Mechanism for VASP barbed end capture. (A) Kymograph showing Cy3-VASP barbed end association in the absence (left) or presence (right) of 2 µM Mg-ATP-actin (+10 µM Latrunculin B). Vertical bar, 1 s; horizontal bar, 2.5 µm. (B) Barbed end localization of single Cy3-VASP tetramer (arrowhead) in the presence of 2 µM Mg-ATP-actin/LatB. Bar, 2.5 µm. (C) Binding probability for Cy3-VASP at different filament positions in the presence or absence of 2 µM Mg-ATP-actin/LatB and/or 5 nM MmCP. V, VASP; A, actin; CP, capping protein. Pixel position 1 is the apparent barbed end; positions 2–10 are actin filament sides. For experiments including MmCP, filaments were capped for 5 min before adding Cy3-VASP and 5 nM MmCP (≥200 events scored per condition). (D) Dwell times for 0.25 nM Cy3-VASP binding to the apparent barbed end versus the sides of actin filaments in the presence of 2 µM actin/LatB ± 5 nM MmCP. (E) Life history of a single actin filament elongating in the presence of 0.75 µM actin (30% Alexa Fluor 488). Bar, 10 µm. (F) The binding frequency of 0.25 nM Cy3-VASP (n = 1,045 molecules) is independent of the probability of the ADP-P_i nucleotide state at each position. (G) Single molecule dwell times for 0.25 nM Cy3-VASP binding to regions of F-actin with p(ADP-P_i) ≥ 0.5 or p(ADP-P_i) ≤ 0.5 were fit to a single exponential (p(ADP-P_i) ≥ 0.5, τ₁ = 0.93 ± 0.01 s, n = 235; p(ADP-P_i) ≤ 0.5, τ₁ = 1.0 ± 0.01 s, n = 1,135). (H) Localization of 2.5 nM Cy3-VASP bound to an ADP-P_i/ADP actin filament (3–17 min old). Bar, 10 µm. (I) Intensity profile of Cy3-VASP bound to actin filament in H, overlaid with probability distribution of the ADP-P_i nucleotide state. The probability distribution of each nucleotide state was solved numerically in MatLab (Fig. S3 E).