INTRAFLAGELLAR TRANSPORT IN CAENORHABDITIS ELEGANS: IDENTIFICATION OF NOVEL PROTEINS AND BEHAVIOURAL FUNCTIONS by Peter Nicholas Inglis B.Sc., Simon Fraser University, 2004 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Molecular Biology and Biochemistry © Peter Nicholas Inglis 2009 SIMON FRASER UNIVERSITY Summer 2009 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL Name: Peter Nicholas Inglis Degree: Doctor of Philosophy Title of Thesis: Intraflagellar transport in Caenorhabdifis e/egans: identification of novel proteins and behavioural functions Examining Committee: Chair: Dr. Hogan Yu Associate Professor, Department of Chemistry Dr. Michel R. Leroux Senior Supervisor Associate Professor, Department of Molecular Biology and Biochemistry Dr. David L. Baillie Supervisor Professor, Department of Molecular Biology and Biochemistry Dr. Nancy C. Hawkins Supervisor Assistant Professor, Department of Molecular Biology and Biochemistry Dr. Michael A. Silverman Internal Examiner Assistant Professor, Department of Biological Sciences Dr. Jeremy F. Reiter External Examiner Assistant Professor, Department of Biochemistry and Biophysics, University of California, San Francisco, USA Date Defended/Approved: ii SIMON PRASER UNIVERSITY HUN KINO OF THE WORLD DECLARATION OF PARTIAL COPYRIGHT LICENCE The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without the author's written permission. While licensing SFU to permit the above uses, the author retains copyright in the thesis, project or extended essays, including the right to change the work for subsequent purposes, including editing and publishing the work in whole or in part, and licensing other parties as the author may desire. Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. No Digital Copy for SFU Library collection: By special permission of the Dean of Graduate Studies, the author is exempted from granting permission to Simon Fraser University Library to keep or make a digital copy for use in its circulating collection (currently available to the public at the "Institutional Repository" link of the SFU Library website). It is agreed that the author may change this decision at any time, by providing SFU Library with a signed Partial Copyright Licence in standard form. The original Partial Copyright Licence attesting to these terms, and signed by this author, may be found in the original bound copy of this work, retained in the Simon Fraser University Archive. Simon Fraser University Library Burnaby, BC,Canada Partial Copyright Licence_PDF Exemption 2007 ABSTRACT Intraflagellar transport (1FT) is the dynamic bidirectional process required for the biogenesis and maintenance of eukaryotic cilia. Landmark studies exploiting the model organism Chlamydomonas reinhardtii have provided a basic mechanism for the process, although recent research examining 1FT in the nematode Caenorhabditis elegans has revealed a greater complexity to the original model of 1FT described in Chlamydomonas, which includes the orthologues of several human proteins involved in cilium-associated diseases. The wealth of genomic, bioinformatic, and molecular tools available to C. elegans researchers is exploited in this thesis to uncover and characterise a number of novel proteins involved in the process of 1FT, namely DYF-11, MKS-1, MKSR-1, and MKSR-2. DYF-11 localises throughout nematode ciliary structures and acts as a key component of the core 1FT subcomplex B. The latter three proteins are members of a previously uncharacterised family of B9-domain containing polypeptides, all of which appear to localise at the base of cilia (basal body/transition zone), where they are found to regulate a cilium-based insulin signalling pathway. The results of this study contribute to the growing realisation in C. elegans cilia research that a network of transition-zone-specific proteins are participating in ciliary processes ranging from subtle modulation of ciliary signalling pathways to the actual biogenesis and maintenance of the organelle. iii Next, we examine previously isolated nematode strains that are defective in paraquat resistance for impaired retrograde 1FT. We identify a retrograde-1FT deficient strain that likely represents a mutation in an 1FT component that has not been previously characterised in C. elegans; the mutation maps closely to a highly conserved protein identified as a core component of 1FT subcomplex A. Finally, we examine the role played by the 1FT-associated Bardet-Biedl syndrome (BBS) proteins in C. elegans thermosensation. bbs mutant worms appear to be slightly defective in responding to both noxious and physiological temperatures. Furthermore, the nature of the physiological temperature defect correlates with a decreased roaming ability that is not the result of impaired locomotion. Altogether, the studies presented in this thesis offer novel insights into both the molecular makeup and physiological functions of 1FT in C. elegans. iv To the two Kennys in my life: The first, my best friend, recently departed The other, my beautiful baby boy, newly arrived You both inspire me to become something greater than I am v ACKNOWLEDGEMENTS I would like to thank my senior supervisor, Dr. Michel Leroux, for providing an intellectual environment in which I could thrive, and for the many interesting discussions we've had over the years. I also want to acknowledge my labmates: Chunmei Li, Nathan Bialas, Oliver Blacque, Peter Stirling, Victor Lundin, Muneer Esmail, Cheryl Wiens, Lesley Chen, Swetha Mohan, Phanh Nguyen, Michael Healey, Jayden Yamakaze, Eric Yao, Navin Bhopal, Yan Xue, and Anthony Breemo. I would also like to thank our many collaborators, including Drs. Nicholas Katsanis, Peter Swoboda, Jonathan Scholey, and Don Moerman. I also greatly appreciate the support and feedback of my supervisory committee members, Dr. David Baillie and Dr. Nancy Hawkins I would be remiss if I failed to recognise the emotional support from my friends. In particular, I would like to thank Sarah Winton, Kevin Sass, and Brian Bradley for providing me with the occasional brief yet fun escape from laboratory life. Finally, and most importantly, the work presented in this thesis would not have been remotely possible without the overwhelming support of my family. My mom, dad, and sister seemed at times to literally bend over backwards in support of my academic pursuits, and I love them all. My wife, Chrystal, in addition to physically aiding my research as a technician in the lab, has treated me with a love and kindness I am not sure I deserve, but without which I had no chance of success. vi TABLE OF CONTENTS Approval ii Abstract iii Dedication v Acknowledgements vi Table of Contents vii List of Figures xi List of Tables xiv Chapter 1. Introduction 1 1.1 The Cilium: Structure and Function 2 1.1.1 Historical perspectives 2 1.1.2 The cilium in nature 3 1.1.3 General ciliary structure 4 1.1.4 Motile cilia versus non-motile cilia 4 1.1.5 The cilia of Caenorhabditis elegans 5 1.2 Cilium Biogenesis: Intraflagellar transport (1FT) 11 1.2.1 The discovery and basic mechanism of 1FT, as derived from studies in Chlamydomonas reinhardtii 11 1.2.2 Additional complexity of 1FT in Caenorhabditis elegans 13 1.3 Signalling and Sensation in Cilia 18 1.3.1 Localisation of key developmental signalling pathways to cilia 18 1.3.2 Cilia-associated diseases: the ciliopathies 21 1.4 Uncovering the Ciliome: Bioinformatic, Genomic and Proteomic Studies 24 1.4.1 Bioinformatic searches for X boxes in C. elegans promoters 24 1.4.2 Comparative genomic analyses 26 1.4.3 Flagellar regeneration transcriptome analyses 29 1.4.4 Ciliated cell-specific transcriptome analyses 30 1.4.5 Proteomic studies of motile cilia 31 1.4.6 Meta-analyses 35 1.5 Research Objectives 36 1.6 Figures 38 1.7 Tables 49 vii Chapter 2. Ciliary Comparative Genomics Of The Chytrid Fungus Batrachochytrium dendrobatidis 54 2.1 Abstract 55 2.2 Introduction 56 2.3 Results/Discussion 58 2.4 Conclusion 61 2.5 Materials and Methods 61 2.5.1 Comparative genomic survey and orthologue identification 61 2.6 Tables 62 Chapter 3. An Essential Role For DYF-11/MIP-T3 In Assembling Functional Intraflagellar Transport Complexes 68 3.1 Abstract 69 3.2 Introduction ·.. 69 3.3 Results/Discussion 73 3.3.1 The C. elegans MIP-T3 gene ortholog C02H7.1 is disrupted in dyf-11 mutants 73 3.3.2 DYF-11 is required for the formation of structurally intact and functional cilia 74 3.3.3 DYF-11/MIP-T3 is a novel intraflagellar transport (1FT) protein 77 3.3.4 DYF-11 is transported in the cilium in a manner similar to 1FT particle subcomplex B 79 3.3.5 DYF-11 is required for the integrity of the motor-1FT machinery 82 3.4 Concluding Remarks 83 3.5 Materials and Methods 86 3.5.1 Strain construction and maintenance 86 3.5.2 Construction of strains harboring a translational DYF- 11 ::GFP construct 87 3.5.3 Localization of MIP-T3 in mammalian cells 87 3.5.4 Cloning of dyf-11 (C02H7.1) 88 3.5.5 C.