Figure 3. Transformed IAR-6-1 and IAR1170 cells display enhanced motility in a migration assay. IAR-2, IAR-6-1, and IAR- 1170 (clone H5) cells were plated on membranes with 8-mm pores in Bio-Coat migration chambers. After 20 h of incubation, the cells on the lower surface of membranes were fixed and stained. The number of migrated cells was determined from an average of the number of stained cells on membranes in 15 randomly selected fields. The data are presented as means6SEM of triplicate assays for each cell line in three independent experiments. Asterisks indicate the values that differ significantly from control (p,0.001, t-test). doi:10.1371/journal.pone.0008027.g003 nontransformed epithelial cells result in the establishment of stable cell-cell contact followed by contact paralysis at the site of the contact and a decrease of protrusive activity at the free edges of the contacting cells. Neoplastic transformation changes cell-cell interactions, preventing contact paralysis after the establishment of cell-cell contact, leading to overlapping of lamellae and to the extension of lamellipodia at the free edges. ynamics of E-cadherin–based AJs in nontransformed and transformed cells To correlate cell behavior with the dynamics of junctional E-cadherin, we established nontransformed IAR-2 cells and transformed IAR-6-1 cells stably expressing GFP-E-cadherin. Using time-lapse differential interference contrast (DIC) and fluorescence microscopy, we investigated living cells for 3–7 h. In nontransformed IAR-2 cells, GFP-E-cadherin accumulated in stable continuous tangential AJs at the cell-cell boundaries (Figure 5A and Video S6). When IAR-2 cells formed new cell- cell contacts, GFP-E-cadherin assembled in tangential lines along the cell-cell boundaries. In contrast, in transformed IAR-6-1 cells, AJs were dynamic (Figure 5B and Video S7). Transformed cells extended protrusions over the entire periphery. When a protrusion contacted another cell, GFP-E-cadherin began to accumulate in dot-like clusters at the site of the cell-cell contact, which were able to grow. In IAR-6-1 cells, we found the remodeling of AJs. AJs in IAR-6-1 cells could rearrange and relocate in the zone of contact. Unlike stable AJs in IAR-2 cells, AJs in IAR-6-1 cells were often disrupted. As a cell detached from one cell, it could form a contact with another cell, in which GFP- E-cadherin accumulated. Thus, transformed IAR-6-1 cells could move and establish dynamic E-cadherin-based cell-cell contacts with neighboring cells. Adhesive Interactions Using the distance map method [25], we analyzed the dynamics of AJs in IAR-2 cells and IAR-6-1 cells stably expressing GFP-E- cadherin (Figure S4). Time-lapse sequences of sparse cultures were used to calculate the mean rates of AJ movement. We found differences in the dynamics of AJs in IAR-2 cells and IAR-6-1 cells. While the mean rate of movement of tangential AJs in IAR-2 cells was 5963 nm/min (mean6SEM, n = 28), the mean rate of movement of radial AJs in IAR-6-1 cells was 189621 nm/min (n = 30). Using ImageJ software, we also measured the distances traveled by individual AJs, and calculated the mean rates of AJ movement. This analysis showed similar results: the mean rate of movement of AJs in IAR-2 cells was 97617 nm/min (mean6SEM, n = 25), the mean rate of movement of radial AJs in IAR-6-1 cells was 176622 nm/min (mean6SEM, n = 25). To compare the dynamics of AJ assembly in nontransformed and transformed epithelial cells thoroughly, we investigated cell-cell collisions in a narrow wound. Live-cell imaging of IAR-2 cultures showed that during the establishment of the cell-cell contact, a tangential line of GFP-E-cadherin formed at the cell-cell boundary and expanded laterally in two opposite directions (Figure 6A and Video S8). The characteristics of GFP-E-cadherin accumulation at the sites of cell-cell contacts in cultures of IAR-6-1 cells were different (Figure 6B, 6C, and 6D, and Video S9). At the site of the cell-cell contact, GFP-E-cadherin initially aggregated into dot-like clusters. Some of these puncta could disappear. The majority of GFP-E-cadherin clusters grew and transformed into radial strands oriented perpendicularly to the cell-cell boundary. AJs could change their position. During cell displacement, AJs between two adjacent cells in a monolayer often elongated and broke. Thus, our studies revealed two different types of E-cadherin-based AJs: stable tangential AJs in nontransformed epithelial cells and relatively dynamic radial AJs in transformed epithelial cells. Effect of C3 loading on the formation of different types of AJs To elucidate the mechanisms of the formation of different types of AJs and the rearrangement of tangential epithelial AJs into radial ones during neoplastic transformation, we examined the involvement of the Rho family small GTPases, Rho and Rac, in regulating cell-cell adhesion. Earlier, we had described radial AJs in Rat-1 fibroblasts associated with short straight actin bundles. As it has been shown that the formation of radial AJs in fibroblasts depends on myosin II-mediated contractility [26,27], we decided to add comparisons of the effects of inhibition of Rho or Rac signaling on radial AJs in Rat-1 fibroblasts to our studies concerning the effects of this inhibition on radial AJs in transformed IAR-6-1 epithelial cells. First, we investigated the effect of inhibition of Rho activity on AJ formation in nontransformed IAR-2 cells, transformed IAR-6-1 cells, and Rat-1 fibroblasts (Figure 7). C3, which ADP-ribosylates and which inactivates cellular Rho, was introduced into wound- edge cells during the wounding of monolayers. Wounding of a cell monolayer with a hypodermic needle in the presence of C3 and fluorescent dextran (as a marker) resulted in loading of ,50% cells at the wound edge. Control experiments showed that fluorescent dextran alone did not affect the rate of wound healing or the accumulation of adhesive proteins in the cell-cell contacts. Loading of cultured cells with C3 led to a delay of wound healing up to 7– 8 h (3–4 h in controls). Actin bundles in C3-loaded cells were disrupted (data not shown). The introduction of C3 into IAR-2 cells prevented the formation of tangential AJs. In C3-loaded IAR- 2 cells, E-cadherin did not accumulate at the cell-cell boundary. Transformed IAR-6-1 cells loaded with C3 could not assemble radial AJs. E-cadherin aggregated only in dot-like clusters. Similar PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e8027