Inhibition of EphB2 clustering blocks repulsion. (A and B) Co-culture assay of HeLa cells coexpressing wtEphB2-3FKBP-YFP and a membrane-tethered FKBP-associated protein (myr-FRB-mCherry) with HeLa cells expressing wild-type ephrinB2-CFP in the absence (A) or presence (B) of heterodimerizer AP21967. Red box in BF images at starting time point indicates the regions highlighted in the high power images (A’ and B’). EphB2 clusters (red arrowheads) in the contact region of the two cells are turned over in control condition, while remaining static in the presence of the heterodimerizer. Orange arrowheads indicate colocalization of YFP-EphB2 and CFP-ephrinB2. In the presence of heterodimerizer, myr-FRB-mCherry coclusters with EphB2 (B’). Black dotted lines in first and last BF images indicate original position of EphB2+ cell. Purple arrows in BF images indicate the direction of retraction movements of the EphB2+ cell (first stimulated from below and later from the right) away from two ephrinB2+ cells. Red arrow indicates the direction the EphB2 cells moves into. The letter d indicates distance between cells as compared with their initial positions. Red dotted line in last BF image indicates final position of EphB2+ cell. In the presence of the heterodimerizer, adhesion cables form between cells (B’) and retraction is blocked (B). Bars: (A and B) 20 µm; (A’ and B’) 10 µm; n.d., image not taken. (C) Quantification of cell repulsion of EphB2-cells. At time point 0, EphB2+ cell position was set to 0 (as indicated in A) and was used as a reference for the following time points. Distance to reference border is plotted for each time point. Movement in negative micrometers reflects movement away from the ephrin+ cell. Each line represents a separate cell pair from n = 2 different time-lapse experiments. Quantification was done blindly with reference to heterodimerizer stimulation.