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. 
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 
and Ivanov et al. 
, 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 
. 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 transformed 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 α-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 radial AJs. We also propose that, regulated by mechanical forces, actin polymerization like the recently described force-dependent actin polymerization in focal contacts 
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 strengthening, 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 
, 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 () containing myosin II 
, which produce tangential tension in the zone of cell-cell contact in nontransformed epithelial cells. In transformed epithelial cells, disappearance of the marginal actin bundle is a key event that results in the rearrangement of AJs. In these cells, the deficiency of tangential tension and centripetal tension in the zone of cell-cell contact leads to the assembly of straight actin bundles and to the maturation of radial AJs.
Hypothetical scheme for alterations of cell–cell interactions between epithelial cells at the early stages of morphologic transformation.
In the present study using live-cell imaging, we also revealed significant alterations of cell-cell interactions caused by neoplastic transformation. The formation of stable contact between nontransformed epithelial cells results in dramatical inhibition of protrusions at the site of contact (contact paralysis) and in a decrease of protrusive activity at the free edges of contacting cells. In contrast, contact paralysis is not seen in transformed epithelial cells. Analysis of kymographs showed that lamellipodia at the sites of the cell-cell contacts continued to extend. Transformed cells also form lamellipodia at the free edges. We suggest that tangential tension of arc-like bundles in nontransformed epithelial cells may decrease the extension of protrusions both at the site of the cell-cell contact and at the free edges of contacting cells. Inhibition of protrusive activity stabilizes the contact between two cells. Similar suppression of protrusive activity after the application of artificial tangential tension with a micromanipulating needle was described by Kolega 
. Tangential tension also can cause fast lateral expansion of contact and align newly assembled actin filaments in perijunctional bundles, strengthening the adhesion. The role of marginal actin bundles in the creation of tangential tension and the decrease of protrusive activity of contacting nontransformed epithelial cells was also supported by our experiments with Y-27632 and blebbistatin, which abolish marginal bundles. In the presence of these inhibitors, contact paralysis during the formation of cell-cell contact was not developed. We propose that the absence of marginal bundles in transformed epithelial cells leading to the deficiency of tangential tension at the border of contacting cells prevents inhibition of protrusive activity of contacting cells. The experiments performed suggest that the alterations in cell-cell interactions and the remodeling of AJs change motile behavior and migratory activity of transformed epithelial cells.
It should be noted that the cell-cell interactions between transformed epithelial cells described in this paper are very similar to those between cells of mesenchymal origin (fibroblasts). Earlier, we described the absence of contact paralysis and significant overlapping between contacting fibroblasts followed by persisting formation of lamellipodia at the free edges 
. The formation of E-cadherin-based AJs in transformed epithelial cells and their regulation by Rho GTPases is similar to those in fibroblasts. Possibly, these alterations in transformed epithelial cells are manifestations of the initial stages of EMT. This question obviously needs further investigation.
Thus, our studies demonstrate that structural and dynamic coordination between cadherins, actin structures, and actin-regulating proteins can change during neoplastic transformation. In summary, we suggest a hypothetical scheme for the rearrangements of AJs and the actin cytoskeleton at the early stages of morphological transformation (). These alterations are initiated by disruption of the marginal actin bundle. The deficiency of tangential tension as a result of the disappearance of the marginal bundle prevents contact paralysis and leads to overlapping of contacting cells. Dot-like AJs form in the zone of overlapping. Centripetal myosin II-mediated tension converts them into radial AJs, which are very dynamic and less stable than tangential AJs in nontransformed epithelial cells. Changes in the activities of Rho family small GTPases, which are characteristic of transformed cells, can be involved in these rearrangements. Remodeling of AJs and alterations of protrusive activity modulate cell-cell adhesion and motile behavior of cells. It is tempting to speculate that rearrangements of the actin cytoskeleton and of E-cadherin-based AJs observed in the present study may be essential for carcinoma cell dissemination.