Eph/ephrin-mediated cell-cell repulsion, but not adhesion, leads to ephrin-A5 cleavage by a metalloprotease. Alexa⁵⁴⁶ephrin-A5-Fc–coated beads were added to cultures of EphA3/HEK293 cells (A), EphA3/HEK293 cells treated (4 hours) with the metalloprotease-inhibitor 1,10-O-phenanthroline (B), or LK63 cells (C). Cells were imaged by time-lapse confocal fluorescence microscopy to illustrate cleavage and internalization of Alexa⁵⁴⁶ephrin-A5. Representative images at indicated times (minutes) after addition of ephrin-coated beads are shown. In panel A, cleaved Alexa⁵⁴⁶ephrin-A5 outlines the cell perimeter, and at later time points also labels cytosolic compartments. Phenanthroline-treated EphA3/HEK293 (B) and LK63 cells (C), lacking cleaved Alexa⁵⁴⁶ephrin-A5, are not visible by fluorescence microscopy, and merged bright-field (gray) and fluorescence (red pseudocolor) images are shown to illustrate ephrin-A5-Fc–coated beads in relation to the cells. Corresponding videos for EphA3/293T and LK63 cells are provided as Video S3 and Video S4, respectively. Scale bars represent 10 μm. (D) Anti-EphA3 immunoprecipitates from nonstimulated (−) or ephrin-A5-Fc–stimulated (+) LK63 or EphA3/293T cells were analyzed by Western blot with anti-ADAM10 and anti-EphA3 antibodies as indicated. p and m indicate the pro (unprocessed) and mature (processed) forms of ADAM10.