Fig. 3: The CP β tentacle is dispensable for capping of single actin filaments.

A Overview of the location of CP mutations. Colors are as in Fig. 2. Insets highlight the region of the β tentacle deleted (green), and the color scale on the actin surface depicts increasing hydrophobicity from gray to white to yellow. B Scheme of actin filament barbed end re-growth after dissociation of CP via TIRFM (top). Single capped filaments were visualized by Cy5-UTRN261N (10 nM, green) in the presence of profilin-actin (2 μM) and myotrophin/V1 (20 nM) after CP washout. CP dissociation leads to growth of the filament visualized in the kymograph (bottom). Over 50 such kymographs were analyzed per experiment, which was repeated independently three times with similar results. C TIRFM time lapse images of filaments following the washout of either wt CP or mutants, as indicated. Conditions are as in B. Filled red arrowheads indicate the positions of barbed ends at the moment of CP dissociation in each case, and empty red arrowheads follow the positions of (re-) growing barbed ends. The experiment was repeated three times with similar results. D Time traces of the fraction of uncapped barbed ends after washout of either wt CP or mutants, as indicated, at t = 0. Dissociation rate constants (k_off) were derived from mono-exponential fits (see Methods). Source data are provided as a Source Data file. E Scheme of single molecule TIRFM assays (top). Individual surface-attached filaments visualized by Cy5-UTRN261N (10 nM, green) grow from profilin-actin (2 μM) in the presence of TMR-CP (4 nM, magenta). CP association results in the termination of filament growth as visualized in the kymograph (bottom). Over 50 such kymographs were analyzed per experiment, which was repeated independently three times with similar results. F TIRFM time lapse images of filament elongation (green) in presence of wt or mutant capping protein (magenta) as indicated under conditions as in E. Empty yellow arrowheads indicate initial positions of barbed ends and filled yellow arrowheads the positions at the moment of CP association. The experiment was repeated three times for each CP concentration with similar results. G Observed reaction rates (k_obs) for capping protein wt and mutants as a function of CP concentration (Supplementary Fig. 4). The observed reaction rates (k_obs) were derived from the mean of N = 3 independent experiments for each CP concentration. Plotted data also contains the independently measured dissociation rates as y-intercepts. Association rate constants (k_on) are calculated from linear fits to the data with the y-intercepts fixed to the measured off-rates. Error bars are SD. Source data are provided as a Source Data file. H Summary table of dissociation (k_off), association rate constants (k_on) and equilibrium dissociation constants (K_D, calculated from the former rate constants) for the interaction of CP (wt or mutants as indicated) with actin filament barbed ends. Errors are SD of the mean values from 3 independent experiments (k_off) or the SEM of the linear fit (k_on).