| Cell polarization is the process through which cells establish discrete membrane or cytoplasmic domains. Cell polarization allows for specialized cellular function and is essential for a wide range of processes, including asymmetric cell division, axon guidance, cell motility, and epithelial differentiation. Not surprisingly, there is a high degree of evolutionary conservation in the molecules and mechanisms that regulate this fascinating process.;Par6, a downstream effector of Cdc42, is highly conserved and required for asymmetric cell division in the worm, C. elegans. To investigate this further we solved the crystal structure of a complex between Cdc42 and the GTPase-binding domain of Par6. Structural analysis uncovered a novel role for a PDZ domain, as a structural scaffold for the adjacent CRIB domain. This demonstrates that emergent properties can arise from interactions between of two distinct domains. Also, we detected changes in a Par6GBD biosensor, consistent with a conformational change, induced by the binding of Cdc42. In addition we identified several in vivo aPKC phosphorylation sites in Par6A.;Previously, our lab and others have shown the Par complex localizes to epithelial cell-cell contacts and is involved in proper formation of epithelial tight junctions (TJs). We now show that RNA interference silencing of ZO-1, a marker of epithelial TJs, disrupts TJ biogenesis in mammalian epithelial cells. We used rescue experiments with mouse ZO-1 to demonstrate that the SH3 domain is a key determinant for junction assembly.;The cell-cell adhesion molecule E-cadherin is a well studied tumor suppressor and regulator of epithelial TJ formation. In order to study how E-cadherin regulates TJs, MDCK cells were selectively depleted of E-cadherin by RNAi. Surprisingly, this had little effect on the protein levels or localization of several markers of Adherens Junctions (AJs) and TJs. However, E-cadherin knockdown cells failed to properly establish cell polarity in response to calcium switch. These studies indicate that E-cadherin functions in the establishment of cell polarity, but that wild-type levels of E-cadherin are not required for the maintenance of tight junctions and cell polarity. |
