Universidade de Lisboa Faculdade de Medicina Unidade de Biologia do Cancro, Instituto de Medicina Molecular The regulation of Interleukin 7 receptor alpha internalization, recycling and degradation by IL-7 - Possible implications in T-cell homeostasis, migration and leukaemogenesis - Catarina Martins de Oliveira Henriques Doutoramento em Ciências Biomédicas For the degree of Doctor of Philosophy 2009 Universidade de Lisboa Faculdade de Medicina Unidade de Biologia do Cancro, Instituto de Medicina Molecular The regulation of Interleukin 7 receptor alpha internalization, recycling and degradation by IL-7 - Possible implications in T-cell homeostasis, migration and leukaemogenesis - Catarina Martins de Oliveira Henriques (Recipient of a scholarship- SFRH7BD/21940/2005 from Fundação para a Ciência e Tecnologia) Tese orientada pelo Doutor João T. Barata, Prof Doutor.Gerard Graham e Prof. Doutora Leonor Parreira Doutoramento em Ciências Biomédicas, especialidade em Ciências Biopatológicas For the degree of Doctor of Philosophy 2009 As opiniões expressas são da exclusiva responsabilidade do seu autor A impressão desta dissertação foi aprovada pela Comissão Coordenadora do Conselho Científico da Faculdade de Medicina de Lisboa em reunião de 13 de Outubro de 2009. Para a Prof. Filomena Mota. Sem o seu apoio e inspiração há muitos anos atrás, eu não seria hoje uma bióloga nem esta tese teria alguma vez existido… Table of Contents Table of contents……………………………………………………….………………….................. i Aknowledgements…………………………………………………………………………………………….. iii Abbreviation list………………………………………………………………………………………......... v Abstract…………………………………………………………………………………………………............ix Resumo…………………………………………………………………………………………………………......xi Chapter 1: General Introduction …………………………………………………………..........1 1.1 Signalling at the cell surface and beyond …...………………............3 ● The Eukaryotic cell…………………………………………………………....….........3 ● Receptor-mediated endocytosis, recycling and degradation........6 ● Receptor targeting and sorting………….………………………………….......11 ● Receptor trafficking and signalling……………………………......……………13 1.2 The immune system and T-cell migration ………………….......….14 ● T-cell polarization and motility………………………………………......……….15 ● Choosing the right direction………………………………………......……………16 ● T-cell homing……………………..………………………………………......……….….22 ● The adhesion cascade…………………………………………………….......………24 ◦ Rolling……………………………………………………………….….......……….25 ◦ Activation and arrest………………….…….…………......…………….27 ◦ Trans-endothelial migration………………………………......…………28 1.3 T-cell homeostasis: The role of Interleukin 7……….........…….38 1.4. IL-7 and T-cell acute lymphoblastic leukaemia: Proliferation, survival, growth and dissemination ?.................37 1.5 Thesis overview …………………………………………………………………………….41 i Chapter 2: Assessing IL-7 mediated T-cell motility and migration in vitro ……………………………………………………………………………………………………………….….43 2.1 Abstract…………………………………………………………………………………………..45 2.2 Introduction…………………………………………………………….....……………..…45 2.3 Materials and Methods …….....………………………..………….……………….48 2.4 Results …………………………………………………..………………….....……………….50 2.4.1 IL-7 stimulation activates T-ALL cell motility in vitro ………50 2.4.2 IL-7 attracts and retains T-ALL cells in vitro …………….….…52 2.4.3 IL-7 induces transient up-regulation of key adhesion molecules and CCR5 chemokine receptor in T-ALL cells……...........…….54 2.5 Discussion ……………………………………………………………………......……….…56 Chapter 3: Regulation of IL-7Rα internalization, recycling and degradation by IL-7…………………………………………………….…………………..…….…….63 3.1 Abstract …………………………………………………………………………………………..65 3.2 Introduction …………………………………………….....………………………………..65 3.3 Material & Methods……………………………….....…………………………………67 3.4 Results ……………………………………………………….....………………………..…….72 3.4.1 IL-7 induces rapid IL-7Rα internalization in T-cells…………72 3.4.2 IL-7Rα internalization occurs via clathrin-coated pits and is required for IL-7-mediated signalling……………………………………............…76 3.4.3 A pool of internalized IL-7Rα constitutively recycles back to the cell surface……………………………………..…………………………............………………80 3.4.4 IL-7 induces rapid IL-7Rα degradation in a lysosome-dependent manner……………………………………………….………………….........………….....…..…82 3.4.5 IL-7-induced IL-7Rα degradation, but not internalization, is partially dependent on JAK3 activity...........................................85 3.5 Discussion ..........................................................................87 Chapter 4: General discussion and perspectives ……………………........….……91 References …………………………………………......………………………………………………..…107 ii Aknowledgements I would like to thank my supervisor, Dr. João T. Barata, for his support and encouragement throughout these four years of constant challenge. I thank him for the opportunity of developing my PhD at his laboratory, where, under his guidance and with the help of all my colleagues at UBCa and UBH, I have learnt almost everything I know about science. Also for critical guidance in the writing of my PhD thesis and submitted paper. I would also like to thank the insightfull discussions I had with Joana Desterro. A significant part of my PhD was also spent at the University of Glasgow, under the supervision of Prof. Gerry Graham and close guidance by Dr. Robert Nibbs. To them I am truly gratefull, not only for the unique opportunity to work in a state of the art Biomedical Research Centre and laboratory, but also, and most importantly, for their friendship and true concern in providing the best scientific support. I thank you for the inspiring discussions and all that you have tought me about science and life in science. Thank you also to the people at the CRG, who have made me feel welcomed in the lab and were always there to help me in any way they could. A special appreciation to a few people who have truly improved my life during these challenging times: José Rino has helped me extensively throughout these four years, not only with crucial scientific advice, technical “know how” and help in the reviewing and formatting of this thesis, and submitted paper, but also with emotional support at the high and (many) low times. Also a special thanks to Maria Soares, who on the past year has helped me with insightful discussions and in the development of possibly my next scientific step. Thank you also for the last minute essential help in reviewing this thesis! The presence of close friends was of outmost importance, and made all the difference in overcoming the stressfull times. Antonio Ferra, Raul Bento, Anne McGinness and Ana Barros have helped me to hang on and gave me valuable scientific input whenever possible. It is impossible to describe all that I am gratefull for, and I can only hope that in my daily life I have made you all aware of how important you all are to me. Finally I would like to acknowledge the source of funding for this project, the Fundação para a Ciência e Tecnologia PhD scholarship and Startup funds from Instituto de Medicina Molecular. iii Abbreviation list AP: Adaptor-Protein APCs: Antigen Presenting Cells ATP: Adenosine Triphosphate CME: Clathrin-Mediated Endocytosis CNS: Central Nervous System DAG: Diacylglycerol DCs: Dendritic Cells DNA: Deoxyribonucleic Acid DOCK2: Downstream of Crk-180 homologue-2 protein S Dome: Domeless ECM: Extra Cellular Matrix EEA-1: Early Endosome Antigen 1 EGF: Epidermal Growth Factor EGFR: Epithelial Growth Factor Receptor EpoR: Erythropoietin Receptor ESAM: Endothelial cell-selective Adhesion Molecule ESL1: E-Selectin Ligand 1 F-Actin: Filamentous Actin FIPs: Rab11-Familiy Interacting Proteins FRET: Fluorescence Resonance Energy Transfer GAPs: GTPase-Activating Proteins GDP: GTPase Dissociation Inhibitors GEFs: Guanine-nucleotide Exchange Factors GPCRs: G-Protein Coupled Receptors GTPase: Guanosine Triphosphatase HEVs: High Endothelial Venules ICAM-1: Intercellular Adhesion Molecule 1 IEL: Intraepithelial Lymphocytes IL-2R-β: Interleukin-2 Receptor- β IL-5R: Interleukin-5 Receptor IL-7: Interleukin 7 v IL-7Rα: Interleukin-7 Receptor alpha IL-9Rα: Interleukin-9 Receptor alpha InsP3: Inositol 1, 4, 5 triphosphate JAK: Janus Kinases JAM: Junctional Adhesion Molecule JNK : c-Jun N-terminal kinase LAMP-1 and LAMP-2: Lysosomal-Associated Membrane Proteins 1 and 2 LFA-1: Lymphocyte- Function associated Antigen 1 LRP-6: Lipoprotein Receptor related Protein-6 MAC-1: Macrophage Antigen 1 MADCAM-1: Mucosal Vascular Addressin Cell-Adhesion Molecule 1 MAPK: Mitogen-activated Protein Kinase MHC: Major Histocompatibility Complex MLC: Myosin Light Chain MLCK: Myosin Light Chain Kinase mLN: Mesenteric Lymph Nodes MMPs: Matrix Metalloproteinases mTECs: Medullary Thymic Epithelial Cells NE: Neutrophil Elastase NK: Natural killer cells NKT: Natural Killer T-cells PDK1 and PDK2: Phosphoinositide-Dependent protein Kinase 1 and 2 PECAM-1: Platelet/Endothelial-Cell Adhesion Molecule 1 PI3K: Phosphoinositide 3 Kinase PIP2: Phosphatidylinositol-2-Phosphate PIP3: Phosphatidylinositol-3-Phosphate PKB: Protein Kinase B (also known as AKT) PLC: PhosphoLipase C pLN: Peripheral Lymph Nodes PP: Peyer’s Patches PSGL-1: P-Selectin Glycoprotein Ligand 1 PTEN: Phosphatase and Tensin homologue RAC: Ras-related C3 botulinum substrate Ras-MAPK: Ras Mitogen-Activated Protein Kinase vi Rho: Ras homologue Rho-A: Ras homologue gene-family member-A RNA: Ribonucleic Acid ROCK: Rho Kinase S1P: Sphingosine 1-Phosphate SAPK: Stress-Activated Protein Kinase SARA: Smad Anchor for Receptor Activation SCRs: Short Consensus Repeats SDF-1: Stromal-cell