Formin mDia2 and CP bind the opposite end of VopL/F-nucleated filaments. Three-color TIRFM of the assembly of 1.5 µM Mg-ATP-actin (15% Oregon green actin) assembled with 0.2 nM 549 (red)-SNAP-VopL/F and either 2 nM 647 (cyan)-SNAP-mDia2 or 2 nM 647-SNAP-MmCP. (A–D) Merged time-lapse micrographs (in seconds) of individual filaments. Bars, 2 µm. Red and blue arrowheads indicate 549-SNAP-VopL/F and 647-SNAP-mDia2/647-SNAP-MmCP. (E) Percentage of filaments in which 549-SNAP-VopL (gray) or 549-SNAP-VopF (gold) are on the same or opposite end as mDia2 or MmCP. n = 15.