The exocytic and endocytic trafficking of E-cadherin in epithelial cells

Epithelial cells form the boundaries between discrete compartments or organs and between the internal and external environments of a multicellular organism. An important structural and functional feature of epithelial cells is their polarity which provides them with distinct apical and basolateral surface domains. As a key component of the adherens junctions on the lateral surfaces of epithelial cells, Ecadherin functions in both cell-cell adhesion and in epithelial polarisation. E-cadherinmediated adhesion is regulated by the trafficking of E-cadherin and other components of the adherens junction to and from the lateral membrane and by cadherin-catenin interactions at the junctions. This thesis examines the polarised trafficking of Ecadherin and catenins and their interactions during this trafficking. Exocytic pathways carry newly-synthesised E-cadherin to the surface and sorting within the pathway ensures the polarised trafficking of E-cadherin to the basolateral membrane. The polarised trafficking of E-cadherin to the basolateral membrane and the temporal assembly of the cadherin/catenin adhesion complex were studied. Findings reported here show that while β-catenin binds early during synthesis to E-cadherin, these components do not connect with p120ctn or with α-catenin until they reach the basolateral membrane showing that there is temporal and contextual assembly of this junctional complex. Use of a mis-targeted mutant of E-cadherin was an important tool in defining pre- and post-sorting events in this pathway. The E-cadherin targeting mutant was then used in more detail to define the role of Ecadherin targeting in cell polarity. Monolayers of MDCK cells expressing the mistargeted E-cadherin had distinctly altered morphology but retained sorting of basolateral markers. Further, when cells were grown in 3D-epithelial cyst cultures the overexpression and mistargeting of E-cadherin caused loss of cell lumens and prevented formation of apical and basolateral cell membranes. E-cadherin expression levels and missorting were thus identified as a key event in developing epithelial polarity. The endocytosis and recycling of surface E-cadherin was also studied as part of this thesis. A series of assays and conditions for inducing and manipulating the endocytosis of E-cadherin were set up and studied, including growth factors, calcium depletion and temperature blocks. Taken together, these results show that surface E-cadherin is first internalised into early endosomes, a key compartment, from which E-cadherin may then be trafficked either directly or via the recycling endosome to other internal compartments or recycled to the plasma membrane. The findings presented in this thesis have further clarified the temporal assembly, sorting, exocytosis and endocytosis of the E-cadherin-catenin cell adhesion complex. They also demonstrate the importance of E-cadherin expression levels and surface localisation to the development of correct polarity in mammalian epithelia