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

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.

Figure 1.

Comparison of Smac/DIABLO and cyt-c release during apoptosis: effect of Casp-3 and z-VAD-fmk–sensitive caspases. (A) MCF-7/Casp-3 cells and MCF-7 cells were treated with 3 μM STS or 3 μM STS plus 200 μM of the broad-spectrum caspase inhibitor z-VAD-fmk for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results. (B) Immunofluorescence analysis showing the redistribution of cyt-c and Smac/DIABLO during apoptosis. Cells were treated with 3 μM STS, 3 μM STS plus 200 μM z-VAD-fmk or 200 ng/ml, and 1 μg/ml TNF-α/CHX for 6 h. Control cells received vehicle (DMSO). Arrows indicate cells that show a cyt-c and Smac/DIABLO redistribution in response to the agents. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (C) Quantification of cells showing cyt-c or Smac/DIABLO release as judged by immunofluorescence analysis. MCF-7/Casp-3 cells (indicated as Casp-3 +) were treated with 3 μM STS. MCF-7 cells (indicated as Casp-3 −) were treated with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk. Data were collected from n = 200–300 cells per treatment in 11–14 randomly selected image frames from n = 3 independent experiments. There was no statistically significant difference between the three treatment groups or between cyt-c and Smac/DIABLO release at any time point investigated. Error bars equal SEM.

Figure 2.

Effect of caspase and proteasome inhibition on the release of mitochondrial proteins. (A) Western blot analysis of Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h in combination with 200 μM z-VAD-fmk and/or 1 μM of the proteasome inhibitor lactacystin. Release of Smac/DIABLO and cyt-c from mitochondria containing pellet fraction into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. α-Tubulin and porin served as control for equal loading of the samples. Experiment was repeated twice with similar results. (B) Western blot analysis of the cytosol and mitochondria-containing pellet fraction of HeLa D98 cells exposed to 3 μM STS for 8 h, either in the absence or presence of z-VAD-fmk or lactacystin. Experiment was repeated with similar results. (C) Western blot analysis demonstrating the processing of caspase-2, -9, and -7 in Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h and its inhibition by 200 μM z-VAD-fmk. α-Tubulin served as control for equal sample loading.

Figure 3.

Release of Smac/DIABLO during apoptosis can occur independent of Bax. Bax-deficient DU145 cells were treated with 3 μM STS for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.

Figure 8.

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.