Loss of β3 leads to formation of peripheral adhesions and enhanced migration. (A) Confocal images of WT, β3−/−, or β3-GFP cells plated on FN and costained for (top panels) β3 (FITC or GFP, green) and β1 (Alexa Fluor 568, red) or vinculin (bottom; shown in black and white). Bar, 10 µm. (B) Quantification of focal adhesion length, percentage of peripheral adhesions, and area/cell from confocal images. Adhesions were quantified from at least 100 cells over three independent experiments. Data are shown as mean ± SEM. (C, left) Confocal images of WT and β3−/− plated on CDMs and costained for active β1 (9EG7-FITC, green), paxillin (Alexa Fluor 568, red), and FN (Alexa Fluor 633, blue). Insets show enlarged views of the boxed regions. Bar, 10 µm. (right) The bar graph shows quantification of the percentage of peripheral adhesions from confocal images. Adhesions were quantified from at least 30 cells over three independent experiments. Data are shown as mean ± SEM. (D and E) Rate constants of focal adhesion dynamics in WT, β3−/−, or β3-GFP cells plated on 2D FN (D) or CDM (E). Data were quantified from time-lapse videos of cells expressing GFP- or mCherry-talin. Data are expressed as min⁻¹ ± SEM. Data were quantified from a minimum of 200 adhesions per cell type over four experiments. (F) WT, β3−/−, and β3-GFP cells were fixed and stained for cell surface levels of total or active β1 integrin (MB1.2 or 9EG7, respectively) and analyzed by FACS. Data are presented as the mean percentage of active cell surface integrin as compared with WT cells ± SEM. (G and H) Quantification of migration speed (G) and directional persistence (H) of WT, β3−/−, or β3-GFP cells plated on 2D monolayers of FN or CDM. Data were quantified from >120 cells per matrix type over three independent experiments. Data are shown as mean ± SEM (indicated by error bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001.