VopL/F nucleate and then remain briefly associated with the pointed end of an actin filament. (A) Top, domain organization of VopL/F. Orange, proline-rich region (P); blue, WH2 domain (W); green, VCD dimerization domain. Bottom, VopL/F constructs used in this study with SNAP tag (red) for labeling. (B–J) Slow acquisition (every 2 s, B–D) and rapid acquisition (every second, E–J) two-color TIRFM of the assembly of 1.5 µM Mg-ATP-actin (15% Oregon green actin) with 0.2 nM 549(red)-SNAP-VopL/F. (B and C) Length of individual control (dashed black), 549-SNAP-VopL–associated (B, solid red), or 549-SNAP-VopF-associated (C, solid red) filaments over time (n ≥ 20). (D) Kaplan–Meier curves representing the mean residence time of 549-SNAP-VopL/F on actin filaments observed (VopL_Obs, VopF_Obs) or assumed to have been associated because of nucleation (VopL_Calc, VopF_Calc). Error bars indicate 95% CI; n ≥ 90 events. (E and F, left) Merged time-lapse micrographs (in seconds) of individual filaments. White arrowheads and open circles indicate bright and dim filament ends. White arrows indicate 549-SNAP-VopL/F. (E and F, right) Merged kymographs of filament length (y axis; bar, 1 µm) over time (x axis; bar, 10 s) of the corresponding filaments. Bars, 2 μm. (G–J) Linescans of the normalized fluorescence intensity of individual actin filaments measured from their bright to dim (bleached) ends. Red dots indicate position of 549-SNAP-VopL/F or 549-SNAP-mDia2 on the filament traces, and shaded red regions indicate where 100% of VopL/F or mDia2 are bound to the filaments (n ≥ 75).