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Multiple Roles for Unc-53/Nav2 in Cell Migration, Trafficking and Innate Immunity in Caenorhabditis Elegans

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Multiple Roles for Unc-53/Nav2 in Cell Migration, Trafficking and Innate Immunity in Caenorhabditis Elegans MULTIPLE ROLES FOR UNC-53/NAV2 IN CELL MIGRATION, TRAFFICKING AND INNATE IMMUNITY IN CAENORHABDITIS ELEGANS by Kristopher Lee Schmidt M.Sc., University of British Columbia, 2005 B.Sc., Trinity Western University, 2001 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Molecular Biology and Biochemistry Faculty of Science © Kristopher Lee Schmidt 2012 SIMON FRASER UNIVERSITY Summer 2012 All rights reserved. However, in accordance with the Copyright Act of Canada, this work may be reproduced, without authorization, under the conditions for Fair Dealing. Therefore, limited reproduction of this work for the purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately [Type a quotei from the document or the summary of an interesting point. APPROVAL Name: Kristopher Lee Schmidt Degree: Doctor of Philosophy Title of Thesis: Multiple roles for UNC-53/NAV2 in cell migration, trafficking, and innate immunity in Caenorhabditis elegans. Examining Committee: Chair: Dr. Barry Honda, Ph.D. Dr. Eve Stringham, Ph.D. Professor, Department of Molecular Biology Co-Senior Supervisor and Biochemistry Professor, Department of Biology, Trinity Associate Professor, Department of Western University __________________________________ __________________________________ Dr. Nancy Hawkins, Ph.D. Dr. Christopher Beh, Ph.D. Senior Supervisor Supervisor Associate Professor, Department of Molecular Associate Professor, Department of Molecular Biology and Biochemistry Biology and Biochemistry __________________________________ __________________________________ Dr. Nicholas Harden, Ph.D. Dr. Harald Hutter, Ph.D. Supervisor Internal Examiner Associate Professor, Department of Molecular Professor, Department of Biological Sciences Biology and Biochemistry __________________________________ __________________________________ Dr. Nathalie Pujol, Ph.D. External Examiner Project Leader, Centre d’Immunologie de Marseille-Luminy __________________________________ Date Defended/Approved: June 1, 2012 ii Partial Copyright Licence ABSTRACT unc-53 is the Caenorhabditis elegans homolog of Nav2, and a member of the Neuron Navigator protein family, a group of cytoskeletal binding proteins with conserved roles in the guidance and outgrowth of cells and cellular processes. The signalling pathways that employ UNC-53 during development, and the role of UNC-53 after development is complete, are largely unknown. A proteomics screen for interactors of UNC-53 identified that UNC-53 interacts with ABI-1. The Calponin Homology domain unique to the long-isoform of UNC-53 is sufficient to bind ABI-1 in vitro and is required in vivo for longitudinal migration. ABI-1 and UNC-53 are co-expressed, and abi-1 genetic loss causes many of the same migration defects as unc-53 mutants. abi-1 and unc-53 function cell-autonomously and in a common genetic pathway in the posterior migration of the excretory canals, and genetically inactivating abi-1 interactors (nck-1, wve-1, arx-2) resulted in phenotypes similar to unc-53 and abi-1. abi-1 and unc-53 are also required for endocytosis as measured by assaying the in vivo uptake of GFP into coelomocytes and primary oocytes. Lastly, abi-1 mutants have unc-53 independent migration defects, and ABI-1 binds to MIG-10A through its SH3 domain, thereby demonstrating that ABI-1 interacts with two proteins that function cell-autonomously in the longitudinal migration of the excretory canals. To identify a post-developmental role for unc-53, a potential role for unc-53 in innate immunity was assessed. unc-53 mutants are susceptible to the human and nematode pathogen Pseudomonas aeruginosa PA14. unc-53 mutants are hypersensitive to Aldicarb and have increased RNA levels of the daf-16 antagonist ins-7 as well as decreased nuclear DAF-16 localization following recovery from cellular stress, suggesting that unc-53 functions in the daf-16 pathway. Loss of function and null alleles of daf-2 and ins-7 only partially suppress unc-53 in immunity, and a null pmk-1 mutant does not enhance unc-53. Together, this data suggests that unc-53 participates in innate immunity through multiple tissues, isoforms and genetic pathways. These findings expand on the varied functions of unc-53 and the signal transduction pathways that it controls, opening up new prospects for future areas of research. Keywords: C. elegans; UNC-53; Neuron Navigators; ABI-1; cell migration; innate immunity iii QUOTATION “Our modern world has fantastic power and knowledge. Man has conquered the moon, delved into the secret of matter, and discovered immense energies. But the only real knowledge necessary for the survival of the human race is lacking: the knowledge of how to transform violence and hatred into tenderness and forgiveness; how to stop the chain of aggression against the weak; how to see differences as a value rather than as a threat; how to stop people from envying those who have more and incite them to share with those who have less.” ~ Jean Vanier iv DEDICATION ~To my Mom Without your dedication and sacrifice I would have never come this far. v ACKNOWLEDGEMENTS “My Grace is sufficient for you because you are made strong in weakness” (2 Corinthians 12:9) My time during this degree program has been alive with reminders of my dependency on the strength and help of others. I am especially indebted to my thesis supervisor Dr. E. Stringham for steering me towards C. elegans as an undergraduate in 1998, and for offering me patience and grace as I developed into a scientist. Thank you also to my exceptional committee (Dr. C. Beh, Dr. N. Harden, and Dr. N. Hawkins). Your individual and collective insights have sharpened me and contributed substantially to this work. Thank you to my many friends and colleagues at Trinity Western University, Simon Fraser University, the Menno Simons Centre (PCDA), the University of British Columbia, Douglas College, and the C. elegans research community. Your support and contributions are unmatched, and you provide clear evidence that a strong and healthy community brings wellness to its members. I am especially indebted to Dr. A. Adeleye, Dr. N. Marcus, Dr. J. Webber, Dr. D. Baillie, D. Tu, Dr. S. Alper, E. Kreiter, C. Grypma, and Dr. E. Ryder, for either technical assistance or formal collaboration. Finally, thank you to my family, and especially my wife Kathryn. You have supported me with kindness, compassion, and loving care. Thank you for your love. vi TABLE OF CONTENTS Approval ............................................................................................................................ ii Abstract ............................................................................................................................. iii Quotation .......................................................................................................................... iv Dedication ......................................................................................................................... v Acknowledgements .......................................................................................................... vi Table of Contents ............................................................................................................. vii List of Figures .................................................................................................................... x List of Tables .................................................................................................................... xii List of Abbreviations ........................................................................................................ xiv Contributions ................................................................................................................. xviii 1: Introduction to UNC-53 and the Neuron Navigators ................................................ 1 1.1 Prior research on UNC-53 from C. elegans .............................................................. 1 1.2 Insights from the Neuron Navigators, UNC-53 homologs ......................................... 7 1.3 Thesis overview ...................................................................................................... 12 2: The cell migration molecule UNC-53/NAV-2 is linked to the ARP2/3 complex by ABI-1 .......................................................................................................... 15 2.1 Abstract ................................................................................................................... 15 2.2 Introduction ............................................................................................................. 16 2.2.1 Growth cones and cell migration ................................................................. 16 2.2.2 Guidance cues and their receptors ............................................................. 20 2.2.3 Rho family of small GTPases ...................................................................... 26 2.2.4 Regulation of actin polymerization .............................................................. 28 2.2.5 Studies of endocytosis in C. elegans .......................................................... 40 2.2.6 Overview ..................................................................................................... 42 2.3 Results ...................................................................................................................
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