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Polarized epithelial cells form selective barriers between tissues and various body compartments that are essential for normal development and organ function. A mature apical junctional complex (AJC), consisting of tight junctions (TJ), adherens junctions (AJ), and desmosomes is crucial for functional polarized epithelia. Rho-GTPases are key regulatory proteins of many cellular processes, including epithelial adhesion and polarization. These small GTPases are in turn controlled spatially and temporally by guanine nucleotide exchange factors (GEFs) that promote GTP-binding, resulting in their activation; and GTPase activating proteins (GAPs) that promote GDP hydrolysis, resulting in their inactivation. In this thesis I studied a novel junction associated GAP protein known as SH3BP1 in a variety of epithelia. SH3BP1 was identified in a functional siRNA screen that was designed to identify actin regulators of epithelial polarisation and differentiation. I will show that SH3BP1 localises to the early AJC when TJ and AJ are not yet properly separated. SH3BP1 regulation is important for tight junction formation and, its depletion affects polarity and junction integrity. I will demonstrate that SH3BP1 is functionally important in epithelial cell lines from different tissues as well as in organotypic three dimensional cultures. A major part of my thesis will focus on the demonstration that SH3BP1 is a crucial EGF receptor signalling effector that guides morphological alterations and actin dynamics. Using the A431 cell line EGF signalling model, I will demonstrate SH3BP1 is required to regulate both Rac1 and Cdc42 signalling, and subsequently its role in the recruitment of junctional proteins first to dorsal ruffles and then to forming tight junctions. I will provide evidence that SH3BP1 forms a heteromeric complex with the scaffold JACOP/paracingulin and the actin capping regulator CD2AP that has a key role in the regulation of actin dynamics.

The Hippo signalling pathway tightly governs tissue growth. Activation of the Hippo signalling pathway leads to phosphorylation and inactivation of transcriptional coactivators YAP and TAZ and their cytoplasmic retention, whereas inactivation of the Hippo signalling pathway leads to dephosphorylation and hyperactivation of YAP/TAZ and translocation into the nucleus to modulate a diverse range of cellular functions such as cell proliferation,migration differentiation and apoptosis. YAP/TAZ can act as a central hub relaying multiple signals and integrating diverse functions. The extracellular matrix (ECM) plays an important role in the regulation of the Hippo signalling pathway as do YAP/TAZ which can themselves be affected by cellular metabolism. Free fatty acids have been shown to regulate the transcriptional activity of YAP/TAZ. However, a link between the ECM, Hippo signalling pathway and lipids has not been previously descibed. This project shows a direct relationship between the Hippo signalling pathway, the ECM and exogenously supplied lipids. Furthermore, the project provides an understanding of how novel ECM components are involved in this regulation. The Hippo signalling pathway effector YAP was shown to interact with ECM proteins; Talin, CD2AP, PDLIM7 and Ezrin by Co-immunoprecipitation leading to the characterisation of a novel protein complex coupling the Hippo signalling pathway, the ECM and lipids in a CaCo2 and Hep-G2 cell culture model system. Scratch assays were used to observe the effect of complex member proteins on cellular migration. Knockdown of YAP and Talin was shown to slow down the migration rate of CaCo-2 cells. Immunofluorescence (IF) imaging was used to describe the effect of the various complex member proteins on the stabilisation of YAP following siRNA knockdown of CD2AP/Talin/Ezrin to show that translocation of YAP between nucleus and cytoplasm was affected. Depletion of talin resulted in inactivation of YAP by inducing its translocation from nucleus to cytoplasm in both CaCo-2 and Hep-G2 cells whereas depletion of CD2AP and Ezrin had no effect. The availability of oleic acid (OA) and palmitic acid (PA), the most abundant fatty acids, on the Hippo signalling effector YAP and the novel components of the Hippo/ECM complex was described. The results obtained indicated that lipids leads to inhibition of YAP activity by significantly increasing the p-YAP levels. CaCo-2 cells treated with lipids showed a marked decrease in cellular migration. Oleic acid was shown to disrupt the interaction of YAP with CD2AP and the interaction of Talin with CD2AP. Addition of OA was shown to affect the localization of proteins of the identified complex by mmunofluorescence microscopy. Upon OA treatment, nuclear YAP was decreased in both CaCo-2 and Hep-G2 cells. The levels of lipid droplet formation following addition of OA in conjunction with the depletion of proteins within the complex was also evaluated in CaCo-2 and Hep-G2 cells. This provided a new insight into the relationship of lipid metabolism with the ECM and Hippo signalling pathway. Planarians have been used as a model organism in order to assess the effect of lipid metabolism in a physiological relevant system. The results gathered with OA in-vivo are consistent with the results obtained in the tested mammalian cell lines. Finally, the novel protein interactions and expression of specific genes with the Hippo signalling pathway upon addition of fatty acids was analysed by mass spectroscopy (MS) and RNA-Sequencing. Numerous YAP-interactors were obtained by MS analysis and most importantly, OA was shown to abolish the interaction of Ezrin and Radixin with YAP. Thedata obtained from RNA-Sequencing revealed that, expression of downstream target genes of YAP (MYC and ANKRD1) were downregulated whereas key upstream components of the Hippo signalling pathway (FRMD6 and TAOK-1) were upregulated upon addition of oleic acid. The data obtained therefore indicates that fatty acids in part interact/manipulate the Hippo signalling pathway to regulate its activation state through the newly described ECM complex.