PhD degree in Molecular Medicine (curriculum in Molecular Oncology) European School of Molecular Medicine (SEMM), University of Milan and University of Naples “Federico II” Settore disciplinare: Bio/11 Novel functions of the SNARE protein Snap29 in membrane trafficking and cell division Elena Morelli IFOM, Milan Matricola n. R09394 Supervisor: Dr. Thomas Vaccari IFOM, Milan Anno accademico 2013-2014 Parts of this thesis have been published in: Morelli E, Ginefra P, Mastrodonato V, Beznoussenko G, Rusten TE, Bilder D, Stenmark H, Mironov AA, Vaccari T (2014). Multiple functions of the SNARE protein Snap29 in autophagy, endocytic and exocytic trafficking during epithelia formation in Drosophila. Autophagy. DOI: 10.4161/15548627.2014.981913 Figure 20H- I’’ and Figure 26A- N were performed by Valeria Mastrodonato. TABLE OF CONTENTS LIST OF ABBREVIATIONS ................................................................................................ 5 FIGURES INDEX ................................................................................................................. 7 1. ABSTRACT ...................................................................................................................... 9 2. INTRODUCTION ........................................................................................................... 11 2.1 SNARE proteins control vesicle and organelle dynamics ............................................... 11 2.1.1 Membrane fusion and regulation of SNARE complex formation .............................. 11 2.1.2 SNARE proteins in Golgi Apparatus and Endoplamic Reticulum network dynamics ................................................................................................... 16 2.1.3 SNARE function in autophagy ................................................................................. 18 2.1.4 Routes of conventional and unconventional secretion involving SNAREs ................ 21 2.1.5 The SNARE protein SNAP29 ................................................................................... 23 2.2 Trafficking and mitosis ................................................................................................. 27 2.2.1 The cell cycle and the onset of mitosis ...................................................................... 27 2.2.2 Endoplasmic Reticulum dynamics and Nuclear Envelope Breakdown ...................... 29 2.2.3 Golgi Apparatus fragmentation at the onset of mitosis .............................................. 33 2.2.4 Formation of the mitotic spindle ............................................................................... 34 2.2.5 Chromosomes movement by MT motor proteins ...................................................... 36 2.2.6 The KT structure and formation................................................................................ 37 2.2.7 The outer KT plate and KT-MT attachment .............................................................. 39 2.2.8 Aurora B and its role in sensing KT-MT tension ....................................................... 43 2.2.9 The RZZ complex and Spindly at the outer KT ........................................................ 44 2.2.10 The SAC and the onset of anaphase ........................................................................ 46 2.3 Endocytosis and autophagy in mitotic cells .................................................................... 48 2.3.1 Trafficking proteins with moonlighting role during cell division ............................... 49 2.4 NE re-assembly .............................................................................................................. 52 1 3. AIMS OF THE PROJECT .............................................................................................. 53 4. MATERIALS AND METHODS ..................................................................................... 54 4.1 Flies strains, mapping, genetics ..................................................................................... 54 4.2 Generation of transgenic Drosophila lines ..................................................................... 55 4.3 Antibodies production ................................................................................................... 56 4.4 Production of siRNA resistant human snap29 cDNA..................................................... 57 4.5 Cell cultures .................................................................................................................. 58 4.6 Double stranded RNA interference in S2 cell lines ........................................................ 58 4.7 SNAP29 siRNA mediated silencing in HeLa and U2OS-HB2-GFP-mCherry-α Tubulin ....................................................................................................................... 59 4.8 Double thymidine block in HeLa cells ........................................................................... 59 4.9 Immunostainings ........................................................................................................... 60 4.10 Notch trafficking assay ................................................................................................ 61 4.11 Imaging ....................................................................................................................... 62 4.12 Protein extraction and Western Blot ............................................................................ 62 4.13 Immunoprecipitation and LC-MS/MS analysis ............................................................ 63 4.14 Reverse Transcription (RT)-PCR................................................................................. 64 4.15 Electron microscopy and tomography .......................................................................... 65 4.16 Adult Wing preparation ............................................................................................... 65 4.17 Time lapse of U2OS-HB2-GFP-mCherry-αTubulin..................................................... 66 4.18 Bioinformatics............................................................................................................. 66 4.19 List of Primers............................................................................................................. 67 5. RESULTS ........................................................................................................................ 69 5.1 Characterization of B6 mutation in Drosophila epithelial organs ................................... 69 5.1.1 MENE (2R)-B6-21 disrupts epithelial tissue architecture in Drosophila ............... 69 5.1.2 MENE (2R)-B6-21 is a mutant in the gene encoding Drosophila Snap29 ............. 69 2 5.1.3 Snap29 localizes to multiple trafficking organelles in interphase and to KTs in mitosis ............................................................................................................... 74 5.2 SNAP29 is a regulator of autophagy and trafficking pathways in Drosophila epithelial tissues .......................................................................................................... 76 5.2.1 Snap29 mutant tissue display altered autophagy and Golgi Apparatus organization ....................................................................................................... 76 5.2.2 Snap29 controls fusion of autophagosomes to degradative organelles ................... 80 5.2.3 Snap29 is a negative regulator of autophagosomes secretion ................................. 83 5.2.4 Snap29 regulates Notch and Domeless receptor secretion and degradation ........... 84 5.3 SNAP29 is required for KT stability and MT-KT attachment during cell division in Drosophila and mammalian cells .................................................................................. 87 5.3.1 Snap29 localizes at the outer KT platform and is important for cell division in Drosophila S2 cells............................................................................................ 87 5.3.2 Human SNAP29 associates to centrosomes and MTs during mitosis and transiently localizes near KT during prophase .................................................... 88 5.3.3 SNAP29 regulates prophase to metaphase transition ............................................. 92 5.3.4 SNAP29 controls KT stability in HeLa cells ......................................................... 95 5.3.5 Depletion of SNAP29 impairs MTs attachment to the KT platform ...................... 96 5.3.6 SNAP29 depleted cells overcome SAC arrest ....................................................... 97 5.4 The in vivo relevance of the trafficking and cell division functions of SNAP29 for epithelial architecture ................................................................................................ 100 5.4.1 Autophagosome accumulation does not cause the epithelial tissue defects observed in Snap29B6 mutant eye disc .............................................................. 100 5.4.2 Epithelial tissue disruption in Snap29B6 mutant epithelial discs is sustained by JAK/STAT signaling pathway ............................................................................. 102 5.4.3 Cell division is affected in Snap29B6 mutant discs ..................................................... 105 5.5 Snap29 function in autophagy and cell division are independent .......................................... 107 6. DISCUSSION ...............................................................................................................