Calpain 2–mediated proteolysis of cortactin is not necessary for invadopodia formation but mediates disassembly of cortactin from invadopodia. (A) Cell lysates from MTLn3 cells stably expressing control or cortactin siRNA were analyzed by Western blotting and probed for cortactin and ERK as a loading control. MTLn3 cells expressing control or cortactin siRNA were cultured on gelatin/FN-coated coverslips and stained with anti–p34-Arc antibody and rhodamine phalloidin. Quantification of invadopodia is expressed as the mean number of invadopodia per cell. *, P < 0.05 compared with control cells. (B) Wild-type (WT) or calpain-resistant (D28) GFP-cortactin was transiently transfected into MTLn3 control and cortactin siRNA lines, cultured on gelatin/FN-coated coverslips, and stained with rhodamine phalloidin. Quantification of invadopodia is expressed as the mean number of invadopodia per cell. *, P < 0.05 compared with control cells. (C) GFP-cortactin WT or GFP-cortactin D28 expressing cells were plated on FN-coated glass-bottomed dishes and analyzed by time-lapse microscopy. Time-lapse montage of cortactin-deficient MTLn3 cells expressing GFP-cortactin WT or GFP-cortactin D28 demonstrates that GFP-cortactin D28 persists at invadopodia for longer durations than GFP-cortactin WT. Duration measurements and assembly and disassembly rates are shown. Arrows indicate representative invadopodia. Asterisk indicates statistical significance based on t test (P < 0.01). See Videos 5 and 6 . Data are means ± SEM from three independent experiments. Bars, 10 μm.