Identification of novel functions of Crumbs3b and Willin/FRMD6

Abstract

Crumbs3 (CRB3) is a component of epithelial junctions that has been implicated in apical-basal polarity, apical identity, apical stability, cell adhesion and cell growth. CRB3 undergoes alternative splicing to yield two variants: CRB3a and CRB3b. Here, I describe novel data demonstrating that CRB3b promotes the formation of tight junctions. However, significantly the FERM binding motif (FBM) of CRB3b is required for CRB3b functionality and ezrin binds to the FBM of CRB3b. Furthermore, ezrin contributes to CRB3b functionality and the correct distribution of tight junction proteins. Both CRB3 isoforms are required for the production of functionally mature tight junctions and also the localization of ezrin to the plasma membrane. Finally, reduced CRB3b expression in head and neck squamous cell carcinoma (HNSCC) correlates with cytoplasmic ezrin, a biomarker for aggressive disease, and show evidence that whilst CRB3a expression has no effect, low CRB3b and high cytoplasmic ezrin expression combined may be prognostic for HNSCC. Willin/FRMD6 has been demonstrated to regulate the hippo signalling pathway in epithelial cells and fibroblasts isolated from the sciatic nerve of a rat. Here, I describe the novel functions of Willin in a neuroblastoma cell line. Willin is able to negatively regulate the ERK signaling pathway and also control the morphology of SH-SY5Y cells. The levels of Willin present in SH-SY5Y cells predisposes the ability of these cells to undergo ERK dependent differentiation. Preliminary evidence demonstrating that Willin is able to regulate the actin cytoskeleton and the hippo signaling pathway in SH-SY5Y cells is presented in this thesis. This data suggests that Willin acts as an important factor in the regulation of cellular processes and in the control of the ERK and hippo signaling pathway.