Junction disruption induces myosin II activation. (A) Low-magnification view of the indicated Caco2 cell sheets, which are engaging in wound healing, coimmunostained for ppMLC2 and Abi1. ppMLC2 and Abi are cocondensed in ∼40% of multicellular junctions in E-cad KO cells, examples of which are indicated by arrows, whereas the junctions in wild-type cells do not show such features. front, the front regions of the migrating cell sheet. (B) Coimmunostaining for ppMLC2 and actin in αE-cat KO Caco2 cells. The boxed area was enlarged to 2.4× at the bottom, and ppMLC2 and actin were scanned along the line indicated. (C) Western blots for ppMLC2 in the indicated Caco2 lines. (D) Wild-type or αEcat KO Caco2 cells were incubated with 10 µM blebbistatin for 6 h and double-immunostained for Abi1 and actin. Cells located around the inner to submarginal zones were photographed. (E) Distribution of the Rho-GTP localization sensor GFP-AHPH. Wild-type or αEcat KO Caco2 cells were transiently transfected with GFP-AHPH and then double-immunostained for GFP and ppMLC2. Arrowheads indicate a closed junction in an αE-cat KO Caco2 cell. (F) A wild-type cell transiently transfected with a function-deficient mutant of AHPH (AHPHmut) tagged with GFP. Scale bars, 100 µm for A; 10 µm for B and D–F.