Figure 4.
Model prediction: ATP synthase reversal stabilises ΔΨm via ATP consumption. (A) ATP synthase for the modelled cell in Figure 3 (A, B) is stably reversed from ∼10 min after the onset of cyt-c release in the presence (blue solid line) or absence (red dashed line) of caspase-3 feedback to complex I/II. (B) Inhibition of ATP synthase activity in the reversed mode according to the model in (A; and Figure 3) at time 30 min after onset of cyt-c release leads to a further depolarisation of ΔΨm, from −76 to −26 mV (model with caspase-3 cleavage of complex I/II) and from −88 to −65 mV (model without caspase-3 feedback to complex I/II). (C, D) Representative single-cell microscopic imaging of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and 100 μM caspase inhibitor zVAD-fmk 1 h prior to exposure to 3 μM staurosporine (STS). Five μM oligomycin was added as indicated. See text for further details on heterogeneous experimental outcome. (C) Fluorescent images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM of two cells. Cell 1 is shown in (C). (E) Fractions of the cell population with reverse, forward or no ATP synthase activity detected by oligomycin responsiveness (237 cells, n=6 experiments). (F) ATP synthase proton flux before and after cyt-c release (t=10 min) according to the model in (A), with retention of 0.1% up to 2% respiration-accessible cyt-c. Calculations indicate a stable reversal of ATP synthase for <0.7% (blue regions) and ATP synthase in forward mode (light green to yellow) for more than 1.2% remaining respiration-accessible cyt-c. Source data is available for this figure at www.nature.com/msb.
