| E-cadherin has been proposed to be at the top of a molecular cascade that is initiated by cell contact and results in the polarization of epithelial cells. In this thesis, the mechanisms by which the E-cadherin interaction results in cellular reorganization have been examined. Using an unpolarized fibroblastic cell line (L cells), the development of cellular polarity due to E-cadherin was examined. Introduction of E-cadherin into L cells is sufficient to induce a polarization of endogenous cell surface Na;In order to further elucidate the mechanisms by which E-cadherin interactions result in the establishment of epithelial polarity, very early stages in the establishment of cell-cell contacts were studied using retrospective immunohistochemistry. We observed that E-cadherin at the cell contact is in a Triton-soluble form for the first 10 minutes of stable cell-cell contact, suggesting that E-cadherin is able to mediate cell contact independent of interaction with the Triton X-100 insoluble cytoskeleton. E-cadherin then rapidly becomes associated with the cytoskeleton at ;To determine if cell contact modulates intracellular signalling, the distribution of tyrosine phosphorylated proteins in MDCK cells was examined before, during and after the induction of cell contact. A 140kD protein was tyrosine phosphorylated only in contacting cells, as determined by immunoblotting whole cell lysates. Cell contact lead to an accumulation of tyrosine phosphoproteins at cell contacts, as determined by immunofluorescence. To determine if occupation of E-cadherin directly affects intracellular calcium, Fura-2 loaded MDCK cells were stimulated with peptides which mimick the binding region of E-cadherin. In preliminary studies, these peptides induced transient increases in intracellular calcium, suggesting that occupation of E-cadherin may directly affect intracellular signalling. |
