Figure 7.

Experimental validation of the model prediction: Elimination of spatial anisotropies during apoptosis execution. (A) Design of the experimental approach. The release of red fluorescent protein from mitochondria was determined in individual cells. The region of the cell that showed the release first was defined as the near end, whereas the far end represented the side of the cell that the signal travelled to. In parallel, the cleavage of a recombinant effector caspase FRET substrate (CFP-DEVD-YFP) was investigated. Upon cleavage of the FRET probe the blue emission increases, while in the intact probe resonance energy transfer allows for YFP emission upon CFP excitation. The delays between onset of RFP or FRET probe cleavage between near and far ends were determined. (B) Representative traces from a HeLa cervical cancer cell analysed by rapid sampling of apoptotic signalling. The cell was treated with 100 ng/ml TRAIL/1 μg/ml CHX. Mitochondrial permeabilisation was measured by RFP release while effector caspase activation was measured by CFP-DEVD-YFP FRET disruption in regions at the near and far ends of the cell. Black arrows indicate onset of the respective events. The delay in mitochondrial permeabilisation is lost upon FRET substrate cleavage. 4 cells with corresponding results were obtained from n = 4 experiments.