mitochondria of MCF-7 cells judged by the colocalization with cyt-c-GFP (Fig. 6, A–C and G–I). As DsRed is only fluorescent as a tetramer (Baird et al., 2000), the size of the red emitting Smac/DIABLO–DsRed complex is ∼188 kD. Time-lapse confocal imaging experiments of MCF-7 cells cotransfected with Smac/DIABLO-DsRed and cyt-c-GFP revealed that treatment with 3 μM STS triggered the release of cyt-c-GFP, whereas Smac/DIABLO-DsRed retained in mitochondria (Fig. 6, D–F and J–L). Absence of Smac/DIABLO-DsRed release was observed in 16 out of 16 cyt-c-GFP release-positive cells in three separate time-lapse experiments.

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