This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Jellett, Adam Title: Dissecting the molecular and functional interactions of retromer General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Dissecting the molecular and functional interactions of retromer Adam Patrick Jellett A dissertation submitted to the University of Bristol in accordance with the requirements for award of degree of PhD in the Faculty of Life Sciences. School of Biochemistry University of Bristol December 2018 Word count: 44,523 i Table of contents Table of contents .............................................................................................. i List of Figures ................................................................................................. vi List of Tables .................................................................................................... x Abstract ........................................................................................................... xi Acknowledgments ......................................................................................... xii Authors declaration ...................................................................................... xiii Abbreviations ................................................................................................ xiv Publications .................................................................................................. xvii Chapter 1: Introduction.................................................................................... 1 1.1 Overview of membrane trafficking .................................................................. 2 1.1.1 The principles of membrane trafficking ........................................................................ 2 1.1.2 Membrane identity ........................................................................................................ 2 1.1.2.1 Phosphoinositides ................................................................................................. 3 1.1.2.2 Rab GTPases ........................................................................................................ 5 1.2 The endolysosomal pathway ........................................................................... 6 1.2.1 Endocytosis .................................................................................................................. 7 1.2.2 The structure and organisation of the endosomal network .......................................... 8 1.2.3 Endosomal maturation ............................................................................................... 11 1.2.3.1 Endosomal Rab conversion ................................................................................ 11 1.2.3.2 Endosomal phosphoinositide conversion ............................................................ 12 1.2.3.3 The promotion of membrane fusions in the endolysosomal pathway ................. 13 1.3 Endosomal cargo sorting ................................................................................13 1.3.1 ESCRT-mediated degradative cargo sorting ............................................................. 14 1.3.2 Endosomal retrieval and recycling ............................................................................. 17 1.3.2.1 Sequence-dependent vs sequence-independent retrieval and recycling ........... 17 1.3.2.2 Avoiding the degradative fate .............................................................................. 18 1.4 Endosomal retrieval and recycling complexes ..............................................19 1.4.1 The retromer complex ................................................................................................ 19 1.4.2 Cargo recycling by the SNX-BAR complex ................................................................ 22 1.4.3 Cargo recognition by retromer ................................................................................... 23 1.4.3.1 SNX3-retromer .................................................................................................... 24 1.4.3.2 SNX27-retromer .................................................................................................. 25 1.4.4 Spatiotemporal control of retromer ............................................................................. 26 1.4.5 The WASH complex ................................................................................................... 27 1.4.6 Retromer-independent endosomal retrieval ............................................................... 29 1.5 Endosomal cargo retrieval and recycling is neuroprotective .......................32 i 1.5.1 Associations between endosomal retrieval complexes and neurological disorders .. 34 1.6 Aims ..................................................................................................................35 Chapter 2: Materials and Methods ................................................................ 37 2.1 Materials ...........................................................................................................38 2.1.1 List of suppliers .......................................................................................................... 38 2.1.2 Sterilised water ........................................................................................................... 38 2.1.3 Cell lines used in this study ........................................................................................ 38 2.1.4 Reagents for cell culture ............................................................................................ 39 2.1.5 Bacterial cell strains ................................................................................................... 40 2.1.6 Bacterial growth media and antibiotics....................................................................... 40 2.1.7 Buffers and solutions .................................................................................................. 41 2.1.8 Plasmid vectors .......................................................................................................... 41 2.1.9 Oligonucleotides ......................................................................................................... 44 2.1.10 Antibodies ................................................................................................................. 48 2.2 Methods ............................................................................................................51 2.2.1 Bacterial cell culture ................................................................................................... 51 2.2.1.1 Bacterial colony growth ....................................................................................... 51 2.2.1.2 Transformation of competent E. coli ................................................................... 51 2.2.1.3 Bacterial liquid cultures ....................................................................................... 51 2.2.2 Molecular biology methods ........................................................................................ 52 2.2.2.1 Small scale purification of plasmid DNA (Miniprep) ............................................ 52 2.2.2.2 Large-scale purification of DNA (Maxiprep) ........................................................ 52 2.2.2.3 Nucleic acid quantification ................................................................................... 53 2.2.2.4 DNA sequencing ................................................................................................. 53 2.2.2.5 Polymerase Chain Reaction (PCR) for gene amplification ................................. 53 2.2.2.6 Polymerase Chain Reaction (PCR) Site-Directed mutagenesis (SDM) .............. 54 2.2.2.7 RNA extraction and quantitative-reverse transcriptase PCR .............................. 55 2.2.2.8 DNA analysis with agarose gel electrophoresis .................................................. 56 2.2.2.9 Purification of PCR and restriction digest products ............................................. 56 2.2.2.10 Restriction enzyme digestion ............................................................................ 57 2.2.2.11 Ligations ...........................................................................................................