Interaction, Function and Regulation of the Tight Junction Protein Tricellulin Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Jimmi David Cording from Lingen (Ems) 2015 The present thesis was realized in the research group Molecular Cell Physiology at the Leibniz-Institut für Molekulare Pharmakologie (FMP) in Berlin-Buch in the time scale from November 2008 until May 2015 under supervision of PD Dr Ingolf E. Blasig. 1. Gutachter: PD Dr. I. E. Blasig Leibniz-Institut für Molekulare Pharmakologie (FMP) Campus Berlin-Buch Robert-Roessle-Str. 10 13125 Berlin 2. Gutachter: Prof. Dr. Volker Haucke Leibniz-Institut für Molekulare Pharmakologie (FMP) Campus Berlin-Buch Robert-Roessle-Str. 10 13125 Berlin Date of Disputation: 02.11.2015 Content Content 1 List of abbreviations .................................................................................. 4 2 Introduction .................................................................................................. 6 2.1 Organization and function of epithelium and endothelium ................................... 6 2.1.1 Tight junctions ................................................................................................ 8 2.2 Molecular composition of tight junctions .............................................................. 9 2.2.1 Claudins ........................................................................................................ 10 2.2.2 Tight junction associated MARVEL proteins .............................................. 13 2.2.3 Occludin ........................................................................................................ 14 2.2.4 Tricellulin ..................................................................................................... 15 2.2.5 MarvelD3 ...................................................................................................... 18 2.2.6 Tight junction-associated proteins ................................................................ 19 2.2.7 The Angulin-protein family .......................................................................... 20 2.3 Tight junction role in pathological events ........................................................... 21 2.3.1 Tight junctions during alterations of the Redox-state .................................. 22 3 Purpose and objectives .......................................................................... 24 4 Manuscript 1 ............................................................................................. 26 4.1 Synopsis ............................................................................................................... 27 4.2 Experimental contribution ................................................................................... 29 4.3 Manuscript 1 ........................................................................................................ 30 4.4 Supplemental material ..................................................................................... 5947 5 Manuscript 2 ............................................................................................. 51 5.1 Synopsis ............................................................................................................... 52 5.2 Experimental contribution ................................................................................... 53 5.3 Manuscript 2 ........................................................................................................ 54 5.4 Supplemental material ......................................................................................... 85 6 Manuscript 3 ............................................................................................. 69 6.1 Synopsis ............................................................................................................... 70 6.2 Experimental contribution ................................................................................... 72 6.3 Manuscript 3 ........................................................................................................ 72 6.4 Supplemental material ......................................................................................... 73 7 Manuscript 4 .............................................................................................. 88 7.1 Synopsis ............................................................................................................... 89 7.2 Experimental contribution ................................................................................... 91 7.3 Manuscript 4 ........................................................................................................ 92 7.4 Supplemental material ....................................................................................... 108 8 Discussion ................................................................................................. 110 8.1 Conclusion and outlook ..................................................................................... 122 9 Summary ................................................................................................... 124 9.1 Zusammenfassung .............................................................................................. 125 10 References ................................................................................................ 127 11 List of publications ................................................................................ 137 11.1 Enclosed in this thesis .................................................................................... 137 11.2 Other publications .......................................................................................... 137 11.3 Presentations at international meetings .......................................................... 138 12 Appendix ............................................................................................. 86139 12.1 Acknowledgements ........................................................................................ 139 3 List of abbreviations 1 List of abbreviations aa amino acid ATP adenosin-5´-triphosphat BBB blood-brain barrier bp base pair BSA bovine serum albumin bTJ bicellular tight junction CFP cyan fluorescent protein CK1 casein kinase 1 CK2 casein kinase 2 Cld claudin CRFR1 corticotropin-releasing factor receptor DAPI 4′,6-diamidin-2-phenylindol DMEM Dulbecco’s Modified Eagle Medium DNA desoxyribonucleic acid dNTP desoxyribonucleotid-5’-triphosphat E.coli Escherichia coli EDTA ethylendiamintetraacetat ER endoplasmatic reticulum FACS fluorescence activated cell sorting FCS fetal calf serum FRET fluorescence resonance energy transfer FRAP fluorescence recovery after photobleaching G418 geneticin GST gluthathion-S-transferase HA Human influenza hemagglutinin HEK human embryonic kidney cells IB immunblot IgG immunglobulin G IP co-Immunpräzipitation JAM junctional adhesion molecule MDCK madin-darby canine kidney cells MD3 marevelD3 MEM minimum essential medium Occl occludin PAGE polyacrylamidgel- electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PKA cAMP-dependent protein kinase PKC protein kinase C RNA ribonucleic acid RNAi RNA interference ROS reactive oxygen species RT room temperature SDS sodiumdodecylsulfate TEMED N, N, N', N'-Tetramethyethylendiamin TER transepithelial electric resistance TJ tight junctions Tric tricellulin Tris Tris (hydrocymethyl) aminoethan 4 List of abbreviations Triton-X100 t-Oktylphenoxypolyethoxyethanol tTJ tricellular tight junctions Tween20 Polyoxyethylensorbitanmonolaurat U units UV ultraviolet YFP yellow fluorescent protein ZO zonula occludens ZO-1 zonula occludens protein 1 5 Introduction 2 Introduction Multicellular organs rely on compartmentalization, to protect the body and to maintain homeostasis of the compartments, which is guaranteed by epithelial or endothelial cells. Organs, such as the uterus, ovaries, the brain and epidermis are all barrier containing tissues primarily intended to separate organ compartments (1). However, tissue barriers do not simply separate two distinct compartments or fluids; they provide protection against potentially harmful external agents and notably also arrange communication of neighboring cells. An essential feature of such barriers is represented by tissue-specific cellular junctions and cell-cell contacts, maintained and regulated by various multi protein complexes such as adherens junctions, gap junctions and tight junctions (TJs). A broad range of distinct disease conditions in humans is directly connected to alterations in epithelial or endothelial barrier properties. This may lead to a loss of stromal homeostasis and thereby affect fluid and electrolyte balance between compartments, within tumor microenvironment or generation of inflammatory states. Tumor progression or bacterial invasion through tissue barriers is accompanied by epithelial/endothelial barrier dysfunction or tightness modulation, often reflecting tumor- or pathogen-induced alterations of specific structures of these tissues, particularly of the TJs (1, 2). In these cases as in many other, pathological conditions, direct/indirect damage of tissue and TJs are found to relevantly contribute to disease progression. 2.1 Organization and function of epithelium and endothelium The four major tissue types are muscle, nerve, connective and epithelial tissues.