Figure 6.
Glycolysis stabilises ΔΨm subsequent to cyt-c release in the absence of active caspases. (A) Modelled ΔΨm repolarisation via higher glycolytic ATP and increased ATP synthase reversal. Cells were assumed to retain 0.1% respiration-accessible cyt-c after release (which was associated with ATP synthase reversal) and different cytosolic ATP levels were considered to be established by glycolysis (calculations for ATP between 80–99%). Elevated cytosolic ATP led to higher ATP synthase reversal (left panel) causing ΔΨm increase (right panel) over almost the entire range. (B) ΔΨm Repolarisation via higher glycolytic ATP and reduced ATP consumption. Model is presented as in Figure 6a, yet retaining 5% respiration-accessible cyt-c to preserve forward ATP synthase activity after cyt-c release. ATP derived from increased glycolysis led to a reduction in ATP synthase activity (left panel) until it has ceased and thus resulted in less proton influx into the mitochondrial matrix (left panel). As a consequence, decreased proton consumption by ATP synthesis resulted in a partial ΔΨm repolarisation. (C–E) HeLa cyt-c–GFP cells incubated with 2 mM sodium pyruvate in the absence of glucose and exposed to STS in the presence of zVAD-fmk. Following cyt-c-induced mitochondrial depolarisation, 25 mM glucose was added to the medium. (C) Representative traces of a cell with cyt-c–GFP release and recovery of ΔΨm measured by TMRM. (D) Comparison of the average TMRM fluorescence in cell populations subsequent to cyt-c release before and 30 min after addition of 25 mM glucose to medium without glucose supply. The significantly higher TMRM fluorescence in the latter scenario indicates a partial ΔΨm repolarisation (196 cells out of three experiments). (E) Cyt-c–GFP and TMRM fluorescence as well as DIC channel images during depolarisation and recovery for a typical cell. Scale bar, 10 μm. Source data is available for this figure at www.nature.com/msb.
