Fig. 5. Experimental validation of model predictions and Monte Carlo simulations.

(A) Averaged normalized intensities from nuclear regions of interest (ROIs; white dotted lines) recorded in 21 HeLa wt cells expressing MyrPalm-ELQTDG-mCherry (top image row) and MyrPalm-MTISDS-YFP (bottom image row) probes. (B) Averaged nuclear intensities from 16 HeLa wt cells expressing MyrPalm-ELQTDG-mCherry (top image row) and MyrPalm-PREQDS-YFP (bottom image row) probes. The amount of the prodomain site cleavage was calculated from the trajectories in (A) and (B), and the percentage cleaved when half of the BID probes was cleaved (t_BID,50%) is shown for each graph. Error bars, SDs; scale bars, 10 µm; p55 prod. site, p55 prodomain cleavage site. (C) Averaged nuclear intensities from 39 HeLa wt cells expressing NES-ELQTDG-mCherry probes, 16 HeLa wt cells expressing NES-(p55 prodomain sites)-YFP probes, and 23 cells expressing NES-DEVA-YFP probes, with a control sequence that cannot be cleaved. The amounts of probe cleavage at t_BID,50% for the probe with prodomain cleavage sites and for the DEVA probe are shown in the graph. (D) Enzymatic domain cleavage can occur by a trans mechanism. Averaged normalized intensities from nuclear ROIs recorded in 20 HeLa wt cells expressing the two probes, MyrPalm-ELQTDG-mCherry and MyrPalm-PVETDS-YFP. The amount of the probe cleavage at t_BID,50% for each probe was about the same and is shown in the graph. p55 enz. dom. site, p55 enzymatic domain cleavage site. (E) Model fits to two exemplary CD95-HeLa trajectories, indicated in Fig. 4A, at [CD95L] = 5000 ng/ml. Left graph: model fits to cytosolic and ER probe concentrations; middle graph: fits to the p43 cleavage activities; right graph: fits to the p18 cleavage activities. (F) Cytosolic probe cleavage trajectories from random parameter (Monte Carlo) simulations for CD95-HeLa (left plot) and HeLa wt cells (right plot). Trajectories were obtained by randomly sampling kinetic rate constants (N_samples = 10⁴) from large parameter intervals, temporally normalized by time t_5% when 5% of the probes are cleaved, and normalized in amplitude by the total cytosolic NES-probe concentrations [shaded areas: space between 5% and 95% percentiles (u_5% and u_95%); solid lines: medians].