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First published online 11 December 2002
doi: 10.1242/jcs.00236

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Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis

¹ Interdisciplinary Center for Clinical Research (IZKF), Westphalian Wilhelms-University, D-48149 Münster, Germany
² Department of Pharmacology and Toxicology, Westphalian Wilhelms-University, D-48149 Münster, Germany

View larger version (36K): [in a new window]   Fig. 1. Release of cyt-C-GFP triggers a decrease in mitochondrial TMRM uptake. (A) MCF-7/Casp-3 cells stably expressing cyt-C-GFP were equilibrated with 30 nM TMRM and treated with 3 µM STS. Cyt-C-GFP release was followed by a reduction in mitochondrial TMRM uptake. Bars, 20 µm. (B) Individual traces of two typical cells treated with 3 µM STS. The release of cyt-C-GFP was detected as a reduction in the standard deviation of the GFP pixel intensities of single cells. Changes in TMRM uptake were calculated from single cells by determining their average pixel intensity in the TMRM-sensitive channel. Squares and diamonds indicate corresponding TMRM and cyt-C-GFP changes of the two cells, respectively. The onset of the decrease in mitochondrial TMRM uptake occurred simultaneously to mitochondrial cyt-C-GFP release, suggesting an irreversible M depolarization during the time period monitored. (C) Quantification of TMRM fluorescence decrease in the STS-treated cells. The time of cyt-C-GFP release was set to zero to calculate average values from 16 cells in four independent experiments. (D) Individual traces of two typical cells treated with 100 ng/ml TNF and 1 µg/ml CHX. Similar traces were recorded from 12 cells in two independent experiments represented.

View larger version (67K): [in a new window]   Fig. 2. Treatment with the broad-spectrum caspase inhibitor z-VAD-fmk protects cells from apoptotic cell shrinkage and activation of DEVDases but a decrease in mitochondrial TMRM uptake can still be detected. (A) MCF-7/Casp-3 cells stably expressing cyt-C-GFP and treated with 3 µM STS plus 200 µM z-VAD-fmk show a rapid cyt-C-GFP release and a decrease in TMRM uptake. Bar, 20 µm. (B) Individual traces of two cells treated with STS plus z-VAD-fmk. (C) Mean traces of 16 cells in four independent experiments calculated from single cell kinetics by setting the time of onset of cyt-C-GFP release to zero. (D,E) Transmission light and GFP fluorescence images of cells treated with 3 µM STS for the indicated time periods in the absence (D) or presence (E) of 200 µM z-VAD-fmk Bar, 20 µm. Note the absence of apoptotic shrinkage in z-VAD-fmk-treated cells despite cyt-C-GFP release. (F) z-VAD-fmk-insensitive decrease in TMRM uptake in HeLa D98 cells stably expressing cyt-c-GFP. Cells were treated with 3 µM STS in the presence of 200 µM z-VAD-fmk. Similar results were obtained in 14 cells in two separate experiments. (G,H) Simultaneous monitoring of DEVDase activity and TMRM uptake in single MCF-7/Casp-3 cells transiently transfected with the FRET probe CFP-DEVD-YFP and treated with 3 µM STS or 3 µM STS plus 200 µM z-VAD-fmk. The increase in the CFP/YFP ratio indicates the time course of the cleavage of the FRET probe. Similar kinetics of FRET probe cleavage and TMRM uptake could be detected in two independent transient transfection experiments per treatment. Traces in H have different time scales to demonstrate the absence of FRET probe cleavage up to 4 hours after completion of the TMRM fluorescence changes.

View larger version (17K): [in a new window]   Fig. 5. Remodeling of TMRM fluorescence changes in a virtual cell. (A) Remodeling of TMRM fluorescence in a STS-treated cell in the absence of caspase inhibitor z-VAD-fmk. TMRM fluorescence changes were remodeled with a M depolarization of 32 mV and a P depolarization of 10 mV as detected with Dibac4(3). The calculated average intensity of TMRM is plotted in black (scale on the left). The modeling of M and P kinetics is plotted in dark and light gray, respectively (scale on the right). Experimentally determined values from cells treated with 3 µM STS are shown for comparison (open squares). (B) Remodeling of a cell treated with 3 µM STS and 200 µM z-VAD-fmk. TMRM fluorescence changes were remodeled with a M depolarization of 32 mV in the absence of P depolarization. (C) Remodeling of TMRM fluorescence changes in cells treated with 3 µM STS and 5 µM oligomycin. TMRM fluorescence changes were remodeled with a M depolarization of 65 mV and a P depolarization of 10 mV as detected with Dibac4(3) (data not shown). Necrosis was remodeled to occur 83 minutes after the release of cyt-C and was simulated with a total depolarization of both potentials at that time. (D) Experimentally determined values for the steady-state level of TMRM fluorescence intensity after cyt-C-GFP release. The values were calculated using the sigmoidal Boltzmann equation as fit function for the single-cell kinetics of TMRM fluorescence from the experiments presented in Figs 1, 2 and 3. (*P<0.05 compared to STS-treated cells).

View larger version (45K): [in a new window]   Fig. 3. FOF1-ATP-synthase reversal maintains M-cyt-C. (A) Addition of 10 µM FCCP after the release of cyt-C-GFP dissipates TMRM uptake. Cells were treated with 3 µM STS. FCCP was added approximately 2 hours after two cells had released cyt-C-GFP. (B) Total depolarization after the release of the cyt-C-GFP fusion protein in cells treated with 3 µM STS and 5 µM of the FOF1-ATP-synthase inhibitor oligomycin. The cells received 5 µM oligomycin 1 hour after addition of 3 µM STS. Note that mitochondria were not able to retain their TMRM fluorescence after the release of cyt-C-GFP and loss of fluorescence was followed by necrosis. Loss of the diffuse cyt-C-GFP fluorescence owing to plasma membrane rupture is indicated with white arrowheads. (C) Individual traces of two typical cells treated with 3 µM STS and 5 µM oligomycin, demonstrating that mitochondria were not able to retain TMRM following the release of cyt-C-GFP. (D) Mean traces of n=15 cells from three independent experiments were calculated by setting the time of cyt-C-GFP release to zero. (E) Addition of 5 µM oligomycin to cells 40 and 60 minutes after the release of cyt-C-GFP led to a rapid, permanent and total depletion of mitochondrial TMRM fluorescence. Similar results have been obtained in two separate experiments.

View larger version (36K): [in a new window]   Fig. 4. Simultaneous depolarization of mitochondrial and plasma membrane potential during STS-induced apoptosis. (A) Changes in TMRM and Dibac4(3) uptake in a typical MCF-7/Casp-3 cell after and exposure to valinomycin (10 nM, M depolarization) and ouabain (100 µg/ml, P depolarization). Similar traces were recorded in 10 cells in two experiments. (B) Individual traces of the average fluorescence intensity of two typical MCF-7/Casp-3 cells equilibrated with the positively charged TMRM and the negatively charged Dibac4(3) after treatment with 3 µM STS. (C) Mean values of Dibac4(3) and TMRM fluorescence kinetics were calculated by setting the time of onset of decrease in TMRM fluorescence to zero. Data represent 11 individual cells from three independent experiments. (D) Inhibition of caspases protects MCF-7/Casp-3 cells from STS-induced P depolarization. Individual traces of two typical cells treated with 3 µM STS and 200 µM z-VAD-fmk. (E) Mean values of 12 cells treated with 3 µM STS and 200 µM z-VAD-fmk in two independent experiments. (F). Time course of Na+/K+-ATPase ß-subunit degradation during STS-induced apoptosis. Cells were treated with STS and whole cell extracts were analyzed by immunoblotting. (G) Treatment with z-VAD-fmk inhibits the degradation of the Na+/K+-ATPase ß-subunit and the activation of effector caspase-3. Cells were treated with 3 µM STS, 3 µM STS plus 200 µM z-VAD-fmk or vehicle. Whole cell extracts were prepared after 8 hours of treatment and analyzed by immunoblotting.