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The Dynamics and Function of the Endolysosomal/Lysosomal System

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The Dynamics and Function of the Endolysosomal/Lysosomal System The Dynamics and Function of the Endolysosomal/Lysosomal System Luther John Davis University of Cambridge Homerton College This dissertation is submitted for the degree of Doctor of Philosophy September 2018 I Acknowledgements Firstly, I would like to thank Prof. J Paul Luzio for his invaluable guidance and effort towards the production and proofreading of this thesis. I would also like to thank Chloe Taylor, Ian Baines, Theo Dare, and Ashley Broom for their expertise, assistance and reassurance. I am grateful to the BBSRC and GSK for funding my project for four years. I thank Sally Gray, Nick Bright, and Lena Wartosch for their technical assistance and support. Finally I would like to express my gratitude to Matthew Gratian and Mark Bowen for training and assistance with microscopy, as well as Reiner Schulte, Chiara Cossetti, and Gabriela Grondys-Kotarba for their help with flow cytometry and cell sorting. II Preface This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. This dissertation does not exceed the word limit of 60,000 words, as stipulated by the School of Clinical Medicine. III Thesis Summary Lysosomes are intracellular organelles that were considered for a long time to be simply an acidic and hydrolytically active end point of trafficking routes for degradation, in the last 20 years, light has been shed on their functional heterogeneity and striking role in signalling and nutrient homeostasis. While the dynamic nature and variety of lysosomal functions are now better appreciated, the mechanisms governing lysosomal fusion, reformation, signalling, and homeostasis remain to be fully elucidated, and are investigated here. In this study, endolysosomes which formed by fusion of late endosomes with lysosomes and are thought to be the predominant site of hydrolytic activity, were further characterised. Using live cell imaging and fluorescent labelling, the proportion of endolysosomes in the total pool of lysosomes was estimated using probes to their acidity and cathepsin activity, and their larger size compared to storage lysosomes was observed. The endolysosomal membrane was also shown to be marked by Rab7, Rab9, PI(3,5)P2 supporting the role of endolysosomes a highly active and dynamic principal site of hydrolase activity. The contributions of VAMP7 and VAMP8 to endolysosome fusion, measured by delivery of endocytosed cargo from late endosomes to endolysosomes, were analysed by CRISPR-Cas9 mediated knockout. Cells lacking VAMP7 and VAMP8 had no effect on delivery to endolysosomes, however at EM level, they displayed extensive tethering between late endocytic organelles, and accumulated small tethered vesicles. YKT6 knockdown impeded delivery to endolysosomes in VAMP7+VAMP8 knockout cells, which was rescued by VAMP7 expression, suggesting YKT6 substituted for VAMP7 in lysosome fusion. Following the hypothesis that reversible dissociation of V1 and Vo sectors of the V- ATPase may control the increase in pH of reforming storage lysosomes, cells expressing tagged V1G1 and Voa3 were generated. These markers of both sectors are present on endolysosomal membranes, and on the emerging endolysosomal tubules, suggesting the V1 and Vo sectors remain associated at this earliest stage of lysosome reformation, but these markers are still in development. IV Two assays were developed to give a readout of, and assess lysosomal stress. Firstly, an assay measuring TFEB-GFP translocation to the nucleus gave a robust and quantifiable readout of lysosomal perturbation. Secondly, a qPCR assay was developed to measure lysosomal gene upregulation as a downstream reporter of TFEB-activating lysosomal perturbations, however this assay, despite being more lysosome-specific, lacked the consistency and dynamic range of the TFEB translocation quantification. In summary, lysosomes are a heterogeneous collection of organelles, which have been better characterised primarily according to their acidity and hydrolytic capacity. Additionally, more SNAREs appear to be involved in lysosome fusion in cells than suggested by cell free assays, and I have developed tools to trace the V-ATPase during reformation of lysosomes after fusion to form endolysosomes. Lastly, I have developed a robust, reporter for a range of lysosomal stress-inducing conditions, providing a broad indication of their effects on lysosomal signalling and homeostasis. V Abbreviations AKT Protein kinase B AMP Adenosine monophosphate ANOVA Analysis of variance AP Adaptor protein APS Ammonium persulfate Arf ADP-ribosylation factor Arp Actin-related proteins ATP Adenosine triphosphate BafA1 Bafilomycin A1 BAR Bin/amphiphysin/rvs BCA Bicinchoninic acid BHK Baby hamster kidney BSA Bovine serum albumin CCV Clathrin coated vesicle Cdc42 Cell division control protein 42 homolog CFTR Cystic fibrosis transmembrane conductance regulator CLEAR Coordinated lysosomal expression and regulation CLEM Correlative light and electron microscopy CLIC Clathrin independent carrier CME Clathrin mediated endocytosis CORVET Class C core/vacuole tethering CQ Chloroquine CRISPR Clustered regularly interspaced short palindromic repeats CTxB Cholera toxin B-subunit DAB Diaminobenzidine DexA488 Dextran AlexaFluor™ 488 10,000 MW DexA647 Dextran AlexaFluor™ 647 10,000 MW DIC Differential interference contrast DMEM Dulbecco’s Modified Eagle’s Medium DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate DRM Detergent-resistant membrane DTT Dithiothreitol EBSS Earle’s balanced salt solution EEA1 Early endosome antigen 1 EGFR Epidermal Growth Factor Receptor EM Electron microscopy Eps15 Epidermal growth factor receptor pathway substrate 15 Eps15R Epidermal growth factor receptor pathway substrate 15R ER Endoplasmic Reticulum VI ESCRT Endosome sorting complexes required for transport FACS Fluorescence-activated cell sorting FCS Foetal calf serum G418 Geneticin GAP GTPase-activating protein GEEC GPI-enriched endocytic compartment GEF Guanine nucleotide exchange factor GFP Green fluorescent protein GGA Golgi-localised gamma-ear-containing Arf-binding protein GPCR G-protein coupled receptor GPI Glycosylphosphatidylinositol GTP Guanosine triphosphate HA Haemagglutinin HEK Human embryonic kidney HOPS Homotypic fusion and vacuole protein sorting HRP Horseradish peroxidase Hsc70 Heat shock cognate protein 70 IFITM3 Interferon-inducible transmembrane protein 3 ILV Intraluminal vesicle KD Knock down KO Knock out LAMP Lysosome-associated membrane protein LBPA Lysobisphosphatidic acid LC3 Microtubule-associated protein light chain 3 LDL Low density lipoprotein LDLR Low density lipoprotein receptor lgp120 Lysosomal membrane glycoprotein 120kDa LM Light microscopy LRO Lysosome-related organelle LTG LysoTracker® Green DND-26 LTR LysoTracker® Red DND-99 LYST Lysosomal trafficking regulator M6PR Mannose-6-phosphate receptor MCF7 Michigan Cancer Foundation-7 MCS Membrane contact sites MEM Minimum Essential Medium MRB Magic Red™ Cathepsin B-substrate MRL Magic Red™ Cathepsin L-substrate mTOR mechanistic target of rapamycin mTORC1 mTOR complex 1 MVB Multivesicular body Mvb12 Multivesicular body sorting factor 12 MW Molecular weight NAADP Nicotinic acid adenine dinucleotide phosphate NRK Normal rat kidney VII Ostm1 Osteopetrosis associated transmembrane protein 1 PBS Phosphate buffered saline PCR Polymerase chain reaction PERK Protein kinase RNA-like endoplasmic reticulum kinase PFA Paraformaldehyde PI(3)K Phosphoinositide 3-kinase PI(3)P Phosphatidylinositol-3-phosphate PI(3,4,5)P2 Phosphatidylinositol-3,4,5-trisphosphate PI(3,5)P2 Phosphatidylinositol-3,5-bisphosphate PI(4,5)P2 Phosphatidylinositol-4,5-bisphosphate PIP Phosphatidylinositol phosphate PNK Polynucleotide Kinase PS Phosphatidyl Serine PVDF Polyvinylidene difluoride RILP Rab-interacting lysosomal protein RNA Ribonucleic acid RPMI Roswell Park Memorial Institute SDS- Sodium dodecyl sulfate– PAGE polyacrylamide gel electrophoresis SEM Standard error of the mean SERCA Sarco/endoplasmic reticulum Ca2+-ATPase SH3 SRC homology 3 siRNA Small interfering RNA SNAP29 Synaptosome associated protein 29 SNARE Soluble NSF attachment protein receptor Snf7 Sucrose non-fermenting protein 7 Snx Sorting nexin Snx1 Sorting nexin 1 SV40 Simian vacuolating virus 40 TAE Tris-acetic acid-EDTA TBS-T Tris buffered saline-tween 20 TEM Transmission electron microscopy TEMED Tetramethylethylenediamine TFEB Transcription factor EB TfR Transferrin receptor TGN Trans-Golgi Network TLR9 Toll-like receptor 9 TPC Two pore channel TSE Tubular sorting endosomes UIM Ubiquitin interacting motif VAMP4 Vesicle associated membrane protein 4 Vap Virulence-associated protein V-ATPase Vacuolar H+-ATPase VPS Vacuolar protein sorting-associated protein Vti1a Vesicle transport through interaction with t-SNAREs homolog 1A VIII Vti1b Vps10 tail interactor 1b WASH WASP and Scar homolog WAVE WASP-family verprolin-homologous
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