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Functional Analysis of a Rho Gtpase Activating Protein Involved In Functional Analysis of a Rho GTPase Activating Protein Involved in Epithelial Differentiation and Morphogenesis Ahmed Nehad Elbediwy Thesis submitted to University College London for the award of Doctor of Philosophy November 2012 Department of Cell Biology Institute of Ophthalmology University College London 11-43 Bath Street London EC1V 9EL Declaration of Work I, Ahmed Nehad Elbediwy, hereby confirm that the work that is presented in this thesis is my own. Where information has been derived from alternate sources, I confirm that I have indicated this in the correct context within the thesis University College London November 2012 2 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 3 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. Based on my data, I will propose a new model on how a negative regulatory complex guides cytoskeletal dynamics and junction formation and, thereby, promotes epithelial differentiation. 4 Table of Contents Table of Contents ............................................................................................................ 5 Table of Figures ............................................................................................................... 9 Table of Tables .............................................................................................................. 13 List of Abbreviations .................................................................................................... 14 Chapter 1 – Introduction .............................................................................................. 23 Overview ...................................................................................................................... 23 Cell-cell junctional complexes ..................................................................................... 25 Tight junctions .............................................................................................................. 27 Structural basis of tight junctions .......................................................................................... 29 Integral membrane proteins ................................................................................................... 30 Adaptor proteins..................................................................................................................... 34 Signalling proteins ................................................................................................................. 36 Adherens junctions ....................................................................................................... 37 Cadherin-catenin complex ..................................................................................................... 37 Nectin-afadin complex ........................................................................................................... 40 Desmosomes ................................................................................................................. 43 Formation of cell-cell junctions and establishment of cell polarity .............................. 44 Formation of cell-cell contacts............................................................................................... 45 Cell polarization..................................................................................................................... 48 Rho GTPases ................................................................................................................ 53 Cdc42 ..................................................................................................................................... 54 Rac1 ....................................................................................................................................... 57 Rho ......................................................................................................................................... 57 GEFs, GAPs and GDIs ................................................................................................. 58 Guanine nucleotide Dissociation Inhibitors (GDIs) .............................................................. 60 5 Guanine nucleotide Exchange Factors .................................................................................. 60 GTPase Activating Proteins ................................................................................................... 61 Regulation of GAPs ................................................................................................................ 62 BAR domain containing GAPs ............................................................................................... 64 Rho GTPases and junction formation ........................................................................... 66 EGFR signalling ........................................................................................................... 72 Experimental plan ......................................................................................................... 75 Chapter 2 - Materials and Methods ............................................................................ 78 DNA cloning techniques ............................................................................................... 78 Polymerase chain reaction ..................................................................................................... 83 DNA agarose gel electrophoresis........................................................................................... 84 Low melt agarose DNA purification using silica beads ......................................................... 84 Low melt agarose DNA ligation procedure............................................................................ 85 TA cloning kit ligation using PCR 2.1 vector (Invitrogen) ..................................................... 86 Transformation of ligated DNA using competent bacteria .................................................... 86 Preparation of competent E.coli bacteria DH5α or BL21pLysS ............................................ 87 Site-directed mutagenesis ....................................................................................................... 87 Protein analysis ............................................................................................................. 89 Immunoblotting techniques .................................................................................................... 89 Co-immunoprecipitation (Co-IP) ........................................................................................... 90 Fusion protein expression and purification ................................................................... 91 Preparation of pGEX-4T-3 fusion proteins for use in GST pull-downs ................................. 91 pGEX-4T-3 fusion protein purification .................................................................................. 92 GST pull-down ....................................................................................................................... 93 Basic tissue culture techniques ..................................................................................... 93 Cell culture ............................................................................................................................. 93 Stable cell lines generation techniques .................................................................................. 94 Immunofluorescence techniques .................................................................................. 95 Fixation techniques ...............................................................................................................
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