Vinculin establishes a lamellipodium–lamellum border. (A) Cortactin immunofluorescence (purple) and Alexa Fluor 488–phalloidin staining (green) of control and Vcl-KO MEF. Note wider lamellipodium in Vcl-KO (asterisk) and diffuse lamellipodium–lamellum border (arrowhead) compared to control MEF (arrow). Bar, 5 µm. (B) pS19MLC2 immunofluorescence (purple) and Alexa Fluor 488–phalloidin staining (green) of control and Vcl-KO MEF. Note low amount of pS19MLC2 in the distal lamellum of control (asterisk) and abundant pS19MLC2 in the distal lamellum of Vcl-KO (arrowhead) MEF. Bar, 10 µm. (C) pS19MLC2 and Alexa Fluor 488–phalloidin fluorescence intensity distribution along line scans perpendicular to the leading edge (mean of 25 cells/condition). (D) Cortactin fluorescence intensity distribution along line scans, placed perpendicularly to the cell edge through the lamellipodium (mean of 60 scans/condition). Width of the distribution at half maximal intensity (I_max/2) indicated. (E) Box and whisker plot of lamellipodium width at I_max/2 of cortactin line scans, placed through the lamellipodium at 15–20-µm intervals along the leading edge; n = 219 (control) and 243 (Vcl-KO) scans of 25 cells/genotype; means indicated; *, P < 0.0001, Mann-Whitney U test.