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Role of Anillin in Regulation of Epithelial Junctions Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2014 Role of Anillin in Regulation of Epithelial Junctions Gibran Chadha Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medical Genetics Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/3363 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. ©Gibran Kamal Chadha 2014 All Rights Reserved ROLE OF ANILLIN IN REGULATION OF EPITHELIAL JUNCTIONS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Human Genetics at Virginia Commonwealth University. By Gibran Kamal Chadha BS in Biotechnology, University of Mumbai, 2011. Director: Andrei I Ivanov Ph.D. Human Genetics. Virginia Commonwealth University Richmond, Virginia May, 2014 ACKNOWLEDGEMENTS I would like to thank Dr. Ivanov for the opportunity to work in his laboratory and use this project for my thesis. I would also like to thank my friends and colleagues for their patience and support. Lastly but most importantly, I would like to thank my family, both here and back home, for all their endless love, patience and support over the past 2 years. ii TABLE OF CONTENTS List of Figures……………………………………………………………………………………..v List of Abbreviations…………………………………………………………………………….vii Abstract…………………………………………………………………………………………...ix Introduction……………………………………………...………………………………………...1 1. Role of epithelial barrier…………………………………………………………………..1 2. Molecular composition of epithelial junctions……………………………………………4 2.1 Tight junctions………………………………………………………………………...4 2.2 Adherens junctions………………………………………………………...………….6 3. Association of epithelial junctions with actomyosin skeleton…………………………….8 4. Nonmuscle myosin II as essential regulator of epithelial junctions…………………..…13 5. Rho GTPases signaling regulates the actomyosin cytoskeleton………………………....15 6. Anillin as an important F-actin, NM II and Rho-binding protein…………………...…...17 Methods and materials…………….,...…………………………………………………………..21 Results…………………………………………………………………………………………...26 Loss of anillin results in disruption tight junctions and adherens junction……………...26 Anillin is not accumulated at cell-cell contacts in interphase cells……………………...30 Anillin depletion induces EMT-like phenotype in A549 cells, but not in DU145 cell.…32 Anillin depletion does not increase cell motility………………………………………...35 Anillin depletion does not affect expression of different AJ and TJ proteins…………...37 Anillin depletion induces disorganization of the perijunctional F-actin belt in DU145 cells………………………………………………………………………………………39 Anillin depletion induces mislocalization of the peri-junctional nonmuscle myosin II....41 iii Knockdown of Anillin does not affect expression and activation status of NM II motor…………………………………………………………………………………….43 Anillin depletion results in dephosphorylation of ezrin-radixin-moesin (ERM) proteins…………………………………………………………………………………..45 Loss of anillin disrupts adducin-based membrane skeleton at epithelial cell-cell contacts………………………………………………………………………………......47 Anillin depletion does not affect adducin expression at transcription level……………..52 Loss of γ-adducin displaces α-adducin from the site of intercellular contacts…………..54 Loss of γ-adducin disrupts epithelial junctions…………………………………………..56 TGF-β treatment downregulates Anillin expression and disrupts epithelial junctions in A549 cells………………………………………………….………………………….…59 Discussion…………………………..……………………………………………………………61 Anillin promotes epithelial phenotype in A549 cells……………………………………62 Anillin promotes epithelial phenotype in A549 cells……….…………………...………62 Anillin regulates organization of the perijunctional actomyosin belt…………………...63 Anillin depletion disrupts the membrane skeleton…………………..…………………..64 Involvement of anillin cancer and tissue fibrosis……………..………………………....68 Conclusion………………….…………………………………………………………………....70 List of References……………….……………………………………………………………….71 Vita……………………………………………………….……………………………………...86 iv LIST OF FIGURES Figure 1: Junctions in a polarized cell…………………………………………………….3 Figure 2: Tight junctions…………………………………………………………………..5 Figure 3: Adherens junctions……………………………………………………………...7 Figure 4: Organization of the apical actin cytoskeleton in simple polarized epithelia…...9 Figure 5: Reorganization of actin cytoskeleton during apical junction formation……….11 Figure 6: Anillin specific siRNAs result in a marked depletion of targeted protein…….27 Figure 7: Anillin depletion results in disruption of adherens junctions in A549 lung epithelial cells……………………………………………………………………………28 Figure 8: Loss of Anillin results in disassembly of TJs and AJs in DU145 prostate epithelial cells……………………………………………………………………………29 Figure 9: Anillin does not localize at intercellular contacts in interphase cells………...31 Figure 10: Anillin depletion selectively induces epithelial-to myofibroblast transition (EMT) in A549 cells………………………………………………………………….32-33 Figure 11: Anillin depletion does not increase motility of epithelial cells……………...36 Figure 12: Effect of anillin knockdown on expression levels of AJ/TJ proteins in DU145 and A549 cells. ………………………………………………………………………….38 v Figure 13: Anillin depletion induces disorganization of perijunctional F-actin belt in DU145 cell. ……………………………………………………………………………...40 Figure 14: Anillin depletion induces mislocalization of perijunctional NM II motor…..42 Figure 15: Knockdown of Anillin does not affect expression and activation status of NM II motor. ………………………………………………………………………………....44 Figure 16: Anillin depletion results in dephosphorylation of ezrin-radixin-moesin (ERM) proteins. ………………………………………………………………………………….46 Figure 17: Loss of anillin disrupts the adducin-based membrane skeleton at epithelial cell-cell contacts. ………………………………………………………………………...48 Figure 18: Loss of anillin disrupts proper localization of γ-adducin…………………….49 Figure 19: Anillin depletion results in selective downregulation of -adducin………50-51 Figure 20: Anillin depletion does not affect adducin expression at transcription level....53 Figure 21: Loss of -adducin displaces -adducin from the areas of cell-cell contacts…55 Figure 22: Loss of -adducin disrupts epithelial junctions…………………………...57-58 Figure 23: TGF-β treatment down-regulates anillin expression and disrupts epithelial junctions in A549 cells…………………………………………………………………..60 vi LIST OF ABBREVIATIONS AND SYMBOLS ANLN: Anillin. AJ: Adherens Junction. TJ: Tight Junction. NM II: Non-muscle myosin II. F-actin: Filamentous actin. E-cad: E-cadherin. N-cad: N-cadherin. JAM-A: Junctional Adhesion Molecule-A ZO: Zonula Occludens SM-22: Smooth muscle marker 22 α-SMA: Alpha smooth muscle actin α: Alpha β: Beta γ: Gamma EMT: Epithelial Mesenchymal Transition EMyT: Epithelial Myofibroblast Transition TGF-β: Transforming growth factor β ATP: Adenosine Triphosphate GTP: Guanosine triphosphate GAP: GTPase activating protein GEF: GTP exchange factor vii DMEM: Dulbecco’s Modified Eagle Medium RMLC: Regulatory myosin light chain MLCK: Myosin light chain kinase ROCK: Rho-associated protein kinase TNF-α: Tumor necrosis factor alpha MT: Microtubules ERM: Ezrin-Radaxin-Moesin FSGS: Focal segmental glomerulosclerosis RCC: Renal cell carcinoma viii Abstract ROLE OF ANILLIN IN REGULATION OF EPITHELIAL JUNCTIONS By Gibran Kamal Chadha. A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Human Genetics at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Major Director: Andrei I Ivanov, Ph.D. Human Genetics. Adherens junctions (AJs) and tight junctions (TJs) are characteristic features of differentiated epithelial cells and are critical for regulation of epithelial barriers and cell polarity. Integrity and remodeling of epithelial junctions depend on their interactions with underlying actomyosin cytoskeleton. Anillin is a multifunctional scaffold able to interact with different cytoskeletal proteins including F-actin and Myosin II. This project aimed to investigate roles of anillin in regulating epithelial AJs and TJs. Using A549 human lung epithelial and DU145 human prostate epithelial cells, we demonstrated the anillin depletion-induced loss of AJs and TJs. This was accompanied by disorganization of perijunctional actomyosin belt and disruption of the adducin- based membrane skeleton that links actin filaments to the plasma membrane and epithelial junctions. Depletion of anillin decreased protein levels of γ-adducin and downregulation of γ- adducin mimicked effects anillin knockdown on AJ and TJ integrity. These findings suggest a novel role for anillin in the assembly of epithelial junctions. INTRODUCTION 1. Role of Epithelial barrier Epithelium plays a vital role in the survival and development of multicellular organisms. The epithelial layers establish a barrier between the body’s interiors and external environment, thus being crucial in maintaining the various internal compartments to preserve their unique architecture and chemical environment. (1) (2) Generally, simple epithelial linings in the reproductive, respiratory and renal systems are composed of a single layer of specialized cells (1) (3). The epithelium also serves as physical barrier between external pathogens and the immune system (4) (5) and is crucial in the
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