In vivo analysis of the LIN-2/7/10 complex in spatial regulation of LET-23 EGFR signalling in C. elegans Kimberley D. Gauthier Department of Anatomy and Cell Biology Faculty of Medicine McGill University Montreal, Quebec, Canada December 2019 A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Doctor of Philosophy © Kimberley D. Gauthier 2019 Table of Contents Table of Contents ......................................................................................................................... i Abstract ...................................................................................................................................... vi Résumé ..................................................................................................................................... viii Acknowledgements ..................................................................................................................... x Preface ....................................................................................................................................... xii Contributions of Authors .......................................................................................................... xiii Nomenclature ........................................................................................................................... xiv List of Figures .......................................................................................................................... xvi List of Tables .......................................................................................................................... xviii List of Abbreviations ................................................................................................................ xix Chapter 1: Introduction and Literature Review........................................................................ 1 1.1 Spatial organization of signalling networks .......................................................................... 2 1.1.1 Epithelial cell signalling and regulation ............................................................................. 3 1.1.1.1 Establishment and maintenance of epithelial cell polarity .............................................. 6 1.1.1.2 Polarized trafficking in epithelial cells............................................................................ 7 1.2 Epidermal Growth Factor Receptor signalling...................................................................... 9 1.2.1 The Ras/MAPK signalling pathway ................................................................................. 13 1.2.2 Importance of EGFR in human health ............................................................................. 13 1.2.3 Spatial regulation of EGFR signalling ............................................................................. 14 1.3 Caenorhabditis elegans vulval cell fate induction as a model for spatial organization of signalling pathways ................................................................................................................... 18 1.3.1 C. elegans as a model organism ....................................................................................... 18 1.3.2 LET-23, the sole EGFR in C. elegans .............................................................................. 21 1.3.3 C. elegans vulval development as a model for spatiotemporal EGFR signalling regulation ................................................................................................................................................... 21 1.3.4 Applications of VPC induction model for EGFR-related cancers ................................... 26 1.4 LIN-2 (CASK), LIN-7 (Lin7), and LIN-10 (APBA) are conserved regulators of subcellular organization ............................................................................................................................... 27 1.4.1 The protein domain structure of LIN-2, LIN-7, and LIN-10 ........................................... 29 i 1.4.1.1 The PDZ domains of LIN-2, LIN-7, and LIN-10 ......................................................... 30 1.4.1.2 LIN-2 and LIN-7 both have L27 domains .................................................................... 31 1.4.1.3 LIN-2 and CASK are MAGUK proteins....................................................................... 32 1.4.1.4 LIN-10 and APBA1 also share CID and PTB domains ................................................ 32 1.4.2 Localization of LIN-2 CASK, LIN-7 Lin7, and LIN-10 APBA1-3................................. 33 1.4.3 Function of LIN-2, LIN-7, and LIN-10............................................................................ 36 1.4.3.1 Lin7 maintains polarized epithelial membrane composition ........................................ 39 1.4.3.1.1 Lin7 and human EGFRs ............................................................................................. 40 1.4.3.2 CASK regulates synaptic plasticity and basolateral membrane organization ............... 41 1.4.3.2.1 Transcriptional regulation by CASK.......................................................................... 42 1.4.3.3 LIN-10 and APBA1-3 regulate intracellular trafficking ............................................... 43 1.4.3.3.1 C. elegans LIN-10 regulates glutamate receptor trafficking in neurons .................... 44 1.4.3.3.2 Mammalian APBA regulates APP trafficking and processing .................................. 44 1.4.3.3.3 LIN-10 homologues, hypoxia, and transcriptional regulation ................................... 46 1.4.3.3.4 APBA in non-neuronal cells ...................................................................................... 47 1.4.3.3.5 Regulation of LIN-10 APBA ..................................................................................... 48 1.5. Spatial regulation by small GTPases .................................................................................. 51 1.5.1 Membrane trafficking regulation of C. elegans vulval cell fate induction ...................... 52 1.5.2 Arf GTPases and their regulators mediate several steps of cellular organization ............ 55 1.5.2.1 Class I and II Arf GTPases regulate Golgi trafficking .................................................. 57 1.5.2.2 Class III Arf6 GTPase regulates recycling and the cytoskeleton .................................. 57 1.5.2.3 Arf GAPs regulate diverse cellular dynamics ............................................................... 58 1.5.2.3.1 Classification and protein structure of Arf GAPs ...................................................... 59 1.5.2.3.2 Arf GAPs regulate trafficking and actin remodelling ................................................ 63 1.5.2.3.2.1 Arf GAPs in trafficking and recycling pathways .................................................... 63 1.5.2.3.2.2 Arf GAPs regulate adhesion complexes and actin remodelling .............................. 65 1.6. Rationale and objectives..................................................................................................... 68 Chapter 2: Materials and Methods ........................................................................................... 70 2.1 Strains and maintenance ...................................................................................................... 71 2.2 Genomic DNA isolation for genotyping ............................................................................. 71 ii 2.3 Molecular cloning ............................................................................................................... 71 2.4 Generating endogenously-tagged transgenic strains by CRISPR/Cas9 .............................. 72 2.5 Generation of extrachromosomal array lines ...................................................................... 73 2.6 LIN-10 subdomain identification and sequence identity .................................................... 74 2.7 Microscopy and Image Analysis ......................................................................................... 74 2.7.1 LIN-2/7/10 localization analysis ...................................................................................... 74 2.7.2 LIN-2/7/10 fluorescence intensity analysis ...................................................................... 75 2.7.3 Analyzing LET-23 EGFR polarized distribution ............................................................. 75 2.7.4 Colocalization analysis ..................................................................................................... 75 2.8 Analyzing VPC cell fate induction...................................................................................... 76 2.9 Correlation analysis for extrachromosomal array expression and VPC induction ............. 77 2.10 RNAi experiments ............................................................................................................. 78 2.11 Co-immunoprecipitation ................................................................................................... 78 2.12 SDS-PAGE and western blot ............................................................................................ 79 2.13 Statistical analysis ............................................................................................................. 80 Chapter 3: In vivo analysis of the LIN-2/7/10 complex ........................................................... 86 3.1 Preface ................................................................................................................................