FIGURE 3.
Direct real-time observation of actin filament barbed end capping by vinculin tail. Conditions were as follows. 0.6 μm MgATP-G-actin (10% Alexa488-labeled) was polymerized in a flow chamber coated with NEM-myosin in 5 mm Tris, pH 7.8, 100 mm KCl, 0.2 mm ATP, 0.1 mm CaCl2, 1 mm MgCl2, 10 mm DTT, 0.2 mm 1,4-diazabicyclo(2,2,2)-octane, 0.2% methyl-cellulose. A, time lapse of the elongation of single actin filaments in the absence (top) or presence of 0.5 μm Vt (bottom). See also the corresponding supplemental Movies 1 and 2. Scale bar, 5 μm. B, kymographs of the single actin filaments shown in A. Actin filament fluorescence intensity was measured along its length (vertical axis) for each frame of the time lapse (horizontal axis). The filaments are oriented with the non-growing pointed end to the bottom and the growing barbed end to the top. In contrast with the continuous elongation observed for control filaments (top), barbed end growth was occasionally interrupted by pauses in the presence of Vt (bottom). C–F, quantification of the association and dissociation rate constants that characterized the interaction between Vt and actin filament barbed ends using TIRF microscopy. C and E, periods of growth and arrest were measured on kymographs corresponding to the elongation of actin filaments in the presence of 0.6 μm MgATP-G-actin (10% Alexa488-labeled) and 0.5 μm Vt. The exponential fit of the time distribution of the periods of growth (C) and arrest (E) gave the association and dissociation rates, respectively. The association rates (D) and dissociation rates (F) were plotted versus increasing concentrations of Vt. Error bars, S.E. G, measurement of the dissociation rate of vinculin from actin filament barbed ends after dilution by using a fluorescence bulk assay. A reaction in which spectrin-actin seeds have been pre-elongated in the presence of 2 μm actin and 2 μm Vt was diluted 20 times in a solution containing 2 μm MgATP-G-actin, 10% pyrenyl-labeled (red line). As a control, we performed the dilution of the same reaction, but we let Vt dissociate for 15 min before adding G-actin (blue line). H, the subtraction of the dissociation kinetics from the linear fit of the control kinetics shown in G gave the kinetics of dissociation of Vt from the barbed end. The exponential fit of the curve gave a dissociation rate constant k− = 0.014 s−1. All of the experiments were performed three times with the same results. a.u., arbitrary units.