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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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Characterization of MCF-7 cells expressing Smac/DIABLO-YFP. (A) Confocal microscopy showing the colocalization of Smac/DIABLO-YFP (green) and TMRM (red) in mitochondria of untreated cells. The overlay image shows yellow pixels at the sites of colocalization. Extended view calculated from three confocal sections with 0.1 μM steps in vertical direction with 0.67 μm resolution and 0.23 μm resolution in horizontal direction. Bar, 10 μm. (B) Western blot analysis of MCF-7 cells stably expressing Smac/DIABLO-YFP. Cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO, Smac/DIABLO-YFP, and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Control received DMSO for 8 h. Experiment was repeated twice with similar results. (C) Smac/ DIABLO-YFP fluorescence and Smac/DIABLO immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 200 ng/ml and 1 μg/ml TNF-α/CHX. Arrows indicate cells that show a Smac/ DIABLO-YFP or Smac/DIABLO redistribution. Control cells received DMSO. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (D) Smac/DIABLO-YFP fluorescence and cyt-c immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 3 μM STS plus 200 μM z-VAD-fmk. Control cells received vehicle (DMSO). Nuclear morphology was detected by Hoechst staining. Arrows indicate cells that show a Smac/DIABLO-YFP or cyt-c redistribution. Bar, 10 μm.
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fig4: Characterization of MCF-7 cells expressing Smac/DIABLO-YFP. (A) Confocal microscopy showing the colocalization of Smac/DIABLO-YFP (green) and TMRM (red) in mitochondria of untreated cells. The overlay image shows yellow pixels at the sites of colocalization. Extended view calculated from three confocal sections with 0.1 μM steps in vertical direction with 0.67 μm resolution and 0.23 μm resolution in horizontal direction. Bar, 10 μm. (B) Western blot analysis of MCF-7 cells stably expressing Smac/DIABLO-YFP. Cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO, Smac/DIABLO-YFP, and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Control received DMSO for 8 h. Experiment was repeated twice with similar results. (C) Smac/ DIABLO-YFP fluorescence and Smac/DIABLO immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 200 ng/ml and 1 μg/ml TNF-α/CHX. Arrows indicate cells that show a Smac/ DIABLO-YFP or Smac/DIABLO redistribution. Control cells received DMSO. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (D) Smac/DIABLO-YFP fluorescence and cyt-c immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 3 μM STS plus 200 μM z-VAD-fmk. Control cells received vehicle (DMSO). Nuclear morphology was detected by Hoechst staining. Arrows indicate cells that show a Smac/DIABLO-YFP or cyt-c redistribution. Bar, 10 μm.

Mentions: To monitor the release of Smac/DIABLO in real-time, we generated MCF-7 and MCF-7/Casp-3 cells expressing a fusion protein comprised of Smac/DIABLO and YFP. Import of Smac/DIABLO-YFP into mitochondria was confirmed by confocal analysis of Smac/DIABLO-YFP fluorescence and its colocalization with the ΔΨM-sensitive dye tetramethyl rhodamine methyl ester (TMRM; Fig. 4 A). We subsequently examined the release behavior of the fusion protein during apoptotic cell death compared with endogenous Smac/DIABLO and cyt-c. Immunoblotting experiments were performed with subcellular fractions of transfected MCF-7 cells exposed to 3 μM STS for increasing time periods (Fig. 4 B). The redistribution of Smac/DIABLO-YFP from the mitochondria to the cytosol during apoptosis was similar to that of endogenous Smac/DIABLO and cyt-c. Comparable results were obtained in Smac/DIABLO-YFP–expressing MCF-7/Casp-3 cells (unpublished data). Cells that released the Smac/DIABLO-YFP fusion protein in response to STS or TNF-α/CHX also showed a redistribution of Smac/DIABLO immunofluorescence (Fig. 4 C). Similar results were obtained when we analyzed Smac/DIABLO-YFP fluorescence and cyt-c immunofluorescence redistribution during apoptosis (Fig. 4 D). Hence, this system enabled one to reliably monitor Smac/DIABLO release at the single cell level during apoptosis.


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

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

Characterization of MCF-7 cells expressing Smac/DIABLO-YFP. (A) Confocal microscopy showing the colocalization of Smac/DIABLO-YFP (green) and TMRM (red) in mitochondria of untreated cells. The overlay image shows yellow pixels at the sites of colocalization. Extended view calculated from three confocal sections with 0.1 μM steps in vertical direction with 0.67 μm resolution and 0.23 μm resolution in horizontal direction. Bar, 10 μm. (B) Western blot analysis of MCF-7 cells stably expressing Smac/DIABLO-YFP. Cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO, Smac/DIABLO-YFP, and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Control received DMSO for 8 h. Experiment was repeated twice with similar results. (C) Smac/ DIABLO-YFP fluorescence and Smac/DIABLO immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 200 ng/ml and 1 μg/ml TNF-α/CHX. Arrows indicate cells that show a Smac/ DIABLO-YFP or Smac/DIABLO redistribution. Control cells received DMSO. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (D) Smac/DIABLO-YFP fluorescence and cyt-c immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 3 μM STS plus 200 μM z-VAD-fmk. Control cells received vehicle (DMSO). Nuclear morphology was detected by Hoechst staining. Arrows indicate cells that show a Smac/DIABLO-YFP or cyt-c redistribution. Bar, 10 μm.
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fig4: Characterization of MCF-7 cells expressing Smac/DIABLO-YFP. (A) Confocal microscopy showing the colocalization of Smac/DIABLO-YFP (green) and TMRM (red) in mitochondria of untreated cells. The overlay image shows yellow pixels at the sites of colocalization. Extended view calculated from three confocal sections with 0.1 μM steps in vertical direction with 0.67 μm resolution and 0.23 μm resolution in horizontal direction. Bar, 10 μm. (B) Western blot analysis of MCF-7 cells stably expressing Smac/DIABLO-YFP. Cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO, Smac/DIABLO-YFP, and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Control received DMSO for 8 h. Experiment was repeated twice with similar results. (C) Smac/ DIABLO-YFP fluorescence and Smac/DIABLO immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 200 ng/ml and 1 μg/ml TNF-α/CHX. Arrows indicate cells that show a Smac/ DIABLO-YFP or Smac/DIABLO redistribution. Control cells received DMSO. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (D) Smac/DIABLO-YFP fluorescence and cyt-c immunofluorescence of MCF-7 cells treated for 6 h with 3 μM STS or 3 μM STS plus 200 μM z-VAD-fmk. Control cells received vehicle (DMSO). Nuclear morphology was detected by Hoechst staining. Arrows indicate cells that show a Smac/DIABLO-YFP or cyt-c redistribution. Bar, 10 μm.
Mentions: To monitor the release of Smac/DIABLO in real-time, we generated MCF-7 and MCF-7/Casp-3 cells expressing a fusion protein comprised of Smac/DIABLO and YFP. Import of Smac/DIABLO-YFP into mitochondria was confirmed by confocal analysis of Smac/DIABLO-YFP fluorescence and its colocalization with the ΔΨM-sensitive dye tetramethyl rhodamine methyl ester (TMRM; Fig. 4 A). We subsequently examined the release behavior of the fusion protein during apoptotic cell death compared with endogenous Smac/DIABLO and cyt-c. Immunoblotting experiments were performed with subcellular fractions of transfected MCF-7 cells exposed to 3 μM STS for increasing time periods (Fig. 4 B). The redistribution of Smac/DIABLO-YFP from the mitochondria to the cytosol during apoptosis was similar to that of endogenous Smac/DIABLO and cyt-c. Comparable results were obtained in Smac/DIABLO-YFP–expressing MCF-7/Casp-3 cells (unpublished data). Cells that released the Smac/DIABLO-YFP fusion protein in response to STS or TNF-α/CHX also showed a redistribution of Smac/DIABLO immunofluorescence (Fig. 4 C). Similar results were obtained when we analyzed Smac/DIABLO-YFP fluorescence and cyt-c immunofluorescence redistribution during apoptosis (Fig. 4 D). Hence, this system enabled one to reliably monitor Smac/DIABLO release at the single cell level during 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