depend on actin polymerization [11,12]. The Arp2/3 regulators N-WASP, WAVE2, and cortactin are involved in the assembly of AJs [12–15]. Rho activity is required for the formation of actin stress fibers and focal contacts [7]. Rho-kinase (ROCK) and the formin protein mDia1 are the main effectors of Rho [16–18]. Active Rho recruites ROCK, which stimulates acto-myosin contractility by phosphorylation of the myosin II light chain and other targets [19]. Data on the role of myosin II-mediated contractility in the establishment of cadherin-mediated adhesion are controversial. Shewan et al. [20] suggested that myosin II is essential for the accumulation of E-cadherin at cell-cell junctions. In other studies, the ROCK inhibitor Y-27632 and the myosin II ATPase inhibitor blebbistatin had no effect on the assembly of AJs in epithelial cells [12,21]. Formin-mediated actin polymer- ization could also be important for the establishment of AJs. Formin1 is essential for cadherin-mediated adhesion in kerati- nocytes [22]. It has also been shown that mDia1 participates in the formation and maintenance of AJs in MCF7 epithelial cells [23]. In this work, we studied the lines of transformed epithelial cells that retained expression of E-cadherin, displayed slightly changed morphology, and formed monolayers in dense culture. In transformed epithelial cells, we found, however, a disappearance of the marginal actin bundle and dramatic rearrangement of E- Adhesive Interactions cadherin-based AJs into radial AJs that are differently regulated by Rho family small GTPases than are tangential AJs in nontrans- formed epithelial cells. We also described the remodeling of radial AJs and the alterations in protrusive activity that led to reduced cell-cell adhesion and to acquisition of motile behavior and migratory capacity by transformed epithelial cells. Results Actin cytoskeleton and AJs in nontransformed IAR-2 epithelial cells and transformed IAR descendants In this study, we compared the morphology, the actin cytoskeleton, and AJs of nontransformed IAR-2 epithelial cells derived from rat liver with those of the transformed IAR descendants IAR-6-1 and IAR1170. In dense culture, nontrans- formed IAR-2 epithelial cells formed a monolayer (Figure 1A). Individual IAR-2 cells were discoid-shaped. They had a marginal actin bundle, which is typical of epithelial cells and internal straight actin bundles (Figure 1B). Immunofluorescence micros- copy of IAR-2 cells stained with antibodies against E-cadherin showed that AJs were aligned as a continuous belt along the cell- cell boundaries and colocalized with the circumferential actin belt (Figure 1C and 1D). We designated AJs of nontransformed epithelial cells ‘tangential AJs’. Figure 1. Nontransformed and transformed IAR epithelial cell lines. (A) In dense culture, nontransformed IAR-2 epithelial cells form a monolayer. (B) A characteristic feature of the actin cytoskeleton of an IAR-2 epithelial cell is the marginal actin bundle (arrowhead). (C, D) E-cadherin- based AJs (tangential AJs) (arrow) in an IAR-2 monolayer organized into adhesion belts along the cell-cell boundaries and colocalized with perijunctional actin bundles (arrowhead). (E–H) An IAR-6-1 line transformed with dimethylnitrosamine. (E) In dense culture, transformed IAR-6-1 epithelial cells form a monolayer. (F) IAR-6-1 cells have a slightly changed morphology. They are polygonal. Note the disappearance of the marginal actin bundle at the cell periphery. (G, H) Double staining for E-cadherin and F-actin shows that AJs in IAR-6-1 cells appear as radial strands (arrow) that are colocalized with straight actin bundles (arrowhead). (I–L) Ras-transformed IAR1170 epithelial cells (clone H5). (I) In dense culture, IAR1170 cells form a monolayer. (J) The shapes of IAR1170 cells in sparse cultures are polygonal. The actin cytoskeleton is present in the form of randomly oriented actin bundles; the marginal actin bundle disappears. (K–L) Punctate clasters of E-cadherin at the cell-cell boundaries in an IAR1170 monolayer (arrow). Circumferential actin bundles are disorganized. Straight actin bundles oriented perpendicularly to the cell-cell boundaries (arrowhead) are seen. Bar, 10 mm. doi:10.1371/journal.pone.0008027.g001 PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e8027