Regulation of autophagosome formation by the PX-domain proteins SNX18 and HS1BP3 Kristiane Søreng Thesis for the degree of Philosophiae Doctor Department of Molecular Medicine Institute of Basic Medical Sciences UNIVERSITY OF OSLO 2017 © Kristiane Søreng, 2017 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-104-6 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................. 1 LIST OF PUBLICATIONS .................................................................................. 2 ABBREVIATIONS ............................................................................................... 3 INTRODUCTION ................................................................................................. 6 The endomembrane system and membrane trafficking ..................................... 6 Vesicle formation ............................................................................................... 8 Membrane identity in the endomembrane system ............................................. 9 Phosphoinositides ........................................................................................... 9 RABs and SNAREs ........................................................................................ 10 Autophagy ........................................................................................................ 10 Autophagy from a historical perspective ..................................................... 11 Core autophagic machinery and regulation ................................................ 12 The Atg1/ULK1 complex .............................................................................. 12 The PIK3C3/Vps34 complex ........................................................................ 14 Atg12 and Atg8 and their conjugation systems ............................................ 14 ATG9 ............................................................................................................. 15 Membrane input for autophagosome formation .............................................. 16 Phagophore nucleation ................................................................................ 16 Membranes sources contributing to forming autophagosomes ................... 17 Mitochondria ................................................................................................ 17 The Golgi apparatus ..................................................................................... 18 ERGIC and ERES ......................................................................................... 19 Plasma membrane and Recycling endosomes .............................................. 19 Phospholipids and lipid modifying enzymes in autophagy ............................. 20 Autophagic PI-binding proteins ................................................................... 22 AIMS OF THE STUDY ...................................................................................... 24 SUMMARY OF INCLUDED PAPERS ............................................................. 25 DISCUSSION ..................................................................................................... 29 Membrane input from recycling endosomes to forming autophagosomes ..... 29 SNX18 promotes autophagosome formation ................................................... 31 SNX18 binds to PtdIns(4,5)P2 ......................................................................... 33 SNX18 interacts with LC3- and GABARAP-proteins .................................... 34 SNX18-dynamin interaction is important during vesicle formation from recycling endosomes ........................................................................................ 35 HS1BP3 is a negative regulator of recycling endosome-derived autophagic precursors ......................................................................................................... 37 The PX domain of HS1BP3 binds phosphatidic acid ...................................... 37 Autophagy and the actin cytoskeleton ............................................................. 40 SNX18 and HS1BP3 regulate autophagosome formation ............................... 41 EXPERIMENTAL CONSIDERATIONS .......................................................... 44 Cell lines and transfections .............................................................................. 44 CRISPR-Cas9 technology ................................................................................ 45 Confocal microscopy, electron microscopy and quantitative image analysis . 46 Autophagy monitoring assays .......................................................................... 47 Interaction studies ............................................................................................ 48 Lipidomics analysis ......................................................................................... 49 The use of model organisms ............................................................................ 50 FUTURE PERSPECTIVES ................................................................................ 51 REFERENCES .................................................................................................... 53 ORIGINAL PUBLICATIONS ............................................................................ 65 ACKNOWLEDGEMENTS The work of this thesis was performed in the laboratory of Professor Anne Simonsen, at the Department of Molecular Medicine, Institute of Basic Medical Sciences at the University of Oslo from 2011 to 2017. The period included a 25 % teaching position that has provided me with the useful experience of being a PBL/LSB-teacher. First I want to express my gratitude to my supervisor, Professor Anne Simonsen, for giving me the opportunity to be a part of this group. Thank you for all your help and valuable guidance and for always taking the time to answer questions and allowing me to develop as a researcher. Your enthusiasm, broad scientific knowledge and concern for your group have inspired me and given me a genuine interest for scientific research. Thanks to Tore Jahnsen at the section of Biochemistry, for being my co-supervisor. To the past and presents members of the group, you have all contributed to this work by sharing your knowledge and taking time to discuss both scientific and non-scientific matters, which I am very grateful for. Thanks to Christian Bindesbøll, my “office-mate”, for our nice conversations. Special thanks to Alf Håkon Lystad for being a good friend, bearing with my ups and downs, and to Gunnveig Toft Bjørndal for your continuous encourage and support! Many thanks to Helene Knævelsrud for everything you taught me during my first years, for great teamwork and collaboration. I would like to thank all co-authors for your collaboration, input and feedback. Thanks to Sven Carlsson for your contribution, help and scientific input on all three papers. Especially I want to thank Sharon Tooze and her lab members at the Francis Crick Institute, London, for giving me the opportunity to come and spend six very nice and exciting months in your autophagy lab. To my dear family, thank you for your care and support during these years, and for your extremely valuable help in order for me to finish this thesis. A special thanks to Kristoffer, for your love, patience and for believing in me. I could not have done this without you. Finally, a big thanks to my two beautiful children, Helle and Eivind, for providing times of distractions filled with love and joy. Oslo, May 2017 Kristiane Søreng 1 LIST OF PUBLICATIONS I. Knaevelsrud, H.*, Soreng, K.*, Raiborg, C., Haberg, K., Rasmuson, F., Brech, A., Liestol, K., Rusten, T.E., Stenmark, H., Neufeld, T.P., Carlsson, S.R. & Simonsen, A. (2013), Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. Journal of Cell Biology, 202, 331-49. II. Holland, P.*, Knæveslrud, H.*, Søreng, K., Mathai, B.J., Lystad, A. H., Pankiv, S., Bjørndal, G. T., Schultz, S. W., Lobert, V. H., Chan, R. B., Zhou, B., Liestøl, K., Carlsson, S. R., Melia, T. J., Di Paolo, G. & Simonsen, A., (2016), HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels. Nature Communications, 7, 13889. III. Søreng, K., Munson, M. J., Lamb, C. A., Tooze, S., Carlsson, S. R. & Simonsen, A., SNX18 regulates ATG9 trafficking from recycling endosomes by recruiting dynamin-2. Manuscript in preparation * Contributed equally Related publications not included in this thesis: Søreng, K., Neufeld, T., Simonsen, A., (2017), Membrane trafficking in autophagy. Review article, IRCMB, in press. Søreng, K., Knævelsrud, H., Holland, P. & Simonsen, A. (2017), HS1BP3 inhibits autophagy by regulation of PLD1, Autophagic punctum, Autophagy, 1-2 2 ABBREVIATIONS 3MA 3-methyladenine ALFY Autophagy-linked FYVE protein ALR Autophagic lysosome reformation AMP Adenosine monophosphate AMPK AMP-activated protein kinase AP Adaptor protein APP Amyloid precursor protein Arp Actin related protein ATG Autophagy-related BafA1 Bafilomycin A1 BAR Bin/Amphiphysin/Rvs-homology Cas CRISPR-associated CMA Chaperone-mediated autophagy CME Clathrin-mediated endocytosis CRISPR Clustered Regularly Interspaced Short Palindromic Repeats DFCP1 Double FYVE containing protein