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A Dissertation Entitled SGEF Forms a Complex with Scribble and Dlg1 A dissertation entitled SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility by Sahezeel S. Awadia Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biological Sciences __________________________________________ Dr. Rafael Garcia-Mata, Committee Chair ___________________________________________ Dr. Ann Miller, Committee Member ___________________________________________ Dr. Tomer Avidor-Reiss, Committee Member ___________________________________________ Dr. Deborah Chadee, Committee Member ___________________________________________ Dr. Katherine Eisenmann, Committee Member Dr Cyndee Gruden, Dean College of Graduate Studies The University of Toledo May 2019 iv An Abstract of SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility by Sahezeel S. Awadia Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biological Sciences The University of Toledo May 2019 The establishment of epithelial cell polarity is essential for growth, differentiation and morphogenesis. Conversely, loss of cell polarity is a hallmark of many disease states, including cancer. The concerted action of three conserved protein complexes – PAR, Crumbs and Scribble – controls the establishment of cell polarity. In addition, the Rho- family of small GTPases, which regulate actin dynamics, plays a key role in the development of polarity. How polarity complexes regulate Rho GTPase function remains unknown. iii The members of the Scribble polarity complex Scribble, Lgl and Dlg1 are known to interact genetically but the direct interaction between Scribble and Dlg1 is not known completely. Here, we show that SGEF interacts with Scribble, a member of the Scribble polarity complex. A novel internal PDZ binding motif in SGEF interacts with PDZ1 of Scribble. We also obtained a crystal structure of PDZ1 of Scribble in complex with the peptide comprising internal PDZ binding motif of SGEF. An unstructured 30 amino acid motif at the N-terminus of SGEF interacts with GUK (Guanylate Kinase) domain located at C-terminus of Dlg1. SGEF interacts with Scribble and Dlg1 through different regions in SGEF. This allows simultaneous binding of both Scribble and Dlg1 to SGEF forming a ternary complex of Scribble-SGEF-Dlg1 in epithelial cells. The Scribble polarity complex has been implicated in regulation of junctional architecture in epithelial cells. Our finding shows that SGEF localizes to apical junctions of Xenopus embryo and lateral junctions of MDCK (Madin Darby Canine Kidney) epithelial cells in Scribble dependent fashion. Scribble and Dlg1 are known to be involved in Adherens Junctions (AJ) establishment and maintenance. Here we show that knockdown of SGEF in MDCK cells leads to disruption of AJ characterized by almost a complete loss of E-cadherin, diffused localization of E-cadherin binding partners -catenin, -catenin and p120-catenin at lateral cortex. Disruption of lateral junctions leads to a collapse of lateral wall of epithelial cells leading to flattening of epithelial cells silenced for SGEF. We also show that silencing of SGEF leads to straightening of otherwise curvilinear tight junctions stained by ZO-1 (Zonula Occludens). The loss of curvilinear phenotype is accompanied by localization of myosin to apical actin belt forming beautifully organized actomyosin arrays. Apical actomyosin arrays in SGEF KD cells leads to increased iv contractility, increased tension, increased permeability and straightening of apical tight junctions. Our results suggested that the scaffolding activity of SGEF to form a ternary complex with Scribble and Dlg1 is essential for the apical actomyosin contractility and proper architecture of the tight junctions whereas the exchange activity of SGEF is required for the E-cadherin based adherens junctions architecture in epithelial cells. MDCK cells when embedded in a 3D matrix like matrigel form characteristic 3D structures called cysts that are highly polarized and have a single open (fluid filled) lumen. We found that the silencing of SGEF displayed a disorganized cyst phenotype with abnormal lumens formation. Further, we showed that formation of open lumen is regulated by the scaffolding activity of SGEF whereas ability to form a single hollow lumen is regulated by exchange activity of SGEF. Finally, we found that SGEF overexpression alters junctional architecture and dynamics and promotes apical constriction. Xenopus embryos overexpressing SGEF had increased cell constriction due to localization of myosin to apical region of cells. This increased contractility reduced the apical cell area in SGEF overexpressing embryos. Taken together, our results suggested that the scaffolding activity of SGEF to form a ternary complex with Scribble and Dlg1 regulates apical actomyosin contractility and TJ properties whereas its catalytic activity to activate RhoG regulates E-cadherin based AJ architecture in epithelial cells. v To my wife, my son and my parents. This would not have been possible without your unwavering support vi ACKNOWLEDGEMENTS First and foremost, I would like to thank my mentor Dr. Rafael Garcia-Mata for guiding me throughout my PhD journey, be it a scientific or personal, he has been pillar of strength and support for me. I am truly blessed to get an opportunity to work in his lab. It is because of Rafael that I am able to fulfill my dream of becoming a scientist. He has inspired me to become an independent researcher and helped me realize the power of critical reasoning. In addition to this, I am also extremely thankful to my committee members Dr. Ann Miller, Dr. Tomer Avidor-Reiss, Dr. Katherine Eisenmann and Dr. Deborah Chadee. A special thanks to Dr. Silvia Goicoechea. Finally, I also appreciate the support of the Chair of the Biology Department, Dr. Bruce Bamber. I am also extremely thankful to Ashtyn Hoover, Kyle Snyder, Torey Arnold, Farah Huq, Atul Khire, Alan Hammer and for all their help and wonderful memories. I am extremely grateful to my lovely wife Alisha. Without her support and understanding this thesis and my PhD were unachievable. She has been extremely supportive of me throughout this entire process and has made countless sacrifices to help me get to this point. She always motivated, inspired and most importantly never lost faith in me. Her smiling face always provided me with added fuel to work towards this thesis. A special thanks to my parents Shamsuddin and Zubeda Awadia and Kusoom Khoja, Scott and Laurie Hahnlen, my brother Sahil, and sister-in-law Anjum for their unfailing faith and support. A special thanks to kids of our family Ronak, Ijju. Lastly, how can I forget my little angel, my son Aariz. His love and affection always kept me going. vii TABLE OF CONTENTS ABSTRACT……………………………………………………………………………..iii ACKNOWLEDGEMENTS ................................................................................................................... vii TABLE OF CONTENTS ..................................................................................................................... viii LIST OF FIGURES ................................................................................................................................... 1 LIST OF ABBREVIATIONS ................................................................................................................. 4 Chapter 1 Introduction ..................................................................................................................... 1 1.1 The structure of epithelial tissue and its role in cancer ............................................................. 1 1.1.1 Tight junctions as a barrier in epithelial tissue ................................................................ 2 1.1.2 The adherens junction “sticks” cells together ................................................................... 3 1.1.3 The dynamic actin cytoskeleton regulates apical constriction in epithelial tissue ................................................................................................................................................. 5 1.2 Epithelial Cell Polarity .............................................................................................................................. 6 1.2.1 The Scribble complex regulates apicobasal cell polarity in epithelial cells .......... 8 1.3 Rho GTPases are key modulators of epithelial polarity and junctional architecture .. 13 1.3.1 RhoG and its function in actin cytoskeleton regulation .............................................. 14 1.3.3 Structure and function of SGEF, a RhoG exchange factor ........................................... 15 1.4 PDZ domains interaction with PDZ Binding Domain (PBD) ................................................... 16 1.5 Model systems to understand junctions in epithelial tissue .................................................. 17 1.5.1 Fixed MDCK cells for 2D junction analysis ....................................................................... 17 1.5.2 The MDCK 3D cysts embedded in matrigel to understand junction and polarity ............................................................................................................................................................ 18 viii 1.5.3 Xenopus embryo to understand junctions and polarity in live epithelial environment ................................................................................................................................
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