Cortactin tyrosine phosphorylation and cofilin are required for invadopodia elongation. (A) Representative 3D reconstructions of the 1-µm Transwell experiment. Data are based on three or more independent experiments. (B) The number of protrusive structures crossing to the bottom of the membrane (>12 µm) normalized to the cell area on top of the membrane is shown (>10 fields per group, n = 4; ***, P < 0.0001). (C) Quantification of cell protrusion through a 1-µm Transwell without Matrigel. The GM 6001 inhibitor was used to show that cell protrusion was independent of degradation (>10 fields/group, n = 4; ***, P < 0.0001). Cortactin tyrosine phosphorylation regulates Cofilin interaction in invadopodia. (D) Representative cofilin–cortactin FRET efficiency images of cells expressing either WT or 3YF cortactin. The white circle indicates the bleached spot. The top right insets show close-ups of the original images (indicated by the boxed regions; n = 5). (E) Quantification of cofilin–cortactin FRET/donor at mature invadopodia and precursors in MDA-MB-231 cell lines expressing WT or 3YF cortactin (endogenous cortactin KD). Light gray bars represent FRET on mature invadopodia and dark gray bars represent FRET on invadopodia precursors (*, P < 0.002; n = 5, >30 invadopodia/group). Error bars indicate SEM.