Fig. 4. The effective diffusion coefficient of AF488-BSA and apparent activation rate constant of caspase 3/7 decrease exponentially with cytoplasmic concentration.
a Representative fluorescence correlation spectroscopy (FCS) autocorrelation functions for AF488-BSA in extracts with different cytoplasmic concentrations. is the autocorrelation function and is the time delay. b Effective diffusion coefficient of AF488-BSA at different cytoplasmic concentrations. Effective diffusion coefficients were calculated by fitting the autocorrelation data from FCS measurements to a Brownian diffusion model. A 60 s fluorescence intensity time course was registered for each FCS measurement. Means ± 90% CI calculated from 3 measurements (n = 1) are shown. Solid black curve is an exponential curve fitted to the means. c (Z-DEVD)2-R110 cleavage kinetics can be monitored as apoptotic trigger waves sweep through a tube of extract. In this example, fluorescence from cleaved (Z-DEVD)2-R110 at 11 positions (left) are shown on the right. Cleaved (Z-DEVD)2-R110 concentration was inferred from fluorescence. d (Z-DEVD)2-R110 cleavage kinetics in a kymograph (upper) can be represented as a series of traces (lower). e Traces shown in (d) were aligned by time (upper) and the model for caspase 3/7 activation and (Z-DEVD)2-R110 cleavage was fitted to the data. Individual fitted traces are shown in the bottom panel. f The effective positive feedback rate constant at different cytoplasmic concentrations was extracted from the fitted model. Shown are means ± S.E.M. compiled from the same 9 experiments as the ones shown in Fig. 2d. The black solid curve is an exponential curve fitted to the means. Source data are provided as a Source Data file.