EphA2 cleavage promotes single-cell invasion. (A) MDA-MB-231 cells stably overexpressing EphA2, EphA2-D/I, EphA2-G/R, and the kinase activity–deficient EphA2-KD and EphA2-KD-D/I proteins were embedded in 3D collagen and allowed to grow for 4 d. Confocal micrographs show EphA2 and filamentous actin (phalloidin) in representative cell colonies. Boxes indicate the areas included as magnified insets on the top panel. CD44 was used as a cell-surface marker. Arrowheads indicate intracellular EphA2. Bars: (top two rows) 50 µm; (bottom two rows) 200 µm. (B–D) Quantitative assessment of single-cell invasion (B), cell shape (C), and EphA2 localization (D; error bars indicate mean ± SEM; three collagen preparations per cell). *, P < 0.05, Mann–Whitney U test. (E) EphA2-D/I increases RhoA activity within 3D collagen and in 2D. Soluble lysates from 3D and 2D cultures of EphA2 or the mutant protein–expressing cells were incubated with Rhotekin RBD-GST–conjugated agarose beads followed by immunoblotting as indicated (n = 4). Phosphorylated myosin light chain (pMLC) was detected from total cell extracts (n = 3). Normalized mean values of RhoA-GTP with total RhoA and pMLC with GAPDH are indicated below each blot.