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. 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 non- transformed 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 [34]. 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 [26]. 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 rearrange- ments of AJs and the actin cytoskeleton at the early stages of morphological transformation (Figure 11). 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- Adhesive Interactions cadherin-based AJs observed in the present study may be essential for carcinoma cell dissemination. Materials and Methods Reagents The ROCK inhibitor Y-27632 and the myosin II ATP-ase inhibitor (6)-blebbistatin were purchased from Calbiochem-Merck. The following primary monoclonal antibodies were used: anti-E- cadherin, clone 36; anti-N-cadherin, clone 32; anti-mDia1, clone 51 (Transduction Laboratories-BD); and anti-a-tubulin, clone DM 1A (Sigma-Aldrich). Secondary TRITC-conjugated antibodies were obtained from Chemicon; secondary Alexa488-conjugated antibod- ies; and Alexa488- and TRITC-conjugated phalloidin were obtained from Molecular Probes-Invitrogen. Secondary anti-mouse IgG1 and IgG2a antibodies were purchased from Southern Biotech. Horseradish peroxidase-conjugated secondary antibodies for West- ern blotting were purchased from Upstate-Millipore. Other reagents were obtained from Sigma-Aldrich. Cell lines, constructs, and transfections The IAR-2 line of nontransformed rat epithelial cells and the IAR-6-1 line of IAR epithelial cells transformed with dimethylni- trosamine were established by Montesano et al. [35]. The pPS/ hygro-N-RasG12D construct was obtained from B.P. Kopnin (N.N. Blokhin Cancer Research Center, Russia). We obtained the IAR1162 and IAR1170 lines, expressing N-RasG12D, using retroviral infection from IAR-2 cells according to the procedure of retroviral transduction and selection with hygromycin [36]. We established individual clones of Ras-expressing IAR1162 cells (nine clones) and IAR1170 cells (five clones). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37uC in a 5% CO2 humidified atmosphere. E- cadherin- and GFP-E-cadherin-expressing constructs were kindly provided by Dr. S.M. Troyanovsky (Northwestern University, Chicago, USA). We stably transfected Rat-1 fibroblasts with E- cadherin plasmid using the FuGene6 transfection reagent according to the manufacturer’s protocol (Roche). To generate IAR-2 and IAR-6-1 lines that stably expressed GFP-E-cadherin, we transfected IAR-2 and IAR-6-1 cells with GFP-E-cadherin construct using the FuGene6 reagent. Stable transfectants were established after 2 weeks of selection in medium with G-418 (1 mg/ml). Analysis of formation of cell–cell contacts in a narrow wound Cell-cell collisions and the establishment of cell-cell contacts were investigated in narrow wounds. We created narrow wounds by scraping cell monolayers on coverslips with an injection needle. Cultures were incubated for 3–4 h and observed or fixed at the time when the wound started to close. Y-27632 and blebbistatin were added to the growth medium from stock solutions at 20 min after wounding. To evaluate the effects of Rho and Rac inhibition on the establishment of cadherin-mediated AJs in a narrow wound, C3 transferase and dominant negative N17Rac (kindly provided by Dr. A. Hall, Memorial Sloan-Kettering Cancer Center, USA) were used. Recombinant proteins were prepared as glutathione S-transferase fusion proteins in Escherichia coli, purified with glutathione Sepharose beads, thrombin cleaved, dialyzed against microinjection buffer, and concentrated as described previously [37]. C3 transferase and N17Rac were introduced into wound edge cells at the time of wounding, as a modification of the method PLoS ONE | www.plosone.org 14 November 2009 | Volume 4 | Issue 11 | e8027