Regulation of cell migration speed by VASP phosphorylation. (A) Rate constants of focal adhesion dynamics in WT and β3−/− cells or β3−/− cells cotransfected with S153D VASP-mCherry. Data are quantified from time-lapse videos of cells expressing GFP-talin. Data are expressed as min−1 ± SEM. Data were quantified from a minimum of 200 adhesions per condition over three experiments. (B) WT or β3−/− cells were mock transfected or transfected with S153A or S153D VASP-mCherry, plated onto FN-coated plates, and imaged using phase-contrast time-lapse microscopy over 16 h. Frames were taken every 10 min. Cells were tracked and migration speed and directional persistence were quantified as in Fig. 1. Data were quantified from >75 cells per condition over four independent experiments. Data are shown as mean ± SEM (indicated by error bars). (C) Analysis of intensity levels of 9EG7 (active β1) in cells expressing specified constructs. Example images of 9EG7 staining are shown underneath the graph for each sample type analyzed. Bars, 10 µm. 10 Error bars indicate ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Proposed model of β3 regulation of β1 activation through control of VASP, RIAM, and talin. In normal fibroblasts (left), the β3 is located predominantly at the periphery of the cell, where it acts to maintain a local phosphorylated pool of VASP. In the absence of β3 (right), VASP is predominantly dephosphorylated at S153 (or S157), leading to enhanced VASP binding to RIAM. This results in increased RIAM association with talin and enhanced talin binding to β1 integrin, which leads to increased activation of β1 at peripheral adhesion sites, increased cell protrusion, and faster, less persistent cell migration.