ΔAB perturbs vinculin binding to F-actin. (A) Point mutations introduced into full-length vinculin to analyze the role of vinculin–F-actin binding. PA, N773A/E775A point mutations in the vinculin head domain resulting in partial activation of WT vinculin; ΔAB, I997A mutation in the vinculin tail domain perturbing F-actin binding/bundling of WT vinculin; PA-ΔAB, N773A/E775A/I997A point mutations resulting in partial activation of ΔAB vinculin. cDNAs were expressed as EGFP fusion constructs. (B) SDS-PAGE of supernatant and pellet after high-speed cosedimentation of WT and ΔAB vinculin with F-actin at indicated concentrations in the absence or presence of activating peptide IpaA. S, supernatant; P, pellet. (C) Densitometric quantification of F-actin cosedimentations shown in B. Data were fit for single site, saturation binding. Error bars show SEM. (D) Fluorescence micrographs of actin filaments polymerized in the absence or presence of IpaA and WT or ΔAB vinculin. Bar, 10 µm. (E) Bar diagram of F-actin bundling induced by WT and ΔAB vinculin in the absence or presence of IpaA; n = 1 (WT – IpaA), n = 1 (WT + IpaA), n = 3 (ΔAB – IpaA), and n = 3 (ΔAB + IpaA). Error bars show standard deviation. (F) SDS-PAGE of supernatant (S) and pellet (P) after high-speed cosedimentation of WT, PA, and PA-ΔAB vinculin with actin at indicated concentrations in the absence or presence of IpaA. (G) Densitometric quantification of F-actin cosedimentations shown in F. Data were fit for single site, saturation binding. Error bars show SEM.