Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 The role pf Gaf-1b in intracellular trafficking and autophagy Diep, Tu-My Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-68814 Dissertation Published Version Originally published at: Diep, Tu-My. The role pf Gaf-1b in intracellular trafficking and autophagy. 2012, University of Zurich, Faculty of Science. The Role of Gaf-1b in Intracellular Trafficking and Autophagy Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Tu-My Diep von Bern Promotionskomitee Prof. Dr. Peter Sonderegger (Vorsitz) Prof. Dr. Jack Rohrer Dr. Uwe Konietzko Zürich 2012 CONTENTS Contents Summary ................................................................................................................... 1 Zusammenfassung ................................................................................................... 3 Abbreviations ............................................................................................................ 5 Publications .............................................................................................................. 9 1. Introduction ......................................................................................................... 11 1.1 Intracellular Transport ................................................................................................................. 11 1.2 Secretory Pathway ...................................................................................................................... 13 1.3 Endocytic Pathways .................................................................................................................... 15 1.4 Endocytic Pathways in polarized cells ........................................................................................ 18 1.5 Autophagy ................................................................................................................................... 20 1.6 SNAREs ...................................................................................................................................... 23 1.7 Rab proteins ................................................................................................................................ 25 1.8 Rab11 and Rab11-interacting proteins ....................................................................................... 27 1.9 The calsyntenin family ................................................................................................................. 29 1.10 GABARAP-likes and MAP1LC3 family ..................................................................................... 32 1.11 Aims .......................................................................................................................................... 35 2. Results ................................................................................................................. 37 2.1 Gaf-1b is a novel interaction partner of calsyntenin-1 ................................................................ 37 2.2 Study of the interaction of Gaf-1b and calsyntenin-1 .................................................................. 39 2.2.1 Calsyntenin-1 interacts specifically with Gaf-1b ................................................................................... 39 2.2.2 The C2 domain of Gaf-1b is involved in the interaction with calsyntenin-1 .......................................... 40 2.2.3 The acidic stretch of calsyntenin-1 is crucial for the interaction with Gaf-1b ........................................ 42 2.2.4 The formation of Gaf-1b/Rip11 complex disrupts the interaction of calsyntenin-1 and Gaf-1b ............ 44 2.3. Gaf-1b and calsyntenin-1 are proteins of recycling endosomes ................................................ 45 2.3.1 Gaf-1b and Rip11 exclusively interacts with GTP-Rab11 .................................................................... 45 2.3.2 Calsyntenin-1 interacts with the recycling-endosomal protein Rab11 through Gaf-1b ......................... 48 2.3.3 Gaf-1b localizes with endosomal recycling and TGN marker in neurons ............................................. 49 2.4 Gaf-1b is transported in calsyntenin-1 vesicles of the recycling-endosomal pathway................ 50 2.4.1 Gaf-1b and Rip11 are selectively associated with the recycling-endosomal type of calsyntenin-1 transport packages ....................................................................................................................................... 50 CONTENTS 2.4.2 Gaf-1b is coupled to the calsyntenin-1/syntaxin 13 complex. .............................................................. 51 2.4.3 Gaf-1b is transported in calsyntenin-1 organelles devoid of APP ........................................................ 53 2.4.4 Gaf-1b interacts simultaneously with calsyntenin-1 and kinesin-light chain 1 ...................................... 54 2.5 Gaf-1b trafficking is disrupted upon blockage of calsyntenin-1 and KLC1 TGN exit .................. 56 2.6 Gaf-1b and receptor recycling ..................................................................................................... 58 2.7 Overexpression of Gaf-1b and calsyntenin-1 affects the morphology of HeLa cells .................. 60 2.7.1 Gaf-1b knock-down induces stress fibre formation .............................................................................. 60 2.8 Gaf-1b is necessary for cell viability ............................................................................................ 62 2.9 GABARAPL1 and GABARAPL2 interact with Gaf-1b ................................................................. 64 2.9.1 GABARAPL1 and GABRAPL2 are novel interaction partners of Gaf-1b .............................................. 64 2.9.2 Localization of the binding domain of Gaf-1b for the interaction with GABARAPL2 ............................. 66 2.9.3 Gaf-1b colocalizes with GABARARPL1 and GABARAPL2 .................................................................. 67 2.10 Gaf-1b is involved in the autophagic pathway .......................................................................... 69 2.10.1 Gaf-1b associates with membranes of the autophagosomal pathway ............................................... 69 2.10.2 Gaf-1b accumulates in perinuclear aggregates upon starvation ........................................................ 70 2.10.3 Gaf-1b knock-down induces autophagosomes .................................................................................. 72 3. Discussion .......................................................................................................... 73 3.1 Gaf-1b interacts with calsyntenin-1 ............................................................................................. 73 3.2 Gaf-1b is associated with the TGN ............................................................................................. 76 3.3 Gaf-1b is transported in TGN-derived calsyntenin-1 organelles along axons ............................ 77 3.4 Gaf-1b is associated with the endosomal recycling compartment and interacts with endosomal recycling markers .............................................................................................................................. 77 3.5 Gaf-1b has a minor effect on receptor recycling ......................................................................... 81 3.6 Gaf-1b is involved in the autophagic pathway ............................................................................ 82 3.7 Gaf-1b is essential for cell viability .............................................................................................. 84 4. Material and Methods ......................................................................................... 87 4.1 Antibodies and Reagents ............................................................................................................ 87 4.1.1 Affinity-purified antibodies .................................................................................................................... 87 4.1.2 Purchased antibodies .......................................................................................................................... 87 4.1.3 Provided antibodies ............................................................................................................................. 88 4.1.4 Reagents ............................................................................................................................................. 88 4.2 Constructs ................................................................................................................................... 89 4.3 siRNA and shRNA constructs ..................................................................................................... 91 4.3.1 siRNA ................................................................................................................................................... 91 4.3.2 shRNA ................................................................................................................................................. 91 4.4 Molecular cloning .......................................................................................................................