Regulators of E-cadherin trafficking in polarized epithelial cells (2007)

E-cadherin is a basolateral membrane protein and a key member of the adherens junction that mediates cell-cell adhesion between adjacent epithelial cells. The location and function of E-cadherin is essential for establishing and maintaining cell polarity and epithelial integrity. The cell adhesion mediated by E-cadherin-catenin complexes at adherens junctions is regulated by the transport of E-cadherin to and from the plasma membrane. This thesis investigates several aspects of E-cadherin trafficking in exocytic and endocytic pathways using a polarized epithelial cell line, MDCK cells, as the primary model. The Rho GTPase family members, Rac1 and Cdc42, have many roles in cells and both have been implicated in various trafficking pathways. Here we investigated potential roles for Cdc42 and Rac1 in the exocytosis of E-cadherin and its delivery to the basolateral cell surface. Overexpression of Cdc42 and Rac1 and of functional mutants of these proteins was performed by transfecting or microinjecting cDNAs into MDCK cells. We documented the subsequent effects on E-cadherin surface delivery by staining endogenous E-cadherin or a GFP-tagged recombinant E-cadherin expressed in the cells. Rac1 and Cdc42 mutants disrupted E-cadherin surface delivery and caused an intracellular accumulation of newly-synthesized E-cadherin. Co-localization experiments pinpointed post-Golgi trafficking at or near the trans-Golgi network as the likely site of action for Cdc42 and Rac1. Additional experiments included the use of toxin treatments to inactivate Rho GTPases and to depolymerize actin filaments. Taken together, these studies showed novel roles for the Rho GTPases and actin filaments in post-Golgi trafficking of E-cadherin Cholesterol rich lipid rafts are membrane domains known to be important for functional clustering of surface molecules and for trafficking. Here we investigated a potential role for lipid rafts in E-cadherin regulation by using cholesterol-depleting drugs to affect E-cadherin localization. These experiments resulted in the accumulation of E-cadherin in the endosomal pathway and further analysis showed that this resulted from abnormal internalisation of surface E-cadherin. Biochemical separation of detergent resistant domains also showed a proportion of E-cadherin is in rafts. Use of endosomal markers for colocalization revealed that cholesterol depletion altered the integrity of endosomes and accumulated E-cadherin in a late endosome-like compartment, as defined also by the added effects of Rab5 and Rab7 mutants. Our findings show that cholesterol-rich lipid rafts are a component of the normal trafficking and surface retention of E-cadherin. Further studies will be needed to fully define the role of rafts in E-cadherin-mediated protein interactions and in adhesion. Lastly, studies were initiated to develop bioinformatic and high-throughput screening approaches for future studies focussed on large-scale analysis of E-cadherin trafficking. Bioinformatics and literature mining were used to construct a Golgi proteome based on subcellular localization of Golgi-associated proteins. The Golgi proteome contains a cohort of proteins associated with the Golgi complex which will include further regulators of E-cadherin trafficking. These data are published as part of an on-line resource the LOCATE database (http://locate.imb.uq.edu.au/), and will now be available for future selection and validation of trafficking proteins involved in E-cadherin transport through the Golgi complex. These studies were complemented by work done to establish a new transfection procedure that will be amenable for high throughout analysis of gene expression. The ‘reverse transfection’ technique was set up and optimised. The expression of Cdc42 and Rac1 mutants was used as proof-of-principle for this approach in showing perturbation of E-cadherin trafficking. The results are consistent with our previous findings and provide strong support for the feasibility and availability of this novel transfection-based cell assay system. Generally, the findings in this thesis implicate new components of trafficking pathways, including Rac1, Cdc42, actin filaments and lipid rafts, as being essential for exocytic and endocytic trafficking and regulation of E-cadherin. New approaches are made ready for larger discovery projects aimed at fully documenting the itinerary and molecules involved in moving E-cadherin through the cell. These findings are relevant to our understanding of E-cadherin and epithelial biology in general.