Direct and indirect inhibition of NMII leads to expansion of lamellipodial overlaps and separation of linear AJs. (A and B) Treatment of cells with 50 µM blebbistatin for 1 h. (A) A field of view with pseudocolored individual cells and cell overlaps. The boxed region is enlarged in B. (B) VE-cadherin immunogold (18 nm, yellow) labels cell–cell contacts but not gap edges. (C–E) Treatment of cells with 50 µM Y27632 for 1 h. (C) VE-cadherin immunogold (18 nm, yellow) at a linear AJ with gaps. Regions outlined in cyan are enlarged in D and E. (D) A preserved linear AJ is evenly labeled with VE-cadherin immunogold. (E) Oval gaps in the linear AJ have VE-cadherin only at remaining cell–cell junctions but not at gap edges. (F) Time-lapse sequences of GFP–VE-cadherin (green) and mCherry-LifeAct (red) at AJs in the course of Y-27632 treatment. Y-27632 was added at time 0. Arrows mark Y-27632–induced intercellular gaps. Bars: (A) 5 µm; (B) 200 nm; (C) 2 µm; (D and E) 500 nm; (F) 10 µm.