VopL/F on the ends of nucleated and preassembled filaments in the presence of profilin. Three-color TIRFM visualization of the addition of 1.5 µM cyan-labeled Mg-ATP-actin monomers (15% Alexa Fluor 647 actin) to preassembled green actin filaments (10% Alexa Fluor 488 actin), in the presence of 0.2 nM 549(red)-SNAP-VopF/L or 549-SNAP-mDia2, with 0, 3.0, or 7.0 µM profilin HsPRFN1. (A) Cartoon of the experiment and potential outcomes. (B) Flow (time zero) of 549-SNAP-VopF in the presence of 7.0 µM profilin. Left, merged time-lapse micrographs (in seconds) of actin filaments. White arrows indicate 549-SNAP-VopF. Bar, 2 µm. Right, kymographs of filament length (y axis; bar, 2 µm) over time (x axis; bar, 10 s) of the corresponding filaments. Top, examples of the predominant filament population, association of 549-SNAP-VopF with the pointed end of nucleated filaments. Bottom, example of 549-SNAP-VopF binding to the barbed end of a preassembled filament. (C) Percentage of 549-SNAP-mDia2 or 549-SNAP-VopL/F associated with nucleated filaments (blue; pointed end for VopL/F, barbed end for mDia2) or preassembled filament barbed ends (green) for different profilin:actin ratios (0:1, 2:1, 5:1; n ≥ 50). (D) Mean association time of 549-SNAP-VopL/F or 549-SNAP-mDia2 with the barbed end of preassembled filaments with a 5:1 profilin:actin ratio. Error bars indicate ± SEM; n ≥ 20 events. (E) Normalized elongation rates of 549-SNAP-mDia2- or 549-SNAP-VopL/F-associated filaments with a 0:1 (purple) or 5:1 (yellow) ratio of profilin/actin. B/P, barbed/pointed end. Error bars indicate ± SEM; n = 20 filaments.