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The Roles of Lipids in Intercellular Adhesion and Cell Movement

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The Roles of Lipids in Intercellular Adhesion and Cell Movement The Roles of Lipids in Intercellular Adhesion and Cell Movement Guang Yang This thesis is submitted to University College London for the Degree of Doctor of Philosophy Supervised by Prof. Ulrike Eggert Prof. Guillaume Charras Dr Victoria Sanz-Moreno Department of Cell and Developmental Biology University College London February 2018 Declaration I, Guang Yang, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Cells actively regulate their lipid composition and localisation during cell division, with both signalling roles and structural roles likely. In this study, I investigate the specific roles of lipids in the formation and maintenance of cell-cell junctions, and in cell migration. Although massive membrane rearrangements occur during both processes and lipids are fundamental building blocks of cell membranes, the roles of lipids remain unclear. After perturbing lipid metabolism enzymes with an siRNA library targeting 260 lipid biosynthetic enzymes in HaCaT (immortalized human keratinocytes), junctions were visualised using the adherens junction (AJ) marker α-catenin, and the resultant junction morphologies were examined. The primary screen revealed a potential role for 16 enzymes with two distinct junctional phenotypes: intercellular gaps and abnormal junction morphology. A secondary migration screen based on wound healing assays was conducted with a subgroup of these hits. I found that depletion of some enzymes results in defects in migration speed and cohesiveness, which suggests potential important roles for those enzymes and the lipids they make in cell migration. AGPAT2 (1-Acylglycerol-3-Phosphate O-Acyltransferase 2) is one of the hits being prioritised for further experiments to understand the role of related lipids. The depletion of AGPAT2 changes cell-cell junction morphology dramatically. In HaCaT, AJs expand on the neighbouring cells, while in Caco-2 (human colorectal adenocarcinoma cells), tight junctions (TJs) show an undulating morphology. Lipidomic analysis conducted following AGPAT2 depletion identified increases in triacylglycerols (TAGs), decreases in ether phosphatidylcholines (PCs) and reduced membrane fluidity indices. Further experiments are being performed to better characterise the phenotypes and identify the exact roles of the lipids. Overall, my study provides evidence that lipids may be involved in cell migration and cell-cell junction formation and maintenance and suggests that proper lipid metabolism is essential for regulating cell-cell junction and migration. 3 Acknowledgements I would first like to express my sincere gratitude to my supervisors: Professor Ulrike Eggert, Professor Guillaume Charras and Dr Victoria Sanz-Moreno for their guidance, expert knowledge and continuous support throughout this incredible adventure in the world of lipid, cell junctions and migration. It is an honour for me to be under their guidance and to grow as a scientist. Next, I wish to thank Dr Stephen Terry, Dr Elisabeth Storck, Dr Cagakan Ozbalci, Federico Doná, Lisa Watson, Dr Amy Wilson, Dr Antonia Booth, Dr Eleonora Muro and Dr Oscar Lancaster in Eggert lab, Dr Jose Orgaz, Dr Irene Rodriguez, Dr Eva Crosas, Bruce Fanshawe, Mirella Georgouli, Dr Gaia Cantelli, Dr Pahini Pandya, Anna Perdrix and Rimple Dalmeida in Sanz-Moreno lab, Dr Thomas Wyatt, Anna Bove and Dr Jonathan Fouchard in Charras lab, and all the other present and past lab members for their help and advice during my PhD. I was very super lucky to have a chance to meet and work with these brilliant people. Particularly, I appreciate Cagakan's splendid expertise in mass spectrum and exotic humorous, Steve's insights in adhesions and investments, Elisabeth's and Irene's expertise in chemistry and statistics, and valuable critics from Jose. And of course, a huge and warm thank you to my fellow PhD comrades, Amy, Fede, Bruce, Mirella, Victoria, Gaia and Pahini for filling these years with more fun and joys. I am also grateful for people who helped and taught me these years: Dr Jeremy Carlton, Dr Snezhana Oliferenko, Dr Ritu Garg, Dr Camilla Cerutti, Dr Sofia Tirados, Dr Ricardo Henriques, Professor Alison Lloyd, Professor Buzz Baum, Professor Michael Duchen, Laura McLaughlin, Isma Ali and many many others I cannot possibly fit here. I would also like to say thank you to Ed, Arjun, Pedro and all my fellows in BBSRC LIDo programme. It has been amazing to progress through with you. Also, this work was not possible without the funding from the BBSRC. On a personal note, I would like to thank all my friends. Particularly, Wei Gu, Yunxiao Zhang, Wenke Wang, Liyuan Zhu and Pan Deng for weekly recreations and chats across the globe. I am exceptionally grateful to my family for always being supportive throughout my studies. The encouragement from my parents and my little brother helped me a lot. Finally, but most importantly, I wish to thank my lovely wife Siyao for her unconditional support and complete faith in me. 4 Table of content Abstract ....................................................................................................................... 3 Acknowledgements .................................................................................................... 4 Table of content .......................................................................................................... 5 List of Figures ............................................................................................................. 9 List of Tables ............................................................................................................ 12 List of Abbreviations ................................................................................................ 13 Chapter 1 Introduction ............................................................................................. 16 1.1 Lipids ................................................................................................................. 17 1.1.1 Lipids Overview .......................................................................................... 17 1.1.2 Different approaches to study lipids ............................................................ 21 1.2 Cell-cell Junctions ............................................................................................. 29 1.2.1 Overview of cell-cell junctions ..................................................................... 29 1.2.2 Adherens junctions ..................................................................................... 35 1.2.3 Functions of lipids in adherens junction dynamics ....................................... 53 1.3 Cell Migration .................................................................................................... 55 1.3.1 Cell migration overview ............................................................................... 55 1.3.2 Collective cell migration .............................................................................. 57 1.3.3 Functions of lipids in cell migration .............................................................. 61 1.4 Adherens junctions and collective migration ...................................................... 63 1.4.1 Massive membrane rearrangements occur during cell-cell junction formation and collective cell migration ................................................................................. 63 1.4.2 Small GTPases regulate formation and maintenance of cell-cell junctions and collective migration .............................................................................................. 65 1.4.3 Adherens junctions connect cells physically and chemically during collective migration .............................................................................................................. 68 1.5 Hypothesis and Aims ......................................................................................... 70 Chapter 2 Methods and Reagents ........................................................................... 71 2.1 Cell culture ........................................................................................................ 72 2.2 Screen ............................................................................................................... 73 2.2.1 Lipid biosynthetic siRNA library ................................................................... 74 2.2.2 Controls used in the screen ........................................................................ 74 2.2.3 Screen workflow ......................................................................................... 74 2.3 siRNA transfection ............................................................................................. 76 2.4 Western Blotting ................................................................................................ 77 2.4.1 Preparation of Lysates ................................................................................ 77 2.4.2 Gel running and transfer ............................................................................. 78 5 2.4.3 Blocking and Immunoblotting ...................................................................... 78 2.5 Immunofluorescence and microscopy ............................................................... 79 2.5.1 Fixation ....................................................................................................... 79 2.5.2 Immunofluorescence
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