ZO-1 down-regulation reduces endothelial cell–cell tension. (A and B) Cells were transfected with siRNAs and, after 2 d, with a VE-cadherin–based FRET tension sensor containing (TS) or lacking (Tminus) the β-catenin binding site. FRET activity was then imaged by gain of donor fluorescence after acceptor bleaching from confluent monolayers. (A) FRET efficiency maps and images taken from venus fluorescent protein before bleaching. (B) Images were quantified by calculating the FRET efficiencies at cell–cell contacts. The values were then normalized to the FRET efficiency obtained with the tail-minus construct, which does not sense tension and hence provides the FRET signals that can maximally be expected (shown are means ± 1 SD [error bars]; n = 12). (C–E) Cells expressing GFP–α-catenin were plated and transfected with siRNAs as in A. The cells were then analyzed by ablating single cells (marked with an asterisk) within the monolayer with a laser and recording the movement of cell–cell contacts in the GFP channel for 1 min. The images in C show overlays of frames taken before ablation in red, after 30 s in green, and after 45 s in blue (see also Videos 1 and 2). The increase in the surface area of the ablated cells and the contraction of the neighboring cells were then analyzed (D and E, shown are means ± 1 SD [error bars]; n = 11). Bars, 20 µm.