Mechanisms regulating the interaction between VASP and F-actin. (A) Localization of single Cy3-VASP tetramers binding to phalloidin-stabilized Alexa Fluor 488 actin filaments. Intensity differences are due to photobleaching of Cy3-VASP. Bottom panel shows the maximum intensity projection of 0.25 nM Cy3-VASP (500 frames) bound to F-actin. Bar, 5 µm. (B) Multi-step photobleaching of a Cy3-VASP tetramer (τ₁ = 11.5 ± 0.07 s for a single Cy3 fluorophore, n = 229 molecules; not depicted). (C) Kymograph of 0.25 nM tetrameric Cy3-VASP bound to an actin filament in TIRF buffer plus oxygen scavenger (see Materials and methods). Vertical bar, 2 s; horizontal bar, 5 µm. (D and E) Dwell times for Cy3-VASP binding to F-actin. (D) Double exponential fit of 1-cumulative frequency (CF) for all binding modes. (E) Single exponential fit of molecules exhibiting diffusive binding mode. (F) Localization of 0.25 nM Cy3-VASP, wild-type, and mutants, binding to phalloidin-stabilized Alexa Fluor 488 actin filaments. Bar, 5 µm. (G) Kymographs of 0.25 nM Cy3-VASP, GAB mutants (Cy3-VASP^{L226A,I230A,L235A} and Cy3-VASP^R236E,K237E), and Cy3-VASP plus 2 µM Mg-ATP-actin (+10 µM Latrunculin B) binding to actin filaments. Vertical bar, 2 s; horizontal bar, 5 µm. (H) Double exponential fits of 1-cumulative frequency for GAB mutants and Cy3-VASP plus 2 µM Mg-ATP-actin (+10 µM LatB). Percentage of molecules with each characteristic dwell time are printed in parentheses.