MLC localization increases at heterotypic contacts. (A) Example images from live imaging experiments (Video 5) of EPHB2 homotypic conditions at low density. EPHB2-GFP (green) cells were mixed with EPHB2-GFP-MLC-Cherry (magenta). Line scan analysis of cell pair at various time points shows no change in MLC localization upon contact. (B) Example images from live imaging experiments (Video 6) of EPHRIN-B1 homotypic conditions at low density. EPHRIN-B1-MLC-Cherry (magenta). The asterisk indicates the analyzed cell. Line scan analysis of the cell pair at various time points shows no change in MLC localization upon contact. (C) Example images from live imaging experiments (Video 7) of heterotypic conditions at low density. EPHB2-GFP-MLC-Cherry (green) cells were mixed with EPHRIN-B1-MLC-Cherry (magenta) cells. Yellow arrows at t = 30 min indicate localized increase in MLC. Line scan analysis of the cell pair at various time points shows that MLC localized to cell contact in EPHB2 cells upon contact. (D) Example images from live imaging experiments (Video 8) of heterotypic, with unclustered-EPHRIN-B1-Fc, conditions at low density. EPHB2-GFP-MLC-Cherry (green) cells were mixed with EPHRIN-B1-MLC-Cherry (magenta) cells; unclustered-EPHRIN-B1-Fc was added to prevent signaling. Line scan analysis of the cell pair at various time points shows no change in MLC localization upon contact. (E) Quantification of the change in MLC fluorescence from before contact (t = –15 min) to after contact (t = 30 min). EPHB2 cells show an increase in MLC localization at heterotypic interface upon contact with EPHRIN-B1–expressing cell. This effect is blocked by the addition of unclustered-EPHRIN-B1-Fc. Column heights represent mean, and error bars represent SD. White arrow at t = 0 min indicates point of contact. Toward indicates toward contact, while away indicates away from contact. Scale bars, 20 µm.