Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Microinjection of fluorophore-tagged cytoskeletal proteins has been a usefu l tool in studies of formation of focal adhesions (FA), We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (T J) of epithelial Madin-Darby bovine kidney cells. We chose alpha -actinin a nd vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterize d, Isolated FITC-labelled chicken alpha -actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade w ithin an hour after injection and the cells were joined to polykaryons havi ng 5 to 13 nuclei. Short fragments of cell membranes containing injected pr oteins, actin, beta -catenin, cadherin, claudin, occludin and ZO-1 were vis ible inside the polykaryons indicating that both AJs and TJs were disintegr ated as a single complex, Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidl y induced the detachment of the cells from the substratum probably due to h igh affinity of vinculin head to talin. Vinculin tail domain had no apparen t effect on the cell morphology, Since small GTPases are involved in the bu ilding up of AJs, we injected active and inactive forms of cdc42 and rac pr oteins together with vinculin to see their effect, Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect, We suggest that excess actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskelet on which leads to fragmentation of junctional complexes and subsequent cell fusion, The results show that cell-cell adhesion sites are more dynamic an d more sensitive than FAs to an imbalance in the amount of free alpha -acti nin and intact vinculin.