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Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961–974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell–cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin–membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin–membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role. © 2004 Wiley-Liss, Inc.

We have studied the effect of intracellular pH on the establishment and maintenance of the cellular polarity in MDCK cells by utilizing nigericin which causes lowering of the cytoplasmic pH. At pH below 6.5, MDCK cells lost their polarized morphology and became roundish, with an increased apical area and shortened and unstable lateral walls. The lateral wall marker proteins uvomorulin and Na,K-ATPase remained segregated to the lateral walls in the acidified cells, as shown by immunofluorescence microscopy. Fodrin, on the other hand, was released from its normal basolateral residence and was found in the cytoplasm. Actin, which normally co-localizes with fodrin along the basolateral walls, showed a dotty distribution in the cytoplasm of acidified cells, while stress fibers remained intact. Microtubular network appeared flattened, but the Golgi complex retained its apical position. The pH change-induced alterations were readily reversible, as the normal basal-apical polarity (columnar shape, distinct apical and lateral domains with apposing and stiff lateral membranes) was reformed within 10 minutes after restoring the normal pH gradient across the cell membrane. This coincided with the translocation of fodrin from the cytoplasm to the lateral walls. The results show that lowering of intracellular pH leads to temporary segregation of fodrin from the other components of the membrane skeleton assembly, and that association of fodrin with the lateral walls seems to be a prerequisite for their close apposition and for the maintenance of normal basal-axial polarity.

The effects of Src tyrosine kinase activation in subconfluent temperature sensitive (ts)-Src-transformed Madin-Darby canine kidney (MDCK) cells were analyzed by shifting them from nonpermissive (40.5 degrees C) to permissive (35 degrees C) temperature. Already, in 15 minutes, adherens junction components were released from the lateral walls and accumulated to basal surfaces. Simultaneously, membranous actin staining vanished, actin bundles appeared at the basal surface, and the cells flattened. The only component phosphorylated and translocated after the shift to 35 degrees C was p120ctn. The epithelial-mesenchymal transition could be inhibited by a specific inhibitor of Src kinase, PP2, or by inhibiting endocytosis. Therefore, Src activation was responsible for the transition, but not because of phosphorylation of adherens junction components but by way of activation of endocytic machinery and RhoGTPase. Expression of an RacGEF, Tiam-1 (T-lymphoma invasion and metastasis gene 1), prevented flattening of Src-transformed MDCK cells at 35 degrees C and resulted in accumulation of cadherin to lateral membranes. In the case where the Src-MDCK cells were cultivated at 35 degrees C and shifted for short time periods to 40.5 degrees C, cadherin rapidly returned to lateral membranes, whereas actin and p120ctn followed hours afterward. This further supports the view that cadherin internalization is the primary target of Src kinase. We also looked at the cell morphology and distribution of cadherin and Tiam-1 in cells grown in three-dimensional gels composed of collagen and laminin or in Matrigel. At nonpermissive temperature, both Src-MDCK and Tiam-1-transfected Src-MDCK cells exhibited nonpolarized morphology in collagen I, a loose cluster in the mixture of collagen I and laminin, and a differentiated cyst in Matrigel. In growth factor-depleted Matrigel, the Src-MDCK cells grew in nondifferentiated clusters, whereas Tiam-1-transfected cells went to apoptosis. The differentiated phenotype of both cell lines could be rescued by Matrigel-conditioned medium, platelet-derived growth factor, or cholera toxin. Concomitantly, both cadherin and Tiam-1 were recruited to lateral membranes. Therefore, cadherin and Tiam-1 seem to be the key players in the differentiation process of MDCK cells.