assembly of tangential AJs required Rac activity. In contrast, radial AJs in transformed epithelial cells and fibroblasts did not depend on functions of Rac. Collectively, the functional studies demon- strated the similarities in regulation of assembly of radial AJs in transformed epithelial cells and radial AJs in fibroblasts by Rho family small GTPases. Discussion This study is the first to demonstrate that transformed epithelial cells can use the disappearance of the marginal actin bundle and the remodeling of E-cadherin-based AJs to weaken cell-cell adhesion and to acquire motile behavior and migratory activity. When we studied transformed IAR-6-1 and IAR1170 epithelial lines, we found that while cells of these lines retain E-cadherin expression, have a morphology slightly changed from the morphology of the parent nontransformed IAR-2 cells, and form monolayers in confluent culture that are similar to IAR-2 monolayer, immunofluorescence staining demonstrated dramatic rearrangements of the actin cytoskeleton and AJs. In transformed IAR-6-1 and IAR1170 cells, the adhesion belt is broken and replaced by E-cadherin-based puncta or strands resembling radial AJs of fibroblasts. AJs in transformed epithelial cells are associated with short straight actin bundles. Our data demonstrated that in contrast with stable tangential AJs in nontransformed epithelial cells, radial E-cadherin-based AJs in transformed cells are very dynamic. To elucidate the mechanisms of formation of tangential and radial AJs, we next analyzed the involvement of small Rho GTPases and their effectors in AJ assembly. Our functional studies revealed the differences in the main driving forces of AJ formation in nontransformed and transformed epithelial cells. We found that mDia1 and Rac signaling are essential for the assembly of tangential AJs only in nontransformed epithelial cells. In contrast, Rac activity and mDia1 are not required for the assembly of radial AJs in transformed epithelial cells and fibroblasts. Earlier, Braga et al. [30] proposed that E-cadherin may be regulated by Rac differentially depending on cellular background (physiological context). Our data demonstrate the involvement of Rac signaling in the establishment of cadherin-mediated adhesion depending on the spatial organization of AJs. We propose that these differences are related to differential regulation of actin polymerization in the areas of tangential and radial AJs. Regulation by Rac1 and mDia1 of actin polymerization in tangential AJs is important for their establishment and stability. We also showed that ROCK-stimulated myosin II activity is not required for the formation of tangential AJs in nontransformed epithelial cells. These findings confirmed the data of Sahai and Marshall [21] and Ivanov et al. [12], which established that myosin II motor activity is not essential for AJ assembly in epithelial MDCK and T84 cells. In contrast, myosin II-mediated contractility is a key regulator of the formation of radial AJs in transformed epithelial cells. As we and others discovered earlier, the formation of radial AJs in fibroblasts depends on myosin II- mediated contractility [26,27]. Radial AJs in transformed epithelial cells are very similar to radial AJs in fibroblasts. Our study involving live-cell imaging of GFP-E-cadherin in trans- formed epithelial cells showed that AJs initially appear as dot-like clusters that grow into puncta or radial strands. We propose that the maturation of initial cadherin-based dot-like clusters into radial junctions depends on (i) association of an adhesion plaque with actin filaments, (ii) myosin II and a-actinin cross-linking of actin filaments into straight actin bundles, and (iii) centripetal myosin II- mediated tension of straight actin bundles, leading to growth of Adhesive Interactions radial AJs. We also propose that, regulated by mechanical forces, actin polymerization like the recently described force-dependent actin polymerization in focal contacts [31] plays an important role in the formation of radial AJs. Local balance between myosin II- mediated contractile forces, adhesive interaction of cells with neighboring cells, and the generation of traction in the direction of lamellipodia extension contribute to growth, adhesion strengthen- ing, and disruption of radial AJs. We hypothesize that the rearrangement of AJs in epithelial cells during neoplastic transformation into radial AJs is defined by reorganization of the actin cytoskeleton, leading to alterations of directions of tension in the area of cell-cell contacts. As we found earlier [32], the formation of tangential E-cadherin-mediated cell- cell contact in nontransformed epithelial cells leads to disruption of marginal bundles at the site of contact and to inhibition of retrograde actin flow. The remaining segments of marginal bundles of adjacent cells form two arc-like bundles (Figure 11) containing myosin II [33], which produce tangential tension in the zone of cell-cell contact in nontransformed epithelial cells. In transformed epithelial cells, disappearance of the marginal actin Figure 11. Hypothetical scheme for alterations of cell–cell interactions between epithelial cells at the early stages of morphologic transformation. The actin cytoskeleton (green) and AJs (red) in nontransformed and transformed epithelial cells. The characteristic feature of the actin cytoskeleton in nontransformed epithelial cells is marginal actin bundles at the free edges (blue arrowhead). In nontransformed cells, the formation of a tangential E- cadherin-based cell-cell contact (red line) leads to the disruption of marginal bundles at the site of the contact. The remaining segments of marginal bundles of adjacent cells form two arc-like bundles. The tangential tension of arcs (blue arrows) decreases protrusive activity both at the site of the cell-cell contact (contact paralysis) and at the free edges of contacting cells. In transformed cells, the crucial alteration of the actin cytoskeleton is the disappearance of marginal actin bundles. Upon the formation of the cell-cell contact, the deficiency of tangential tension as a result of the disappearance of marginal bundles prevents contact paralysis and leads to overlapping of lamellae of contacting cells. Radial AJs assemble in the area of overlapping. These AJs are associated with straight actin bundles. The enlargement of radial AJs depends on centripetal myosin II-mediated tension (blue arrows). AJs in transformed cells are very dynamic and unstable. Reorganization of the actin cytoskeleton and remodeling of AJs caused by neoplastic transformation results in a decrease in cell-cell adhesion and alters motile behavior of epithelial cells. doi:10.1371/journal.pone.0008027.g011 PLoS ONE | www.plosone.org 13 November 2009 | Volume 4 | Issue 11 | e8027