Inhibition of cell migration by blocking cortactin tyrosine phosphorylation. Tracks of live-cell recordings. Bar, 50 μm. The migration of HT1080 cells overexpressing EGFP (GFP), GFP-tagged cortactin (Cttn), the phosphorylation-incompetent cortactin mutant [Cttn(Y/F)], the cortactin phosphorylation mimic mutant [Cttn(Y/E)] or their N-terminal truncated forms [CT, CT(Y/F), CT(Y/E)] were tracked. The inserts are EGFP fluorescence picture of cells taken at 360 min. (B) Plot of migration speed. The numbers are the EGFP positive cells plotted. The statistical analysis was made against the results of cells overexpressing EGFP. *P < 0.05; ***P < 0.001. (C) FAK and its autophosphorylation in cells overexpressing N-terminal truncated cortactin mutants. FAK and its autophosphorylation at Tyr397 in HT1080 cells overexpressing EGFP or GFP-tagged cortactin C-terminus mutants were analyzed by western blotting. (D) Centrosome orientation in migrating cells expressing GFP-tagged cortactin C-terminus mutants. HT1080 cells transfected with vector expressing GFP protein (EGFP), GFP-tagged cortactin C-terminus (GFP-CT), phosphorylation-incompetent C-terminus (Y421/466/475/482F) [GFP-CT(Y/F)] or phosphorylation mimicking C-terminus (Y421/466/475/482E) [GFP-CT(Y/E)] were subjected to wound-healing analysis. The centrosome (stained with anti-pericentrin antibody) in the GFP-positive cell at the migration front was analyzed. Cells with centrosomes located within 120° of the migration direction were counted as polarized, and 200 cells were plotted for each cortactin mutant.