Adhesive Interactions Figure 6. E-cadherin dynamics during cell–cell collision in a narrow wound. Nontransformed IAR-2 cells and transformed IAR-6-1 cells were stably transfected with GFP-E-cadherin. (A) IAR-2 cells. DIC image shows lateral expansion of the cell-cell contact (arrows). During cell-cell contact formation, GFP-E-cadherin accumulates in a tangential line (arrowheads) at the boundary between two cells (see also Video S8). (B, C, D; see also Video S9) IAR-6-1 cells. (B) During cell-cell contact formation, there is overlapping of lamellae in the area of the contact (arrows). Radial AJs (arrowheads) assemble in the zone of overlapping. (C) Time-lapse images of the same cell-cell contact acquired at 1-min intervals. GFP-E-cadherin initially aggregates into dot-like clusters. A majority of GFP-E-cadherin dots grow and elongate (asterisks). Other AJs can disappear (arrowheads). (D) Because of cell movement, pre-existing AJs (bracket in B) elongate and break (arrowheads). Interval time, 2 min. Bar, 10 mm. doi:10.1371/journal.pone.0008027.g006 of transformed IAR-6-1 cells, 30 mM Y-27632 abolished actin bundles. Radial E-cadherin-based AJs did not assemble at the sites of cell-cell contacts. We saw staining of E-cadherin only in dot-like clusters. Y-27632 (30 mM) destroyed actin bundles and completely prevented the formation of radial AJs in Rat-1 fibroblasts. We were able to visualize only a few dots of N-cadherin at the sites of cell-cell collisions. It is now well-recognized that Rho/ROCK signaling controls the creation of myosin II-driven forces. We examined the effect of the myosin II ATPase inhibitor blebbistatin [28] on the formation of tangential and radial AJs (Figure 8A). Our experiments showed that tangential contacts can assemble without myosin II activity. In the presence of 50 mM blebbistatin, which completely destroyed marginal and straight actin bundles in IAR-2 cells, E-cadherin accumulated in a wavy line oriented along the cell-cell boundary. Blebbistatin (50 mM) abolished actin bundles and prevented the formation of radial AJs in IAR-6-1 cells. In these cells, we revealed only dot-like and fragmented linear junctions at the sites of the cell-cell contacts. In Rat-1 cells, 50 mM blebbistatin had quick and strong effect on cell morphology, the actin cytoskeleton, and AJs PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e8027