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This study attempted to analyse in detail the effect of src kinase on the growth and differentiation of MDCK cells in different extracellular matrix (ECM) environments. A method was developed to label the membrane proteins in situ and the distribution of cytoskeletal and junctional proteins was visualized in three-dimensional cell complexes, using optical sections generated by confocal microscopy. Independently of the ECM, non-transformed MDCK cells formed differentiated cell cysts with one or a few lumina, with the apical side facing the lumen; ZO-1 was expressed at the tight junctions close to the apical side and beta-catenin, E-cadherin and fodrin along the entire lateral walls. The phenotype of src kinase activated MDCK cells was strongly dependent on the ECM and varied from an irregular cluster in collagen I, to tubular structures in laminin or proteoglycans, and finally to a polarized cell cyst in Matrigel. In collagen I, E-cadherin and beta-catenin were seen partially along the lateral walls and partially in the cytoplasm of src-transformed MDCK cells; fodrin was released into the cytoplasm and ZO-1 was not visualized. When the src-transformed cells were cultivated in Matrigel, their junctional proteins were recruited to the cell membranes and ZO-1 reappeared at the apical face. Thus, the components of Matrigel could overcome the deleterious effect of src on the polarity of MDCK cells. TGFbeta1, together with its receptors and other soluble factors in Matrigel, were responsible for the induction of differentiation. The results show that tyrosine phosphorylation sensitizes the epithelial MDCK cells to ECM and TGFbeta1., (Copyright 2001 John Wiley & Sons, Ltd.)