Figure 4.
S3D cofilin severs weakly and inclusion of competitors enhances severing activity. A, normalized equilibrium average lengths (Lavg) of Alexa-labeled actin filaments at various WT (black squares) or S3D (white circles) binding densities, n ≥ 200 per condition. Solid black lines though WT and S3D cofilin binding data represent fits of the data to a discrete, site-specific severing model (Equation 1) (14) with fixed cooperativity values of ω = 5 and 47, respectively. The top horizontal dotted line with the value Lavg = 1 denotes average length of bare actin filaments. The dashed black line shows a simulation of average filament lengths (Equation 1) with WT cofilin apparent severing rate constants but with S3D cooperativity (ω = 47), demonstrating that the increased cooperativity alone cannot account for S3D severing deficiency. Error bars, S.E. B, binding competition of phalloidin (black circles) or non-muscle tropomyosin Tpm3.1 (white circles) with S3D for pyrene-labeled actin filaments. Smooth lines though data are fits to the Hill equation and are strictly for visualization purposes. C, equilibrium mean lengths (open squares in boxes) of Alexa-labeled actin filaments that are either bare, half-decorated with S3D cofilin, or partially decorated with S3D cofilin and a competitor (Tm or Ph). Equilibrium distribution of filament lengths (n ≥ 200) is indicated by a 25 or 75% marker (horizontal line) connected by vertical dashed lines. The marker × indicates percentile 1 or 90, whereas the floating marker (heavy horizontal line) represents the maximum or minimum of filament length.