Adhesive Interactions Figure 5. E-cadherin dynamics at the sites of cell–cell contacts. Nontransformed IAR-2 cells and transformed IAR-6-1 cells were stably transfected with GFP-E-cadherin. (A) In IAR-2 cells, GFP-E-cadherin accumulates in the zones of cell-cell interactions, forming stable tangential AJs (see also Video S6). (B) IAR-6-1 cells form lamellipodia along their entire edges. When a protrusion contacts another cell, E-cadherin begins to aggregate in punctate AJs in the zone of contact (arrows). AJs can grow, rearrange, and relocate (asterisks). The disruption of AJs results in detachment of one cell from another (arrowheads) (see also Video S7). Bar, 10 mm. doi:10.1371/journal.pone.0008027.g005 dot-like clusters of N-cadherin were seen at the site of the contact between C3-loaded Rat-1 fibroblasts. These cells failed to form radial AJs. Thus, Rho activity is required for the formation of tangential and radial AJs. Effect of the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin on the formation of AJs To assess the impact of Rho effectors in the establishment of AJs of different types, we first compared the effect of the specific ROCK inhibitor Y-27632 on the formation of tangential AJs in nontransformed IAR-2 cells and of radial AJs in transformed IAR- 6-1 cells and Rat-1 fibroblasts (Figure 8A). Addition of 30 mM Y- 27632 to culture medium for 2–3 h resulted in disassembly of both marginal and straight actin bundles in IAR-2 cells. Nevertheless, in the presence of Y-27632, E-cadherin accumulated in tangential AJs that had a more wave-like shape than AJs in control IAR-2 cells. In the presence of Y-27632, the establishment of cell-cell contact was not accompanied by contact paralysis of colliding cells. Treated with Y-27632, IAR-2 cells continued to form lamellipodia at the sites of cell-cell contacts (Figure S5). In the case PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e8027