In the absence of actin monomers, VopL/F bind preassembled filament ends. Two-color TIRFM visualization of the addition of 0.6 nM 549-SNAP-VopL/F to preassembled actin filaments (10% Alexa Fluor 488 actin) in the absence and presence of 1.5 µM Mg-ATP-actin (10% Alexa Fluor 488 actin) monomers. (A–D) Merged time-lapse micrographs (in seconds) of filaments upon flow-in (time zero) of 549-SNAP-VopL or formin 549-SNAP-mDia2 with and without actin monomers, as indicated. White arrows mark VopL and mDia2. Bars, 5 µm. (E) Length distribution of bound filaments in the presence (red) and absence (black) of actin monomers. Blue lines indicate mean filament length. n ≥ 50 events. (F) Percentage of filaments that are bound on one (purple) or two (orange) ends. Error bars indicate SD; n ≥ 50. (G) Mean association time of 549-SNAP-VopL/F on actin filament ends in the absence of monomer. Error bars indicate SEM; n ≥ 100 events. (H) Percentage of 549-SNAP-VopL binding events that are on the barbed (B) or pointed (P) end (gray columns), and mean association time of 549-SNAP-VopL on either the barbed or pointed end (red columns). Error bars indicate SEM; n ≥ 100 events.