EphA2 processing by MT1-MMP enhances cell repulsion. (A) Representative time-lapse microscopy images of control, EphA2, or MT1-MMP knockdown MDA-MB-231 cells at the indicated time points. Black arrows indicate the moving direction of cells marked with black asterisks toward the cells marked with white or blue. White arrows indicate the moving direction of both cells after collision. Representative track plots are shown as colored lines (right; n = 25 cells, 5 h). See Videos 2–4. Bars: (time-lapse panels) 10 µm; (tracking panels) 300 µm. (B) Control, EphA2-expressing, and EphA2-D/I–expressing cells were subjected to immunoblotting as indicated (n = 3). Ponceau red staining served as a loading control. The arrowhead indicates ∼60-kD EphA2 fragments. (C) Control or MT1-MMP knockdown cells were followed by time-lapse microscopy for 12 h. The cells coexpressing GFP and EphA2 or EphA2-D/I were tracked. Pie charts show the percentages of non-detaching and detaching cells after collision (90 min). (D) Contact acceleration indices (Cx) of free-moving and colliding cells (see Fig. S2 C). P-values were determined with a Mann–Whitney U test; (E) Representation of velocity vectors of free-moving and colliding cells in compass plots. The heavy red line represents the scaled displacement of all cells before collision, and thin black lines show the scaled displacement of each cell after collision. The thin red line is a reference line that marks the angle of 90° relative to the displacement before contact (heavy red line). (F) Representative time-lapse microscopy images at the indicated time points. White arrows indicate the moving direction of cells marked with white asterisks, and red arrows indicate a newly formed leading edge. See Videos 5–7. Bar, 10 µm.