Elbediwy, AN; (2013) Functional Analysis of a Rho GTPase Activating Protein Involved in Epithelial Differentiation and Morphogenesis. Doctoral thesis , UCL (University College London).

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Abstract

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.

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