INTRAFLAGELLAR TRANSPORT IN CAENORHABDITIS ELEGANS: IDENTIFICATION OF NOVEL 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: 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

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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 -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 (/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 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 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. elegans phenotypic analyses 88 3.5.6 Analysis of sensory neuron structure and cilia length measurements 90 3.5.7 Visualization of 1FT and rate measurements by time-lapse microscopy 91 3.6 Acknowledgements 91 3.7 Figures 93 Chapter 4. Functional Interactions Between The Ciliopathy­ Associated Meckel Syndrome 1 (MKS1) Protein And Two Novel MKS1-Related Proteins 109 4.1 Abstract 110 4.2 Introduction 111 4.3 Results 116

viii 4.3.1 An evolutionarily-conserved family of 89 domain-containing proteins in ciliated organisms 116 4.3.2 All three 89 domain-containing proteins localize to basal bodies and/or cilia 118 4.3.3 Interdependent localization of MKS/MKSR proteins to basal bodies 120 4.3.4 The C. elegans MKS/MKSR proteins do not appear to be essential for transition zone positioning, cilium formation, or IFT function 123 4.3.5 The C. elegans mks/mksr control lifespan via the insulin signalling pathway 127 4.4 Discussion 131 4.4.1 Evolutionary conservation of 89 domain-containing proteins in ciliated organisms 131 4.4.2 MKS1, MKSR1 and MKSR2 are associated with basal bodies and cilia 132 4.4.3 Function of the MKS/MKSR proteins in ciliogenesis and cilium-associated signalling 133 4.4.4 MKSR-1 and MKSR-2 as potential Meckel syndrome gene candidates 138 4.5 Concluding Remarks 139 4.6 Materials and Methods 139 4.6.1 C. elegans strains and genetic crosses 139 4.6.2 Characterisation of the C. elegans mks-1, mksr-1, and mksr- 2 alleles 140 4.6.3 Subcellular localisation of the human and C. elegans MKS-1, MKSR-1 and MKSR-2 proteins 140 4.6.4 Mammalian RNA interference 142 4.6.5 Phenotypic assays for ciliary structure, chemosensation and lipid content. 143 4.6.6 Lifespan assays 143 4.6.7 DAF-16 nuclear localisation analyses 144 4.6.8 Intraflagellar transport assays 144 4.6.9 8ioinformatic and phylogenetic analyses 145 4.7 Acknowledgements 145 4.8 Figures 147 4.9 Tables 168 Chapter 5. Analysis Of Intraflagellar Transport Subcomplex A (1FT· A) In C. e/egans 170 5.1 Abstract 171 5.2 Introduction 171 5.3 Results/Discussion 173 5.3.1 Dye-filling analysis of paraquat-resistant mutant library 173 5.3.2 Ciliary defects in dye-filling defective strains 174 5.3.3 Progress in the cloning and characterisation of the qa5054 allele 175

ix 5.4 Concluding Remarks 177 5.5 Materials and Methods 179 5.5.1 Strains and genetic crosses 179 5.5.2 Dye-filling (Dyf) assays 180 5.5.3 Microscopy 180 5.5.4 Mapping 181 5.6 Figures 182 Chapter 6. Control of C. e/egans Thermosensation And Locomotion By Bardet-Biedl Syndrome Proteins 187 6.1 Abstract 188 6.2 Introduction 188 6.3 Results 193 6.3.1 C. elegans bbs mutants are thermosensory defective 193 6.3.2 C. elegans bbs mutants demonstrate reduced roaming on isothermal gradients in a manner unrelated to rate body bends 196 6.4 Discussion/Conclusion 196 6.5 Materials and Methods 200 6.5.1 Shallow gradient thermotaxis assay 200 6.5.2 Thermal avoidance assay 201 6.5.3 Analysis of OSM-9 localization 201 6.5.4 C. elegans body bend assay 202 6.6 Figures 203 Chapter 7. Conclusion 209 7.1 Identifying strong candidate ciliary genes using existing ciliomic data 210 7.2 Future directions of 1FT research in C. elegans 212 7.2.1 The role of protein modifiers in 1FT 212 7.2.2 The basal body-transition zone module 214 7.3 Placing the novel C. elegans 1FT proteins into the core 1FT complex 216 7.4 General conclusions 218 7.5 Tables 220 Appendices 222 Appendix A. CD-ROM Data 222 Reference List 223

x LIST OF FIGURES

Figure 1-1. Generalised structure of the eukaryotic cilium 38 Figure 1-2. Generalised structure of a motile cilium (flagellum) 39 Figure 1-3. Ultrastructures of cilia and relative positions of all known ciliated neurons (cell bodies and associated dendrites) in the C. elegans hermaphrodite 40 Figure 1-4. Structure of a typical C. elegans amphid cilium 41 Figure 1-5. Generalised mechanism of intraflagellar transport (1FT), as based on research in Chlamydomonas reinhardtii 42 Figure 1-6. Hypothetical model for intraflagellar transport (1FT) in the amphid sensory cilia of Caenorhabditis elegans 43 Figure 1-7. Experimental observations of Caenorhabditis elegans intraflagellar transport (1FT) in mutants deficient in Kinesin-2, OSM-3, or IFT- mutants 45 Figure 1-8. Experimental observations of Caenorhabditis elegans intraflagellar transport (1FT) in mutants deficient in 1FT subcomplex A, 1FT subcomplex B, or BBSome components 47 Figure 3-1. The C. elegans dyf-11 strain contains a mutation in the gene e02H7.1, the MIP-T3 ortholog 93 Figure 3-2. Amino acid alignment of C. elegans DYF-11 (C02H7.1) with MIP T3 protein orthologs from C. reinhardtii and H. sapiens 95 Figure 3-3. dyf-11 (mn392) mutants display ciliary structure defects, behavioural phenotypes indicative of ciliary anomalies, and a lipid accumulation phenotype 97 Figure 3-4. DYF-11 is a component of the intraflagellar (1FT) transport machinery 100 Figure 3-5. C. elegans DYF-11 ::GFP may be functionally associated with 1FT particle subcomplex B 103 Figure 3-6. Fluorescence images indicating the possible presence of DYF 11 ::GFP in the distal segments of AWe neuron cilia 105 Figure 3-7. C. elegans DYF-11 is required for the assembly and function of many components of the motor-1FT machinery, including Ki nesin-II 107

xi Figure 4-1. Phylogenetic analysis showing that 89-domain-containing proteins from ciliated organisms belong to a family of proteins consisting of three clades or family members, namely Meckel Syndrome 1 protein (MKS-1), MKS-1-related protein 1 (MKSR-1) and MKS-1-related protein 2 (MKSR-2), and sequence comparisons of different 89 domains 147 Figure 4-2. MKS-1, MKSR-1 and MKSR-2 proteins localize to centrosomes or basal bodies in human cells and transition zones in C. elegans 149 Figure 4-3. V5-tagged human MKS1, MKSR1 or MKSR2 transiently expressed in non-ciliated IMCD3 cells colocalizes with y-tubulin...... 151 Figure 4-4. Mutations in the C. elegans mks-1, mksr-1 and mksr-2 genes and analysis of the corresponding transcripts 152 Figure 4-5. Interdependent localization of C. elegans MKS-1, MKSR-1 and MKSR-2 proteins to transition zones 154 Figure 4-6. Single, double and triple C. elegans mkslmksr mutants exhibit normal transition zone positioning, ciliary axonemal structures, intraflagellar transport and chemosensory behaviors 156 Figure 4-7. mkslmksr mutant animals do not display dye-filling defects indicative of abrogated ciliary structures, or Nile Red (lipid content) phenotypes 159 Figure 4-8. mkslmksr mutant animals display wild-type localization of intraflagellar transport (1FT) marker proteins and normal 1FT rates along the middle and distal segments of cilia 161 Figure 4-9. Transmission electron microscopy (TEM) analyses of wild-type (N2) and mks-1;mksr-1 animals cross-sectioned through their amphid sensory neurons, showing their ciliary transition zones, cilia middle segment (doublet microtubules) and cilia distal segment (singlet microtubules), as indicated 163 Figure 4-10. Genetic interactions between the C. elegans mks-1, mksr-1 and mksr-2 genes revealed by an increased lifespan phenotype...... 164 Figure 4-11. MKS/MKSR proteins appear to function upstream of DAF-2 and DAF-16 in the insulin signaling/lGF-1 pathway to regulate lifespan 166 Figure 5-1. Mapping of qa5054 allele to the centre of C. elegans III 182 Figure 5-2. Dye filling-defective (Dyf) paraquat-resistant strains with defects in retrograde intraflagellar transport 184 Figure 6-1. Experimental approaches used in Chapter 6 203

xii Figure 6-2. C. elegans ciliary mutants show thermotaxis and thermal avoidance defects as well as mislocalized OSM-9 205 Figure 6-3. C. elegans bbs mutants show defective roaming in isothermal tracking assays but appear to have normal body movement.. 207

xiii LIST OF TABLES

Table 1-1. Description of individual ciliated neuron types and their reported functions 49 Table 1-2. Components and available C. elegans mutants of the conserved intraflagellar transport machinery 51 Table 2-1. Established ciliary genes found in the comparative genomics study 62 Table 2-2. Genes identified in the comparative genomic study that encode proteins with TPR domains and WD repeats 66 Table 2-3. Genes identified in the comparative genomic study that encode small GTP-binding proteins 66 Table 2-4. Genes identified in the comparative genomic study that encode proteins which likely interact with tubulin 66 Table 2-5. Genes identified in the comparative genomic study that have putative human orthologues implicated in disease, but have yet to be associated with ciliary dysfunction 67 Table 4-1. Distribution of 89 domain-containing proteins in ciliated organisms and absence from non-ciliated species 168 Table 5-1. Dye-filling results from paraquat-resistant mutant library 186 Table 7-1. Currently uncharacterised top ciliary candidate genes based on various ciliomic analyses 220 Table 7-2. Putative ciliary genes based that fit stringent X-box criteria 221

xiv CHAPTER 1. INTRODUCTION

Note regarding contributions: Setions 1.1.5 and 1.2.2 of this chapter were derived from the following review article: Inglis PN, Ou G, Leroux MR, Scholey JM. (2007). The sensory cilia of Caenorhabditis elegans. WormBook (www.wormbook.org): 1-22. The majority of this article was written by me, with editorial assistance from M. Leroux. The sections that deal with C. elegans 1FT (Chapter 1.2.2 in this thesis) were written in collaboration with G. Ou and J. Scholey. Section 1.4 of this chapter was taken directly from the following review article: Inglis PN, Boroevich KA, Leroux MR. (2006). Piecing together a ciliome. Trends Genet. 22: 491-500. The sections included in this chapter were written by myself and M. Leroux. K. Boroevich developed the website, www.ciliome.com. referenced later on in the chapter. Figures 1-6, 1-7, and 1-8 are part of a manuscript recently submitted to Methods in Cell Biology (Inglis PN, Blacque OE, Leroux MR. Functional genomics of intrafJagellar transport-associated proteins in C. elegans), and were designed by myself, with editorial input from M. Leroux.

1 1.1 The Cilium: Structure and Function

1.1.1 Historical perspectives

In the last decade, the eukaryotic cilium - once thought to be associated only with motility - has emerged as a central player in many essential cellular processes. This fascinating microtubule-based organelle is now known to be the site of key developmental signalling pathways, the origin of virtually all extracellular sensory processes, and may even be a regulator of cell division. A myriad of human diseases, now collectively referred to as ciliopathies, have been connected to the dysfunction of non-motile, sensory cilia, and represent conditions that result in symptoms ranging from blindness to obesity.

Remarkably, since the emergence of Caenorhabditis elegans as a prominent model organism nearly 35 years ago, researchers of this tiny nematode have been amassing an enormous library of mutants defective in cellular processes essential for the structure and function of sensory cilia. C. elegans, in fact, is one of the few metazoans that possess solely non-motile cilia.

Virtually every physiological process in the adult nematode has some dependence on the cilium, including metabolism, reproduction, mobility, behaviour and lifespan. It is for these reasons that C. elegans has emerged as a robust and powerful system in the exploding field of ciliary research. The research described in this thesis employs C. elegans as a model system to uncover several novel, highly conserved cilium-associated proteins, as well as

2 gain insight into a hitherto essentially unstudied sensory function of non-motile cilia.

1.1.2 The cilium in nature

Ciliary structures are broadly grouped into two categories, based entirely on their motility. Motile cilia, also known also as flagella (although this term is

being employed less frequently to avoid confusion with the unrelated bacterial flagella), have been well studied, and are best known for their roles in cellular

movement, such as in the unicellular algae Chlamydomonas reinhardtii and

mammalian sperm cells. However, motile cilia are not always associated with the

movement of individual cells; in various metazoans, these structures are frequently involved in the movement of materials across epithelial layers.

Examples of this specific function include the movement of mammalian oocytes

during ovulation towards the uterus, and the process of mucociliary clearance

within the bronchi (reviewed in Pazour & Rosenbaum, 2002).

Non-motile cilia, also referred to as primary cilia, are found on virtually all

vertebrate cell types (Satir & Christensen, 2008), and are broadly associated with

the sensation of extracellular stimuli. These stimuli can be mechanical, chemical,

electromagnetic, or thermal (Sharma et aI., 2008; Tan et aI., 2007). The two

classic examples of primary cilia function are the vertebrate photoreceptor cells,

whose so-called connecting cilia and outer segments, which are responsible for

receiving visual cues, are in fact specialized ciliary structures, and the epithelium

of the renal nephron, in which primary cilia respond to the flow of nascent urine

through the tubule system by mechanical stimulation (Praetorius & Spring, 2003).

3 Chapter 6 of this thesis describes the role played by the non-motile cilia of

Caenorhabditis elegans in the sensation of thermal cues (see also Tan et aI.,

2007). It is important to note that, in all known cases, the cilium is acting solely as the medium for sensory perception; there are specific proteins that localise to the cilium that are responsible for receiving and transducing stimuli.

1.1.3 General ciliary structure

The core structural feature of the cilium is referred to as an , which protrudes from the surfaces of cells, and essentially represents the

"skeleton" of the organelle (Figure 1-1). The axoneme is a circular array of nine doublet microtubules, each created by polymerized dimers of a- and p- tubulin.

Each doublet consists of two microtubules: the complete A-tubule comprised of

13 protofilaments, and the incomplete B-tubule containing 11 protofilaments.

Axonemes are nucleated by a basal body, a centriole-derived structure typically composed of triplet microtubules, including the A- and B- tubules that define the axoneme itself, along with a third, the incomplete C- tubule. The axoneme is

surrounded by the ciliary membrane, which although continuous with the cell

membrane, may possess markedly different biophysical properties.

1.1.4 Motile cilia versus non-motile cilia

In addition to the general structural components already alluded to, motile

cilia possess additional complexities associated with their specific movements

(see Figure 1-2). In virtually every known example, motile cilia are found to

possess a central pair of microtubules in the centre of their . This

4 central pair possess poorly characterised, electron-dense appendages that seem to interact with complexes projecting towards the centre of the axoneme from the A-tubules of the outer doublet microtubules. This interaction regulates the highly intricate coordinated movement of two axonemal dynein complexes that also project from each of the A-tubules: the outer dynein arms

(ODAs), which consist of two dynein heavy chains and intermediate and light chains, and the inner dynein arms (IDAs), which consist of at least seven dynein heavy chain isoforms (reviewed in Satir & Christensen, 2008). Although this thesis focuses almost entirely on the mechanisms of non-motile cilium biogenesis, it should be emphasized that the process is similar in motile cilia, and, in fact, may be slightly more complicated, due to the greater number of axonemal components that need to be transported in order to build the structure.

1.1.5 The cilia of Caenorhabditis e/egans

Unlike many organisms, including humans, the only ciliated cell type in C. elegans is the sensory neuron, and none of the cilia in the nematode are motile.

Of the 302 neurons found in the adult hermaphrodite, a substantial number (60) possess cilia at the ends of their dendritic processes (reviewed in Inglis et aI.,

2007; see Figure 1-3 for the distribution of ciliated neurons in the worm). There are a number of intriguing differences between the previously described general cilium structure and the sensory cilia of the nematode.

Firstly, C. elegans basal bodies have been described as more

'degenerate' and termed 'transition zones' by Perkins et al. (1986).

Ultrastructurally, the C. elegans transition zone, also termed 'proximal segment,'

5 typically possesses a circular array of doublet microtubules (as opposed to the triplet microtubule arrangement most often associated with basal bodies in other organisms), although most, if not all nematode orthologues of known basal body proteins have been found to specifically localise to the basal body/transition zone region, including gamma-tubulin (Bobinnec et aI., 2000). In amphid and phasmid cilia, the transition zone is followed by a so-called 'middle segment' characterized by a canonical arrangement of 9 doublet microtubules, and this middle segment transforms into a 'distal segment' built of singlet microtubules (Figure 1-4); notably, in some ciliated neurons, these ultrastructural features may be somewhat divergent (Ward et aI., 1975; Ware et aI., 1975; Perkins et aI., 1986).

Additional singlet microtubules are often present in the central region of the C. e/egans cilia, but these microtubules are likely distinct from the central pairs observed in motile cilia. On the whole, this organization of doublets transitioning to singlets at the distal end is very similar to that seen in the flagella of mating

Chlamydomonas cells (Mesland et aI., 1980) and may be a general property of sensory cilia, as it has also been observed in several vertebrate cell types (e.g., pancreatic, renal and olfactory cells; Reese 1965; Webber & Lee, 1975; Hidaka et aI., 1995). The nature and positions of the C. elegans ciliated cell bodies and of representative dendritic ciliated endings are shown schematically in Figure 1­

3. A more detailed description of each C. elegans cilia type can be found below, and are also summarized in Table 1-1.

Amphids/Phasmids

6 The primary chemosensory organ of C. elegans is built from a collection of amphid neurons whose cell bodies are located in the anterior region of the pharyngeal bulb and possess axons that associate with the nerve ring. The dendrites of these neurons extend to the anterior end of the animal and terminate with diverse ciliated structures (Figure 1-3). The proximal regions of amphid cilia are typically protected by a sheath cell and extend through a channel created by socket cells to become partially exposed to the external environment. The majority of amphid neurons possess cilia shaped as single rods (ASE, ASG,

ASH, ASI, ASJ, ASK) or pairs of rods (ADF, ADL). Other amphids boast cilia that have membrane elaborations and possess unusual shapes; these are the wing neurons (AWA, AWB, AWC) and the amphid finger neuron (AFD), in which a small cilium is surrounded by approximately 50 villi. Both the wing and AFD neuron cilia terminate within a sheath cell, and thus are not exposed to the external environment. The role played by the AFD neuron in cilium-based thermosensation will be discussed in Chapter 6. The lengths of amphid cilia range from -7.5 I-Im (in the ASE, ASG, ASH, ASI, ASJ and ASK neurons) to

-1.5 I-Im for the AFD cilium (Ward et aI., 1975; Ware et aI., 1975; Perkins et aI.,

1986). Similar in structure to the single rod-like cilia found in amphids are the

PHA and PHB phasmid cilia. These are located slightly posterior to the anus of the worm and are exposed to the external environment (Hall & Russell, 1991). It should be noted that, unless stated otherwise, the cilia studied in this thesis belong to the amphid and phasmid neurons.

Inner/outer labial, cephalic neurons

7 The inner labial neuron types (ll1, Il2) are both arranged symmetrically in sets of 6 cells; ultimately terminating in the 6 "lips" that surround the mouth of the worm. Originating from a position anterior to the amphids, the dendrites of these

neurons terminate in shorter cilia with a more degenerate structure than seen in the amphids. While the Il1 cilia consist ofaxonemes made up of 7 doublet

microtubules, those of Il2 neurons are more variable (ranging from 5-7 doublets). These neurons are further distinguished by the fact that, while the Il1

cilia ultimately terminate, or embed, in the subcuticle, the Il2 cilia are exposed to

the external environment via openings in the cuticle (Ward et aI., 1975; Ware et

al.,1975).

The outer labial (2 lateral outer labial, or Oll neurons, and 4 quadrant

outer labial, or Ola neurons) and cephalic (CEP; 4 neurons) neurons similarly

terminate, albeit in a more restricted fashion, in the cuticle near the sub-dorsal,

sub-ventral, and lateral lips of C. elegans. The cilia found at the dendritic termini

of CEP neurons possess a degenerate axoneme (6-8 doublet microtubules),

while those found in the OlL/Ola neurons have an unusual square axonemal

arrangement of 4 microtubule doublets. The cilia of CEP neurons are unusual in

that, -11..1m from the basal body (within the subcuticle), the axonemal

microtubules associate with additional non-axonemal microtubules, generating

an electron-dense structure difficult to reconstruct via EM (Ward et aI., 1975;

Ware et aI., 1975).

Interestingly, the Il1, Oll and Ola neurons are unique in the fact that

they have striated rootlet structures descending from their transition zones (Ward

8 et aI., 1975; Ware et aI., 1975). Ciliary rootlets are prominent fibrous polymers of the protein Rootletin that emanate from the proximal end of the basal body (Yang et aI., 2002). Rootlets have been implicated in the maintenance and longevity of vertebrate sensory cilia (Yang et aI., 2005), as well as in providing scaffolding for kinesin-1-based intracellular transport (Yang & Li, 2005). It should be noted that very little is known about the rootlets of C. elegans; even a rootletin homolog has yet to be clearly identified.

Pseudocoelomic ciliated neurons

Two unusual ciliated cell types, AQR (located near the pharynx) and PQR

(found posterior to the phasmids in the tail), are found, along with their cilia, to be directly exposed to the pseudocoelomic cavity of the worm. Extremely little is

known about the ultrastructure of the cilia of these neurons, although they can be

identified under a compound microscope using, for example, the GCY-36 protein fused to GFP (Cheung et aI., 2004).

Ciliated deirid neurons

The 4 lateral, cervical deirid neurons are found in pairs, at the posterior

end of the pharyngeal bulb (AOE) and slightly anterior to the anus (POE). Like

many of the other neurons discussed in this review, their ciliated dendritic

endings are in a channel formed by a socket cell and an invaginated sheath cell.

The cilia of both AOE and POE terminate in the subcuticle, and thus are not

exposed to the external environment. These AOE/POE cilia are remarkably

similar to those found in the 4 CEP neurons, and, interestingly, these 8 neurons

collectively constitute the complete dopaminergic neuron set for the

9 hermaphrodite worm (Sulston and Brenner, 1975; Ward et aI., 1975; Ware et aI.,

1975).

Additional ciliated neurons

BAG and FLP are two relatively uncharacterized ciliated neurons whose cilia both terminate in or near the lateral lips of the worm. Unlike many of the other neurons described in this review, their cilia are not surrounded by support cells. Furthermore, their ultrastructures are quite complex, appearing via EM reconstruction as "bags" (BAG) or "flaps" (FLP) (Ward et aI., 1975; Ware et aI.,

1975; Perkins et aI., 1986). Recently, Zimmer et al. (2009) demonstrated that the

BAG neuron was involved in oxygen sensation in a manner similar to that observed in the pseudocoelomic neurons.

Male-specific ciliated neurons

C. elegans males have 52 additional ciliated sensory neurons, the majority of which are found in the male tail rays/hooks, where the cilia perform sensory functions (Peden & Barr, 2005). It should be noted, however, that only 48 of these 52 neurons are confirmed by EM to have cilia (Sulston et aI., 1980).

General descriptions of the structure and function of male-specific cilia are described in Table 1-1. It should be noted that, while in many organisms spermatozoa possess motile cilia, those of C. elegans are aflagellar, relying on amoeboid locomotion to reach and fertilize oocytes (Nelson et aI., 1982).

10 1.2 Cilium Biogenesis: Intraflagellar transport (1FT)

1.2.1 The discovery and basic mechanism of 1FT, as derived from studies in Chlamydomonas reinhardtii

Intraflagellar transport (referred to herein as 1FT), the dynamic bidirectional process required for building eukaryotic cilia, was first observed as a submembranous flagellar motility in the unicellular green algae Chlamydomonas reinhardtii (Kozminski et aI., 1993; a general model of 1FT is shown in Figure 1-

5). Subsequent analyses determined that tip-directed, or anterograde 1FT, was driven by heterotrimeric Kinesin-2, and was required for cilium biogenesis

(Kozminski et aI., 1995; Cole et aI., 1998; Orozco et aI., 1999). The reverse, basal-body directed, retrograde 1FT, was determined to be driven by a complex of Dynein proteins, most notably a homologue of cytoplasmic Dynein Heavy

Chain 1b (cDHC1b; Pazour et aI., 1998; Pazour et aI., 1999). Comparative biochemical analyses of wild-type and Kinesin-2-defective (fla10)

Chlamydomonas flagella resulted in the identification of two large protein subcomplexes associated with 1FT, termed 1FT subcomplex A (1FT-A) and 1FT subcomplex B (1FT-B), which collectively consist of at least 15 proteins (Cole et aI., 1998). These proteins are highly conserved in all eukaryotic organisms possessing cilia, including C. elegans (Cole, 2003; a comprehensive list of all conserved 1FT-associated polypeptides is shown in Table 1-2).

The specific roles played by each of the core 1FT proteins also appear to be highly conserved. Mutations in genes encoding components of 1FT-dynein

(including mutants defective in Chlamydomonas FLA14 and DHC1b, and C. elegans CHE-3, XBX-1 and DYLT-2) consistently result in severely abrogated

11 cilia, with significant protein accumulations observed at the distal tips, as well as between the outer doublet microtubules and ciliary membrane (Pazour et aI.,

1998; Pazour et aI., 1999; Signor et aI., 1999; Schafer et aI., 2003; unpublished observations; Figure 1-7). This result makes intuitive sense, as, in the absence of retrograde 1FT, 1FT particles transported to the distal tip of cilia by Kinesin-2 would have no mechanism for transport back to the base.

Perhaps more surprising, however, was the observation that mutations in components of IFT-A consistently mimic IFT-dynein mutants in organisms including Chlamydomonas, C. elegans, Tetrahymena thermophila, Trypanosoma brucei, and Mus musculus (Piperno et aI., 1998; lomini et aI., 2001; Collett et aI.,

1998; Perkins et aI., 1986; Qin et aI., 2001; Schafer et aI., 2003; Absalon et aI.,

2008; Tsao & Gorovsky, 2008; Tran et aI., 2008; Figure 1-8). These results would suggest that IFT-A may in fact be responsible for activating retrograde 1FT, either directly, or by carrying the IFT-dynein complex to the distal tip during anterograde 1FT (reviewed in Pedersen & Rosenbaum, 2008).

While IFT-A has been associated with retrograde 1FT, it appears that IFT­

B is associated with anterograde transport. IFT-B mutants consistently demonstrate an inability to generate ciliary structures, in a manner similar to those observed in IFT-Kinesin mutants. The observations were first made in the

IFT-B mutants of C. elegans, including che-2, che-13, osm-1, osm-5 and osm-6, long before the discovery of 1FT (Perkins et aI., 1986; Figure 1-8), and has subsequently been demonstrated in numerous organisms, ranging from

Chlamydomonas to mammals (Pazour et aI., 2000; Haycraft et aI., 2001; Schafer

12 et aI., 2003; Follit et aI., 2006; reviewed in Cole et aI., 2003; Pedersen &

Rosenbaum, 2008).

As already described, abrogation of anterograde 1FT via deletion of any of the three components of heterotrimeric Kinesin-2 in Chlamydomonas (for example, fla10 mutants), results in an inability to build or (as seen in the case of temperature-sensitive mutants) maintain ciliary structures (Kozminski et aI.,

1995). However, unlike the other general components of 1FT as described above, there appears to be a greater degree of complexity within the anterograde motor

machinery of cilia with non-canonical distal segments. These distal segments

range from simple, such as the microtubule singlets of the non-motile C. elegans

amphid cilia and the motile cilia of human respiratory/ovarian epithelia (Kubo et

aI., 2008), to elaborate, as seen in the winged amphids of C. elegans and vertebrate photoreceptor outer segments. It appears that an additional kinesin

motor, homodimeric Kinesin-2, is essential for the development of these non­

canonical cilia types (Jenkins et aI., 2006; Insinna et aI., 2008; Awan et aI.,

2004). The model of homodimeric Kinesin-2 function in the biogenesis of non­

canonical cilia is based on studies in C. elegans, which are described in detail

below.

1.2.2 Additional complexity of 1FT in Caenorhabditis e/egans

As in all other ciliated organisms, ciliogenesis in the nematode depends

on the intraflagellar transport (1FT) of ciliary precursors from the transition zone,

which sits at the junction between the dendrite of the sensory neuron and the

cilium, to the growing ciliary structure (Figure 1-6). The many known

13 components of the 1FT machinery, some of which were first identified in C. elegans (Scholey et aI., 2004 and see below) are listed in Table 1-2. Using time­

lapse microscopy it has been shown that in C. elegans, two 1FT motors of the

kinesin-2 family, namely heterotrimeric kinesin-2 and homodimeric OSM-3, move

1FT-particles (also consisting of two multi-protein subcomplexes, A and B; Cole et

aI., 1998) and presumably ciliary precursor proteins from the base of cilium to their sites of incorporation. This anterograde 1FT-machinery, and presumably

turnover products, are then transported back to the base of the cilium using the

IFT-dynein motor (Figure 1-6; Orozco et aI., 1999; Signor et aI., 1999; Snow et

aI., 2004). These two anterograde motors cooperate to build the middle and

distal segments of cilia. In the middle segment, kinesin-II and OSM-3-kinesin

function redundantly to move the same 1FT-particles and to assemble the middle

segment of the axoneme. In this segment, the slower-moving kinesin-II (0.5 J.lm s­

1) reduces the speed of the faster-moving OSM-3 (-1.2 J.lm S-1) to give rise to the

intermediate rate of motor-1FT-particle transport observed (-0.7 J.lm S-1).

Subsequently, at the middle-distal segment boundary, kinesin-II returns to the

base of the cilium, liberating OSM-3, which now moves 1FT-particles and bound

cargo to the distal tip at its own faster velocity (-1.2 J.lm S-1) to extend the distal

singlets of the axoneme (Snow et aI., 2004; Figure 1-6). Thus, animals lacking

functional kinesin-2 (e.g., kap-1 or klp-11 mutants) build a full-length cilium due to

the redundant function of OSM-3, osm-3 mutants specifically lack the distal

segment, and osm-3; kap-1 double mutants fail to make cilia because of the

absence of functional Kinesin-2 or OSM-3 (Snow et aI., 2004). It should be noted

14 that OSM-3 alone specifically extends distal singlets on some axonemes, but not others (Evans et aI., 2006).

The two sequential anterograde 1FT-pathways are coordinated by several types of regulator proteins. Three C. elegans homologs of human Bardet-Biedl

Syndrome (BBS) proteins (BBS-1, BBS-7 and BBS-8) have been shown to stabilize the 1FT-particle subcomplexes A and B which are bound to the Kinesin-2 and OSM-3 1FT-motors, respectively (Blacque et aI., 2004; Ou et aI., 2005a;

Snow et aI., 2004; Ou et aI., 2007). Abrogation of BBS protein function results in slightly truncated cilia and chemosensory or lipid accumulation defects (Blacque et aI., 2004; Mak et aI., 2006). The implications for these observations are of interest given that BBS, which is characterized by a diverse array of ailments, including obesity, cystic kidneys, and retinal degeneration, is one of a growing number of known ciliopathies (Beales, 2005; Blacque & Leroux, 2006). At least eight genes encoding BBS proteins are present in C. elegans (Table 1-2). A second modulator of the sequential 1FT pathway, a conserved ciliary protein also first characterized in C. elegans, DYF-1, specifically docks the OSM-3 kinesin onto 1FT-particles and simultaneously activates its motor activity; a dyf-1 mutant therefore specifically lacks the distal segment singlet microtubules (Ou et aI.,

2005a). Finally, the MAP kinase DYF-5 has been implicated in the undocking of

Kinesin-2 from the 1FT particles at the ends of middle segments and the overall association of OSM-3 Kinesin with 1FT particles (Burghoorn et aI., 2007).

The ability to analyze strains bearing GFP-tagged 1FT proteins by time­ lapse microscopy in C. elegans has provided researchers with a powerful means

15 to dissect 1FT function and study ciliary mutants (Orozco et aI., 1999). Until now, this technique to study cilia function has distinguished C. e/egans from the other

prominent ciliary model organism, Chlamydomonas. In addition to providing

crucial information about BBS and various 1FT-associated proteins such as DYF­

1, such in vivo studies are complemented by the fact that in many C. e/egans

Ciliary mutants, abnormal 1FT causes defects in sensory cilia structures and

sensory behaviour. For example, osm-3 and che-3 mutants possess defects in

the functions of the anterograde IFT-kinesin and retrograde IFT-dynein,

respectively, and display structural defects in the sensory cilia and corresponding

deficiencies in osmotic avoidance and chemotaxis (Signor et aI., 1999a; Wicks et

aI., 2000). Notably, the first evidence that biochemically-fractionated 1FT-particle

subunits identified in Chlamydomonas are essential for ciliary assembly was

based on the phenotypes of the corresponding C. elegans mutants, such as

osm-1/1FT172, osm-6/1FT52, osm-5/IFT88, che-2/1FT80, che-11/IFT140 and che­

13/IFT57 and daf-10/IFT122 (Brazelton et aI., 2001; Cole et aI., 1998; Qin et al.

2001; Scholey et aI., 2004). In addition, other components of the 1FT machinery

present in the Chlamydomonas flagellar proteome but not specifically identified in

biochemical fractionations of 1FT-particles (Pazour et aI., 2005) have first been

described in C. elegans, namely the aforementioned DYF-1 (Ou et aI., 2005a),

DYF-2, a protein that may help bridge the 1FT subcomplexes A and B (Efimenko

et aI., 2006), DYF-3, a protein associated with polycystic kidney disease that is

likely part of 1FT subcomplex B (Ou et aI., 2005b), DYF-13 (Blacque et aI., 2005),

and IFTA-1 (1FT-Associated protein 1), a likely subcomplex A protein (Blacque et

16 aI., 2006). Each of these mutants are characterized phenotypically as having cilia structure and chemosensory defects.

Although our understanding of the 1FT transport process has matured significantly in the last few years, very little is known about the nature of the proteins that require 1FT-mediated transport to reach their ciliary destination (i.e., cargo). Indeed, only radial spoke proteins had been found to be bona fide 1FT cargo proteins in Chlamydomonas (Qin et aI., 2004); now, several have surfaced in C. elegans. One class of 1FT-cargo are the cilia-localized TRP-type channels

OSM-9 and OCR-2, which are implicated in various chemosensory responses

(Tobin et aI., 2002). Both have been shown to undergo 1FT (Qin et aI., 2005), marking the first account of a non-axonemal component being visualized to move along a cilium. Interestingly, OSM-9 and OCR-2 depend on each other for their ciliary localization, and ectopic expression of OCR-2 in AWC neurons is sufficient to drive OSM-9 to the cilia in this neuron (Tobin et aI., 2002). Another apparent

1FT cargo is TUB-1, the C. elegans homolog of the mammalian protein Tubby, which is associated with an obesity phenotype in mice (Kleyn et aI., 1996;

Noben-Trauth et aI., 1996). TUB-1 motility has been reported in both dendrites and cilia, and its function is required for normal lipid homeostasis, life span, and chemotaxis (Mukhopadhyay et aI., 2005). Lastly, IFTA-2, a RAB-like protein expressed exclusively in ciliated cells has been shown to undergo 1FT, but interestingly, is not required for building an intact cilium. Instead, IFTA-2 may represent a signalling molecule that is required for ciliary function; consistent with this notion, the ifta-2 mutant has an extended lifespan and dauer formation

17 defects that have previously been ascribed to cilia dysfunction (Schafer et al.

2006).

In summary, proteins that affect 1FT in C. elegans can be divided into a number of distinct modules: 1FT-A, IFT-B, Kinesin-2-associated, OSM-3­ associated, IFT-Dynein-associated, and the complex of BBS proteins. Both the transport profiles of GFP tagged proteins, along with the behaviours of representative ciliary markers in mutant backgrounds can expedite the assignment of a potentially novel 1FT protein to a specific 1FT module (examples of cilium structure/function defects for each 1FT module can be seen in Figures

1-7 and 1-8). Chapter 3 describes the discovery of a novel and conserved 1FT

protein, DYF-11, which, using the approach described above, appears to be associated with 1FT-B. Chapter 5 describes a genetic screen for mutants specifically defective in IFT-AlIFT-Dynein, based on mutant phenotypes that are typical for those modules. Finally, Chapter 4 represents the first steps towards defining an additional 1FT module, namely the transition zone module, in which

member proteins specifically complex in or around the ciliary transition zones and

regulate cilium-based signaling and/or ciliogenesis in a manner that has yet to be

clearly identified.

1.3 Signalling and Sensation in Cilia

1.3.1 Localisation of key developmental signalling pathways to cilia

A slew of studies over the last decade have connected cilia to key

developmental signalling pathways. In most cases, the connection was originally

18 made based on the similarity of phenotypes seen in murine signalling and

1FT/cilium structure mutants. It appears that, in at least some cases, key proteins

in each ciliary signalling pathway require either localization to the cilium or transport within the cilium to function (three cilia-based signalling paradigms are described in more detail below). In Chapter 4 of this thesis, our analyses in C.

elegans hint at an association of another key signalling pathway (Insulin/IGF) with ciliary structure/function.

Hedgehog Signalling

Individuals lacking components of the Hedgehog (Hh) signalling pathway

are characterized by a number of clinical manifestations, including skeletal and

cranial malformations (reviewed in Fliegauf et aI., 2007). These phenotypes are

also observed in murine 1FT-defective mutants, offering the first indication that

cilia playa role in the Hh pathway (Zhang et aI., 2003; Huangfu et aI., 2003). This

notion was strongly supported by the recent observations that key components of

the Hh pathway localise to cilia, including Smoothened (Smo; Corbit et aI., 2005)

and the transcription factors Gli2 and Gli3 (Haycraft et aI., 2005; reviewed in

Singla & Reiter, 2006). Further analyses revealed that 1FT acts downstream of

the receptor Patched-1 (PTCH1), but upstream of Hh-regulated GLI transcription

factors (Li et aI., 2005; Haycraft et aI., 2005). It has been proposed (Haycraft et

aI., 2005) that following binding of Hh signalling proteins to Patched-1,

Smoothened (SMO) is released and transported to the ciliary tip, presumably by

1FT, ultimately terminating GLI processing, and resulting in the release of active

GLI proteins to the nucleus.

19 Canonical/Non-Canonical Wnt Signalling

The protein Inversin, implicated in nephronophthisis type 2 (an established ciliopathy), appears to act as a switch between the canonical (~-catenin­ dependent) and non-canonical (~-catenin-independent)Wnt signalling pathways

(reviewed in Gerdes & Katsanis, 2008). Specifically, Inversin targets cytoplasmic

Dishevelled (DSH) for proteasomal degradation, thus inhibiting non-canonical

Wnt signalling, while concurrently activating the canonical pathways (Simons et aI., 2005). The ciliary connection to Wnt signalling has been further fleshed out by the recent discovery that loss of murine , , and bbs6/ result in increased stability of cytoplasmic ~-catenin, pointing to an overall increase in canonical Wnt activity (Gerdes et aI., 2007). Similar results have been observed in Kif3a (which encodes a component of the heterotrimeric kinesin-2 anterograde

1FT motor) mutant mice, directly implicating 1FT in the regulation of Wnt signalling

(Corbit et aI., 2008). Similar to the results observed in the aforementioned analyses of Shh signalling, key components of the Wnt pathway specifically localise to cilia, including ~-catenin (Corbit et aI., 2008), although several key players, including Frizzled (FRZ) and DSH, do not (reviewed in Gerdes &

Katsanis, 2008).

It was recently discovered (Park et aI., 2006) that the non-canonical

Wnt/planar cell polarity (PCP) effector proteins Fuzzy (Fz) and Inturned (In) accumulated at the base of cilia, where they were found to control the orientation of axonemal microtubules. Further studies revealed that the PCP protein

Dishevelled (Dsh) was essential for the docking of the basal body to the apical

20 plasma membrane (Park et aI., 2008). One model posits that ciliary signalling, in fact, encourages the cell to activate the PCP pathway in lieu of the canonical Wnt pathway, resulting in the expected post-mitotic PCP-associated differentiation and not Wnt-induced mitosis (Gerdes et aI., 2007; Corbit et aI., 2008; reviewed in

He, 2008; Fliegauf et aI., 2007).

PDGF Signalling

In certain cell types (specifically mouse embryonic fibroblasts), the

Platelet-Derived Growth Factor Receptor (PDGFRa) protein is found to localize to primary cilia (Schneider et aI., 2005). PDGF-dependent activation of PDGFRa at the membrane of the cilium results in the activation of AKT, and the phosphorylation of MEK1/ERK1, both within the cilium or basal body (Schneider et aI., 2005). These data collectively implicate cilia for the first time in cell growth and proliferation.

1.3.2 Cilia-associated diseases: the ciliopathies

Dysfunction of motile cilia has long been associated with human disease.

Loss of ciliary motility can result in both male and female infertility, significant respiratory inflammation, and loss of left-right body axis asymmetry (reviewed in

Badano et aI., 2006). A body-wide loss of cilium-based cellular motility results in a condition known as Primary Cilia Dyskinesia, or PCD. PCD can result from deficiencies in any number of components of the motile cilium, including the

IDAs, ODAs, central pair apparatus, and radial spoke complex (Satir &

Christensen, 2008).

21 The connection between non-motile (primary) cilia and human health has only recently emerged. The role played by primary cilia is less obvious than their motile counterparts; for the most part, the physiological functions of these organelles occur at a molecular level, where key sensory proteins reside in the ciliary membrane and depend on 1FT for trafficking of signalling proteins. The major breakthrough in understanding the role played by primary cilia in human health came following the discovery that the gene encoding the IFT-B component

IFT88 was mutated in the oak ridge polycystic kidney mouse line (orpk; Pazour et aI., 2000). Although the ORPK mouse was originally isolated as a model for polycystic kidney disease (PKD), it is also an excellent example of ubiqiuitous loss of cilia in mammals (reviewed in Lehman et aI., 2008). In addition to the phenotypes characteristic of PCD, ORPK mice also develop kidney, liver, and pancreatic cysts, as well as hydrocephalus, retinal degeneration, ataxia, and a number of skeletal abnormalities (Lehman et aI., 2008; Sharma et aI., 2008).

Since the association between IFT88 and orpk mutations in mice, dozens of human disorders have been attributed to loss of ciliary function. Of particular relevance to this thesis are the conditions known as Bardet-Biedl syndrome

(BBS) and Meckel-Gruber syndrome (MKS). Many BBS proteins (BBS1, 2,4,5,

7, and 8) complex together to form a so-called BBSome, that localises principally to the base of primary cilia in mammalian cells (Nachury et aI., 2007), and, as described above, are known to regulate the coordination of the Kinesin-2/0SM-3 anterograde 1FT motors in C. elegans (reviewed in Inglis et aI., 2007). Of the remaining six known BBS proteins, BBS3 is an ADP ribosylation factor of the

22 ARL family; BBS6, 10, and 12 are members of the type II chaperonin family;

BBS9 is a relatively uncharacterised yet conserved protein; BBS11 is an E3

Ubiquitin Ligase (reviewed in Zaghoul & Katsanis, 2009). In humans, BBS is a non-lethal disorder that appears to affect only primary cilia, and is characterised by obesity, impaired cognitive function, kidney cysts, hypogonadism, and polydactyly (reviewed in Blacque & Leroux, 2006). Chapter 6 of this thesis examines the thermosensory behaviours of C. elegans in bbs mutant backgrounds.

MKS is a significantly more severe affliction than BBS, resulting in perinatal mortality, and is characterised by severe central nervous system and neural tube defects, polydactyly, and kidney/liver cysts (reviewed in Sharma et aI., 2008). Less is known about the MKS-associated proteins than their BBS counterparts, although MKS1, MKS3, and MKS6 have all been shown to be required for the biogenesis of primary cilia (Dawe et aI., 2007; Tallila et aI., 2008).

Intriguingly, the MKS-associated proteins appear to have a greater degree of conservation amongst all ciliated organisms than BBS proteins, perhaps hinting at a more central role in cilium structure/function; this will be discussed further in

Chapter 2. Of particular interest are three B9 domain-containing proteins, including MKS1, which are found exclusively in ciliated organisms. Chapter 4 of this thesis examines the complementary roles played by each member of the B9

protein family with respect to ciliary function in the nematode.

23 1.4 Uncovering the Ciliome: Bioinformatic, Genomic and Proteomic Studies

1.4.1 Bioinformatic searches for X boxes in C. e/egans promoters

A landmark study by Swoboda et al. (2000) revealed that the expression of C. elegans genes required for cilium biogenesis, normally restricted to ciliated sensory neurons (60 of the 302 neurons in total), is controlled by the regulatory factor X (RFX)-type transcription factor, DAF-19. Several genes encoding 1FT proteins are regulated by DAF-19, which binds to a conserved promoter element termed the 'X box.' Other C. elegans ciliogenic genes, including six bbs genes and novel 1FT genes such as dyf-1, dyf-3 and dyf-13, were later shown to also be regulated by the DAF-19/X boxes (au et aI., 2005b; Fan et aI., 2004; Li et aI.,

2004; Blacque et aI., 2005; Murayama et aI., 2005; Ansley et aI., 2003). This indicated that C. elegans X-box-containing genes expressed exclusively in ciliated cells are likely to encode proteins with important ciliary functions and, notably, murine RFX3 and at least one of the two Drosophila RFX transcription factors also regulate proper cilium assembly and function (Dubruille et al.; 2002;

Bonnafe et aI., 2004). These findings prompted genome-wide searches for such genes in C. elegans.

In one study, Efimenko et al. (2005) scanned C. elegans promoters for a

14-bp 'average' (degenerate) X-box consensus sequence. A set of 758 genes having an occurrence of the motif with up to three mismatches (when sequences were compared with a refined consensus sequence) and within 1000-bp upstream of the start codon were identified. Because bona fide C. elegans X boxes are typically -100-bp upstream of start codons and tend to be conserved

24 (Ansley et aI., 2003; Swoboda et aI., 2000), more stringent criteria (within 250 bp and ::51 mismatch) could be considered to uncover novel ciliogenic and ciliary genes with greater confidence. In all, 164 genes fulfil these criteria, including six previously uncharacterized xbx genes whose expression in ciliated cells was shown to be reduced in a daf-19 mutant background. One of the genes, xbx-2, encodes a Tctex-1 domain-containing dynein light chain that was visualized to undergo 1FT when fused to green fluorescent protein (GFP). Another protein identified, TUB-1, was recently shown to localize to cilia and undergo 1FT

(Mukhopadhyay et aI., 2005); its disruption in C. elegans causes ciliary phenotypes (chemosensory defects and increased life span) and, intriguingly, an increase in lipid accumulation similar to that seen in a mouse knockout of the gene homolog, tubby (Mak et aI., 2006; Noben-Trauth et aI., 1996; Kleyn et aI.,

1996).

In a similar study, Blacque et al. (2005) used a profile Hidden Markov

Model created with 22 X-box sequences to query all C. elegans promoters. The training set included several X boxes from genes previously identified by Fan et al. (2004) that were shown to be expressed strictly in ciliated cells. The search yielded 1572 genes with X-box sequences within 1.5-kb of start codons and with significant human homologs. Canonically positioned X boxes «250-bp from start codon) were found in 293 of these gene promoters. From this data set, the expression of 14 uncharacterized but evolutionarily conserved genes was shown to be restricted to ciliated neurons. One of these (dyf-13) was found to encode a novel 1FT protein required for proper cilium formation (Blacque et aI., 2005).

25 Together, the two bioinformatic studies identified numerous putative novel ciliary genes, including kinases, receptors and transcription factors, many of which seem to be unique to sensory (non-motile) cilia. Although the two data sets probably include a significant proportion of false positives and might miss more divergent but functional X boxes, they are complementary because they only overlap partially. The predictive abilities of the,se data sets to identify ciliary and ciliopathy-associated genes is substantial; for example, C48B6.8 was initially confirmed by Blacque et al. (2005) to be expressed exclusively in ciliated cells, and the human homolog was later shown to be mutated in some patients with

Bardet-Biedl syndrome (Nishimura et aI., 2005), identifying it as BBS9. Similarly, the human homologs of two X-box-containing C. elegans genes (R148.1/xbx-7 and F35D2.4) are now associated with Meckel syndrome (Smith et aI., 2006;

Kyttala et aI., 2006), a putative basal body and ciliary disorder characterized by polydactyly, renal and hepatic cystic dysplasia and developmental defects in the central nervous system (occipital encephalocele).

1.4.2 Comparative genomic analyses

The availability of numerous sequenced genomes from ciliated and non­ ciliated eukaryotes has recently set the stage for powerful comparative genomic studies. In silico subtraction of homologous genes found in non-ciliated organisms from a ciliated genomic dataset should enrich for genes that have unique, cilium-specific functions. Li et al. (2004) successfully applied such an approach by using a panel of genomes from different organisms. One study involved finding the intersection between the ciliated human and

26 Chlamydomonas genomes and subtracting the non-ciliated Arabidopsis genome.

This yielded a so-called flagellar apparatus-basal body (FABB) proteome

containing 688 proteins. The data set included a large percentage of known ciliary proteins (52 of 58 queried), several known basal body proteins (four of six

examined) and a large proportion (-50%) of proteins with no known function or

link to cilia. Similar genomic arithmetic was performed by obtaining the

intersection between the Chlamydomonas and other ciliated organism genomes

(Mus musculus, Ciona intestinalis, C. elegans and Drosophila) and excluding

Arabidopsis gene homologs. This produced data sets that overlapped to a large

degree with the FABB proteome. The smaller amount of overlap with the C.

elegans comparison could be partly explained by the lack of motile cilia in the

nematode. Notable disease-associated proteins present in the FABB proteome

included most known BBS proteins in addition to NPHP4 and , which

are linked to kidney disorders (Hildebrandt & OUo, 2005), and Hydin, which is

implicated in hydrocephalus (Davy & Robinson, 2003).

A similar comparative genomic study by Avidor-Reiss et al. (2004)

comprised six ciliated organisms (Drosophila melanogaster, Homo sapiens, C.

elegans, C. reinhardtii, Plasmodium falciparum and Trypanosoma brucei) and

three non-ciliated organisms (A. thaliana, S. cerevisiae and D. discoideum).

Using Drosophila as an anchor, genome subtractions were employed to reveal

four classes of genes: (i) those conserved in all ciliated organisms (16 genes); (ii)

those conserved in organisms with motile cilia (i.e. in all ciliated organisms

except C. elegans; 18 genes); (iii) those found in organisms with

27 compartmentalized (but not cytosol-assembled) cilia (i.e. all ciliated organisms except P. falciparum; 103 genes); and (iv) those specifically present in organisms with compartmentalized and motile cilia (i.e. humans, Chlamydomonas,

Trypanosoma and Drosophila but not C. e/egans or Plasmodium; 50 genes).

Of the 187 genes identified in total, the authors observed that 30 out of 36

known ciliogenic genes examined were present, including those encoding evolutionarily conserved 1FT and motor proteins. This indicated that the

bioinformatic analyses enriched significantly for cilia-specific genes. Nearly a third (52) of the 187 Drosophila genes identified were found to have an X-box sequence in their promoters, implicating RFX transcription factor(s) as important

regulator(s) of ciliogenesis, as in C. elegans (Dubruille et aI., 2002). Seventeen genes were chosen for expression analysis; remarkably, all transgenes were found to be expressed exclusively in mechanosensory and chemosensory

ciliated neurons, with the exception of one (a dynein light chain), which is also

expressed in sperm cells. Interestingly, two of the genes included small GTP­

binding proteins of the ARF-like family, ARL3 and ARL6. The first has been

associated with cilium biogenesis in Leishmania (Cuvillier et aI., 2000) and the

second was subsequently shown to encode a BBS protein implicated in 1FT,

BBS3 (Fan et aI., 2004 ; Chiang et aI., 2004). These two ARL proteins were both

identified in the bioinformatic search for C. elegans X boxes (Blacque et aI.,

2005; Efimenko et aI., 2005), and a third ARL protein - ARL2L1/ARL­

13b/scorpion - was also uncovered in the X-box studies and results in cystic

kidneys when disrupted in zebrafish (Sun et aI., 2004).

28 One advantage of comparative genomic studies is that cell body or basal body proteins that support cilia function but are not specifically localized to the organelle (e.g. cannot be detected in proteomic analysis of isolated cilia) can be identified. However, the approach used relies almost exclusively on identifying gene counterparts (orthologs) by homology searching. Consequently, bona fide ciliary genes might be excluded because they are members of conserved multigene families with similar but non-redundant functions (e.g. tubulins and molecular motor components), or they are too divergent, small or sequence information-poor (e.g. contain repetitive sequences such as coiled coils) to be

identified reliably. Performing additional comparative genomic analyses with new organisms will help to uncover a larger complement of ciliary genes, some of which might be organism-specific. Chapter 2 of this thesis represents such an

approach, employing >15 ciliated organisms, including the newly sequenced

genome of the chytrid fungus Batrachochytrium dendrobatidis.

1.4.3 Flagellar regeneration transcriptome analyses

In unicellular organisms, cilia are normally resorbed before cell division

and can be shed during certain environmental conditions. Cilia regeneration

would be expected to require substantial upregulation of genes necessary for

building a functional organelle. Using a DNA oligonucleotide microarray, Stoic et

al. (2005) monitored the genome-wide expression of genes from

Chlamydomonas undergoing reflagellation following acid shock-induced flagellar

severing. The authors identified 220 genes that had more than twofold induction

during flagellar regeneration, including 85 genes previously known to encode cilia

29 or basal body components. Using a less stringent criterion of >1.75-fold induction, 35 of 56 genes known to be transcriptionally induced during reflagellation (as shown by Li et al., 2004) were identified, whereas none of 54 uninduced genes and 20 non-flagellar genes had such an increase. These data confirm the low false-positive rate of the approach, but present a relatively high false-negative rate because of the stringent threshold criterion used for selecting upregulated genes.

1.4.4 Ciliated cell-specific transcriptome analyses

Another means of obtaining a transcriptome enriched for ciliary genes is to compare, in a multicellular animal, the transcription profiles of ciliated cells with those lacking cilia. Such an approach was employed by Blacque et al. (2005) by using a panel of embryonic ciliated or non-ciliated cells from C. e/egans combined with serial analysis of gene expression (SAGE). All 60 ciliated sensory neurons were specifically marked using GFP expressed from a bbs-1 promoter, isolated by disrupting the embryo and fluorescence-activated cell sorting, and subjected to SAGE expression profiling. Similarly, specific subsets of cells expressing pan-neuronal-, intestinal- or muscle-specific GFP markers were analyzed by SAGE. When a 1.5-fold or greater level of expression in ciliated cells versus each of the pan-neuronal (mostly non-ciliated), non-ciliated intestinal and muscle cell transcriptomes is considered, 1282 genes are represented. Many of the expected ciliary genes are found in this dataset, including 1FT and bbs genes, as are numerous other genes of unknown function. The prominin 1 membrane protein homolog, F08B12.1, represents an interesting example that deserves

30 further attention given its strong enrichment (13-fold) in ciliated cells. It has not been associated with cilia function, but is found in membrane protrusions and the sperm tail, and its disruption in the mouse causes retinal degeneration (Corbeil et aI., 2001).

The advantage of the microarray and SAGE over proteomic studies is that they can uncover any gene required for the formation or function of cilia, irrespective of the subcellular localization of the encoded protein. For example, in

C. elegans, such studies might help identify specialized genes required for the function or differentiation of all or specific ciliated sensory neurons. A study by

Colosimo ef al. (2004) employed microarrays to assess the difference in expression profiles between C. elegans unsorted embryonic cells and two isolated ciliated sensory neurons, the olfactory AWB neuron and the thermosensory AFD neuron. Overall, however, these approaches can have both high false-positive and negative rates because of low enrichment factors

(particularly for poorly expressed genes), emphasizing the need for additional complementary studies (e.g. proteomic analyses) to define genes important for cilia function.

1.4.5 Proteomic studies of motile cilia

A powerful means to identify ciliary proteins is simply to perform proteomic analyses on isolated cilia. The first attempt to identify a ciliary axoneme proteome, using cilia released from human bronchial epithelial tissue culture cells as starting material, was described by Ostrowski ef al. (2002). The cilia were extracted with detergent to yield ciliary axonemes, and peptides were generated

31 by proteolytic digestion either directly or following 1D or 2D gel electrophoresis separations of the proteins. Liquid chromatography (LC)-mass spectrometric

(MS) analyses of the peptides revealed a total of 214 proteins identifiable on the basis of one or more peptide match(es). Several known ciliary axonemal proteins were identified, including tubulins, dynein components and radial spoke proteins, in addition to flagellar or sperm-specific components, such as the sperm­ associated antigen 6 (SPAG6)/Chlamydomonas PF16 protein. Many of the proteins identified were initially listed as uncharacterized, but as pointed out by

Marshall (2004), several of these are now known to have an ortholog in other organisms, including the cystic kidney-associated DYF-3/qilin 1FT protein (Ou et aI., 2005; Sun et aI., 2004).

More recently, Smith et al. (2005) performed 2D LC separations on tryptic peptides isolated from two independently obtained, membrane-free preparations of Tetrahymena thermophila ciliary axonemes, and analyzed the eluted peptides by MS. A total of 223 proteins were deemed bona fide ciliary components. Of these, 139 matched entries from non-redundant databases. Many of the evolutionarily conserved proteins are components previously localized to motile cilia, such as structural (e.g. tubulins, radial spokes proteins, PF16) and motor

(kinesin and dynein subunits) proteins.

A significant advantage of using isolated cilia is that they can be fractionated biochemically to obtain different sub-organellar protein extracts for proteomic studies. Pazour et al. (2005) used such a strategy to reveal a

Chlamydomonas ciliary proteome enriched for proteins in the membrane plus

32 axoneme, in the membrane plus matrix (the matrix contains components such as

1FT proteins that are not tightly associated with either the axoneme or membrane) or in two differentially extracted axoneme preparations. The complete data set of proteins identified in the preparations included 360 proteins represented by five or more peptides, deemed to represent ciliary proteins with a high level of confidence, and 292 additional proteins with two-to-four peptides that probably includes many bona fide ciliary components but also many false positives. An additional 492 proteins were identified on the basis of a single peptide. Remarkably, 97 of 101 known Chlamydomonas ciliary proteins were present in the complete data set, providing strong evidence for the completeness of the identified ciliome. Several of the genes encoding proteins found in the proteome were chosen for RT-PCR expression analysis during flagellar regeneration. Most of the 87 genes that had an uncharacterized human homolog

(60 of 69 tested) were induced by deflagellation, indicating their potential involvement in ciliary functions.

Several proteins - including tRNA synthethases, ribosomal proteins and histones - were highlighted as being probable contaminants. A key discovery by the authors is that >90 signal transduction proteins (e.g. kinases, phosphatases,

2 many potential Ca +-binding EF hand-containing proteins, receptors, channels and small GTPases) were uncovered, underlining the capacity for sensory perception by motile cilia. The discovery of multiple ciliary proteins associated with cystic kidney disease as also notable, including , fibrocystin,

33 ARL2L1/ARL13b/scorpion, DYF-3/qilin and the NIMA kinase NEK1 (Hildebrandt

& Otto, 2005; Quarmby & Mahjoub, 2005).

In a more recent study, Broadhead et al. (2006) performed a proteomic analysis on the unique flagellum of the bloodstream parasite T. brucei. Both within its vector (the African tsetse fly) and host, T. brucei has a flagellum that is associated with a paraflagellar rod (PFR) structure not found in other cilia discussed earlier. The ciliary axoneme, basal body and PFR were isolated using a standard procedure of treatment with detergent and high salt concentration, and used to derive a ciliary proteome by LC-MS. Of the 331 proteins identified, many (208) are specific to trypanosomatids and although some of these could be attributed to the inclusion of the paraflagellar rod in the proteome, most novel axonemal components might be unique to this group of organisms. Not surprisingly, most of the remaining 123 proteins are motile and cilia-specific, showing a large degree of overlap with the Chlamydomonas and Tetrahymena ciliomes, whereas only a few are homologous to C. elegans and Drosophila proteins.

As seen in other studies, several proteins identified by Broadhead et al.

(2006) are implicated in human or mouse diseases, including Hydin and PACRG.

Notably, the requirement of both of these proteins for cilia function (motility) was validated in this study. Another interesting observation made by the authors is that 34 of the T. brucei genes uncovered have homologs in humans that map to the critical intervals of a diverse set of genetically mapped diseases with potential ciliary involvement. These include primary ciliary dyskinesia (PCD), PKD, retinal

34 dystrophies and BRESEK syndrome (which is characterized by cystic kidneys and polydactyly), among other ailments (Broadhead et aI., 2006).

One drawback of proteomic studies is their limited sensitivity. Thus, true ciliary proteins of low abundance might not be identified, or might be represented with only one peptide and thus cannot be considered bona fide ciliary proteins because the subset of proteins represented with few peptides is more likely to contain contaminating proteins. Another potential difficulty is that biochemical fractionation might remove ciliary components. For example, although all studied

C. elegans BBS proteins have been shown to be components of the 1FT machinery, it is intriguing that none of the homologous proteins from humans,

Chlamydomonas or Tetrahymena were identified in the proteomic studies. Such shortcomings, as with those specific to the bioinformatic and genomic studies, again highlight the need to combine the results of different complementary studies to help in the identification of proteins that have ciliary roles.

1.4.6 Meta-analyses

As described above, each ciliomic approach is characterised by specific

strengths and weaknesses. Because of this, the most effective way of identifying

the most likely and relevant ciliary gene/protein candidates necessarily involves

the cross-comparison of each ciliomic approach. We (Inglis et aI., 2006) and

others (Gherman et aI., 2006), employed such meta-analysis in order to resolve

the list of potentially undiscovered ciliary proteins. These analyses can both be

easily accessed through searchable online databases: www.ciliome.com and

www.ciliaproteome.org. respectively. Top candidates identified in each study

35 have subsequently been characterised, and, indeed, have turned out to play highly conserved roles in cilium biogenesis/function. Examples include DYF­

11/MIP-T3 (Chapter 3) and the family of B9 domain-containing proteins (Chapter

4).

1.5 Research Objectives

The primary goal of my doctoral research has been to employ ciliomic analyses, including existing studies and my own individual approaches, in order to identify/characterise novel proteins that play conserved roles in cilium biogenesis and function. To characterise candidate genes, I employ the nematode C. elegans as a model system, which as described above, has several advantages with respect to rapid analysis of putative ciliary genes, particularly those involved in intraflagellar transport (1FT).

Chapter 2 represents a detailed comparative genomic analysis that takes advantage of the recent sequencing of the Batrachochytrium dendrobatidis genome. B. dendrobatidis represents the sole class of fungi are capable of generating cilia during a specific stage of their life-cycles. A detailed, cilium­ centric analysis of this organism's genome, therefore, represents an opportunity to gain molecular insights into a more ancestral version of the organelle.

Chapter 3 describes the cloning/characterisation of the C. elegans dyf-11 mutant. The gene altered in the dyf-11 strain, C02H7.1, was a strong ciliary gene candidate based on the ciliomic approaches listed above. By observing the transport profiles of GFP-tagged DYF-11, as well as the behaviours of various

36 1FT markers in the dyf-11 mutant background, we were able to determine that

DYF-11 is a likely component of 1FT -B, and is therefore vital for ciliogenesis.

Chapter 4 represents the analysis of a family of proteins that localise to C. elegans basal body/transition zones. Three so-called B9 proteins, named after the B9 domains found in each, were identified in numerous ciliomic studies, and one member, MKS1, is implicated in Meckel-Gruber syndrome, a known ciliopathy. We show that the B9 proteins do not have any overt role in 1FT or the generation of normal ciliary structures, but have a more subtle influence on cilium-based insulin signalling.

Most of the 1FT research in C. elegans has focused largely on the anterograde (tip-directed) portion of the process. Using an existing C. elegans mutant library enriched for strains defective in ciliary genes, I screened for defects specifically in the 1FT subcomplex A and 1FT dynein modules, by looking for distinctive GFP-tagged protein accumulations in the cilia. Chapter 5 describes this study, which includes the identification of a potentially novel allele of a known

1FT subcomplex A component.

Finally, Chapter 6 represents a slight departure from the primary research goals of this thesis. Using a number of ciliary mutants, we assign a novel sensory modality to cilia - thermosensation. Worms defective in BBSome components show a decreased ability to respond to both noxious and physiological thermal stimuli. This chapter also describes the effects of ciliary gene mutation on the roaming abilities of individual worms, specifically with respect to temperature sensation.

37 1.6 Figures

Figure 1-1. Generalised structure of the eukaryotic cilium.

» ox ::J CD 3 CD

c:o (II en (II c:o o a. '<

iiii'iiiiiiiiiiiiiiiiiiiii A-tubule • Triplet microtubules 0 Protofilament ___ B-tubule • Doublet microtubules - Membrane iiii'iiiiiiiiiiiiiiiiiiiii C-tubule

38 Figure 1-2. Generalised structure of a motile cilium (flagellum).

» ox :::J Cll 3 Cll

OJ Ql (fl Ql OJ a a. '<

A-tubule • Triplet microtubules 0 Protofilament B-tubule • Doublet microtubules - Membrane C-tubule

Structures associated with motile cilia:

--- Outer Dynein Arm --- Inner Dynein Arm

--4 Radial Spoke Nexin 00 Central pair microtubules

39 Figure 1-3. Ultrastructures of cilia and relative positions of all known ciliated neurons (cell bodies and associated dendrites) in the C. e/egans hermaphrodite. The worm cartoon (bottom) illustrates the positions of all ciliated cell bodies and their dendritic extensions. The four insets shown (top), are schematics of electron

micrograph reconstructions of known ciliated endings (adapted from Perkins et al. (1986) for amphids and phasmids and Ward et al. (1975) for labial and

cephalic neurons). Cu, cuticle; CR, ciliary rootlet; SCu, subcuticle; So, socket

cell; Sh, sheath cell.

--Cu------

Sh _fl I ,I 'T+:--~-CR I I CR--/fl!t-: I ASE, ,7 I L, ,. ASG. ASH, ,t MD ASI, ASK. I'D'?: }""A. AWB III 112 CEP Oll, OlQ PHA. PHB ASl I'Dl AWC '

OL CEPO POE AFD .... ADL, ASCi. ASI, " ASK. AWA, Awe I FLP " / "" "" "" "" "" . I " CEPV / " " 'I MJF, ASE, ASH, I "OLav ASJ " I -' " BAG ./ " -' -' " -' ",,~ -' -' -' -' -' -' -'

40 Figure 1-4. Structure of a typical C. e/egans amphid cilium.

0 0 0

C/) o 0 co to 3 co o 0 ~ 00

s:: a: a.ro C/) co to 3 co / ~

~ ::J (/l ;:;: o' ::J N o ::J co

;;;;;:;;;;;:;;;:;:; A-tubule • Doublet microtubules o Protofilament __ B-tubule -Membrane

41 Figure 1-5. Generalised mechanism of intraflagellar transport (1FT), as based on research in Chlamydomonas reinhardtii.

3. Tip rearrangement

o 1FT Subcomplex A ® 1FT Subcomplex B I Kinesin-2 2. Anterograde 1FT .It IFTDynein I Cilia Membrane Protein E A-tubule .2 U B-tubule C-tubule

4. Retrograde 1FT

1. Assembly of 1FT raft

ro>­ (/)"0 co 0 COCO

>­ "0o co Q) ()

42 Figure 1-6. Hypothetical model for intraflagellar transport (1FT) in the amphid sensory cilia of Caenorhabditis e/egans Model for intraflagellar transport in C. elegans. Six distinct steps are indicated (1-

6) for the canonical 1FT pathway in amphid and phasmid rod-type cilia, with the individual 1FT modules and ciliary structural elements shown in the legend. BB, basal body; TZ, transition zone. See text for details.

43 4. Tip rearrangement 0 1FT Subcomplex A ® 1FT Subcomplex B I Kinesin-II A 1FT Dynein -..I/) OSM-3 Kinesin E ...... ! N A-tubule ...... c: I - -(l) 3. OSM-3 mediated B-tubule ~ .- E0'1 anterograde 1FT o (l) BBSome (f) -• 1FT Cargo Y-links I Transitional fibers

2. Kinesin-1I/0SM-3 (l)e - (l) mediated "0 E anterograde 1FT :Q0'l 5. Retrograde 1FT ~ (l) (f)

~ [ ~ 1. Assembly of [ 1FT particles

2 ·C 6. Dissociation of "0 c: 1FT particles (l) o

44 Figure 1-7. Experimental observations of Caenorhabditis e/egans intraflagellar transport (1FT) in mutants deficient in Kinesin-2, OSM-3, or 1FT-dynein mutants. Defects in 1FT and ciliary structures observed in kinesin or dynein motor mutants.

Schematics for wild-type, kinesin-2 (klp-11 or kap-1), osm-3, or IFT-dynein (che-3 or xbx-1) mutants use the same conventions depicted in Figure 1-6, and are shown alongside representative fluorescence images of GFP-tagged 1FT-A, IFT-

B or BBS components. In the schematics, the four distinct regions of amphid/phasmid cilia are indicated (BB, basal body/transitional fiber; TZ, transition zone; middle and distal segments) are indicated along with variations

(e.g., absent distal segment in osm-3 and 1FT protein accumulations in the IFT- dynein mutants). Fluorescent images: cil, cilia; *, accumulations.

45 IFT-A::GFP IFT-B::GFP BBS::GFP Wild-type S1'"'"-0.7 pmfs S"- -1.2 I1 mfs • ~'iIliiliiiiiiiii~• ---'-'--- ______a~~ CI~ c{ ,{ J8B'TL JBBrTZ I@ JGB"Z ~cell -- .. bally

kinesin-2 mutant (klp-11, kap-1) s#' -1.2 pmfs. B -1.2 ~lm/s .. ~ ~'iIliiliiiiiiiii~ ==---'______;~~ c{ ,,{ elf JBB!'"Z I@ JEllliTZ JBB'TZ ...... -I

~ en osm-3 mutant -0.5 pmfs • ~~ Clf ~CIi Clf I@ JRB,TZ BOrTZ [ JBB'TZ ...... -I :.L.:

IFT-dynein mutant (che-3. xbx-1)

B}~ ...... , .. .., ~ 'to ...-.:- • **." *J CII "* 1FT protein . ... * * accumulation • , ~------== , ...... -- -jIi

BBITZ MS os Figure 1-8. Experimental observations of Caenorhabditis e/egans intraflagellar transport (1FT) in mutants deficient in 1FT subcomplex A, 1FT subcomplex B, or BBSome components. Defects in 1FT and ciliary structures observed when core 1FT and BBS proteins are disrupted. The schematics and fluorescent images for the indicated wild-type and mutant strains follow the same convention as that shown in Figures 1-6 and

1-7. An 'X' shows that a specific module is disrupted.

47 IFT-A::GFP IFT-B::GFP BBS::GFP

\tVild-type 8 -0.7 ~m/s • 8r-12 ~m1s ~·lIIIlIIiIIiliil~• __=.. i11______._ ~~ CI~ c{ c{ JBBfTZ ] BBrTZ ~@ ]E'S.TZ --..... -- -

-1.2 ~lm~ eil ....

~ f*JBBfTZ ] HHill • some • TZs[ : 1FT protein ; ______.__ . , accumulation't •• 9~ ---.------' lit . JtlBi .... TZ ...... "\ ~ (Xl 1FT subcomplex A mutant (c/le-11. dar-tO) . * 8J ---. ., ., ... ~. J B6ITZ ... .. I1f1/T 1 [ , )eil . ... 1FT protein . * ~: accumulation ]BBfTZ ...... , ., - 1FT subcomplex 8 mutant (che-2, c/Je-13. osm-1, osm-5. osm-6) - B6fTl' BB!TZ[ JBBI BBlrZ [ TZ

BBITZ MS DS 1.7 Tables

Table 1-1. Description of individual ciliated neuron types and their reported functions. Ciliated neuron category lists the designations for each of the ciliated neuron types. l =left; R =Right; D =Dorsal; V =Ventral. Cilium structure describes the overall structure (as seen via electron microscopy) of each cilia type. Exposed cilia are protruding out of the worm to the external environment. Those cilia that are not environmentally exposed are embedded in the structures described.

References: Ward et aI., 1975 (ASE, ADF, ASG, ASH, ASI, ASJ, ASK, ADl,

AWA, AWB, AWC, AFD, Il1, Il2, CEP, OlQ, Oll, ADE, PDE); Ware et aI., 1975

(ASE, ADF, ASG, ASH, ASI, ASJ, ASK, ADl, AWA, AWB, AWC, AFD, Il1, Il2,

CEP, OlQ, Oll, ADE, PDE); Perkins et aI., 1986 (ASE, ADF, ASG, ASH, ASI,

ASJ, ASK, ADl, AWA, AWB, AWC, AFD, Il1, Il2, OlQ, Oll, BAG, FlP, ADE,

PDE); Kaplan & Horvitz, 1993 (ASH, OlQ, FlP); Hart et aI., 1995 (ll1); Sulston

& Brenner, 1975 (ADE, PDE); Hall & Russell, 1991 (PHA, PHB, PQR); Cheung et aI., 2005 (AQR, PQR); Sulston et aI., 1980 (all male neurons).

49 Ciliated Cilium Exposed Embedded? General Role Notes neuron structure ?

ASE (L/R) Single rod Yes Chemoattraction

ADF (l/R) Two rods Yes Dauer entry

ASG (L/R) Single rod Yes Chemoattraction

ASH (L/R) Single rod Yes Nose-touch; chem%smo repulsion

ASI (L/R) Single rod Yes Chemoattraction

ASJ (L/R) Single rod Yes Dauer recovery

ASK (L/R) Single rod Yes Chemoattraction

ADl (L/R) Two rods Yes Chemorepulsion

AWA (L/R) Winged No ~heath cell Chemoattraction

AWB (l/R) Winged No ~heath cell Chemorepulsion

AWC(L/R) Winged No ~heath cell Chemoattraction

AFD (L/R) Single rod No !sheath cell Thermosensation Surr. by villi

Il1 Single rod No !subcuticle Nose-touch Rootlets (DL/DR/L/RN)

Il2 Single rod Yes Putative (DL/DR/L/RN) chemosensory

CEP Single rod No Cuticle Basal slowing Assc'd wi other (DL/DRNlNR) response MTs

OlQ Single rod No Cuticle Nose-touch and Rootlets (DL/DRNlNR) basal slowing

all (L/R) Single rod No ~uticle Putative Rootlets mechanosensory

BAG (L/R) Bag No ~ubcuticle Oxygen sensation shaped

FlP (L/R) Flap No !subcuticle Nose touch shaped

ADE (L/R) No ~ubcuticle Basal slowing

PDE (L/R) No !subcuticle Basal slowing

PHA (L/R) Single rod Yes Chemorepulsion

PHB (L/R) Single rod Yes Chemorepulsion

AQR Single rod No Pseudo- Oxygen sensation

50 ~oelom

PQR Single rod No Pseudo- Oxygen sensation l,;oelom

Male-specific

CEM Yes Putative male (DL/DRNLNR) chemotaxis

RnA (L/R) (n=1 No Structural cell Mating behaviour 9)

RnB (L/R) (n=1 Yes Mating behaviour 9) (except R6B)

HOA No Subcuticle Sensing vulva

HOB Yes Sensing vulva

PCA (L/R) No Sensing vulva

SPD (L/R) Yes Sperm transfer

SPV (L/R) Yes Sperm transfer

Table 1-2. Components and available C. e/egans mutants of the conserved intraflagellar transport machinery. Each protein is grouped into one of the 1FT modules described in the text. For some components first characterized in C. elegans, namely DYF-2, IFTA-1 and

IFTA-2, association with the noted 1FT subcomplexes A (1FT-A) or B (1FT-B) has only been recently confirmed by Cole and Snell (2009). The association of DYF-

1, DYF-3, DYF-11 and DYF-13 with IFT-B has been shown biochemically by

Omori et al. 2008 and Follit et al. (2009). N.D., not detected in C. elegans; *, uncharacterized allele; #, likely not a null mutant, based on preliminary phenotyping and cDNA sequencing (unpublished observations).

51 1FT modules! Descriptions C. elegans C. e/egans Selected C. elegans components sequences proteins Alleles References Kinesin-II 95kD motor F20C5.2 KLP-11 tm324 Show et aI., 2004 85kD motor Y50D7A.6 KLP-20 ok2942* Accessory F08F8.3 KAP-1 ok676 Snow et aI., 2004 OSM-3 Motor M02B7.3 OSM-3 p802 Shakir et aI., 1993 Kinesin IFT-Dynein Heavv chain F18C12.1 CHE-3 e1124 SiQnor et aI., 1999 Light int. F02D8.3 XBX-1 ok279 Schafer et aI., 2003 chain Int. chain C17H12.1 DYCI-1 tm3700* Light chain. D1009.5 XBX-2 tm2097 Efimenko et aI., 2005 IFT-A IFT144 ZK520.3 DYF-2 m160 Efimenko et aI., 2006 IFT140 C27A7.4 CHE-11 e1810 Qin et aI., 2001 IFT139 ZK328.7 gk508¥, qk47-r IFT122 F23B2.4 DAF-10 e1387 Bell et aI., 2006 IFT121 C54G7.4 IFTA-1 nx61 BlacQue et aI., 2006 IFT43 N.D. IFT-B IFT172 T27B1.1 OSM-1 p808 Bell et aI., 2006 IFT88 Y41G9A.1 OSM-5 p813 Haycraft et aI., 2001 IFT81 F32A6.2 IFT-81 tm2355, Kobayashi et aI., tm2356 2007 IFT80 F38G1.1 CHE-2 e1033 Fujiwara et aI., 1999 IFT74!72 C18H9.8 IFT-74 tm2393, Kobayashi et aI., tm2397 2007 IFT70 F54C1.5 DYF-1 mn335 Ou et aI., 2005 IFT57!55 F59C6.7 CHE-13 e1805 Haycraft et aI., 2003 IFT54 C02H7.1 DYF-11 mn392 Kunitomo & Iino, 2008 IFT52 R31.3 OSM-6 p811 Collet et aI., 1998 IFT46 F46F6.4 DYF-6 m175, Bell et aI., 2006 mn346 IFT27 N.D. IFT25 N.D. 1FT22 T28F3.6 IFTA-2 tm1724 Schafer et al., 2006 IFT20 Y110A7A.20 gk548*, Ou et al., 2007 tm2935* Qilin C04C3.5 DYF-3 m185 Murayama et aI., 2005 putative C27H5.7 DYF-13 mn396 Blacque et aI., 2005 IFT-B BBSome BBS1 Y105E8A.5 BBS-1 ok1111 Blacque et aI., 2004 BBS2 F20D12.3 BBS-2 tm3231*, Blacque et aI., 2004 tm3044*, ok2053* BBS3 C38D4.8 ARL-6 ok3472* Fan et aI., 2004 BBS4 F58A4.14 BBS-4 tm303~

52 BBS5 R01H10.6 BBS-5 qk507* Ou et aI., 2007 BBS7 Y75B8A.12 OSM-12 n1606 Blacque et aI., 2004 BBS8 T25F10.5 BBS-8 nx77 Blacque et aI., 2004 BBS9 C48B6.8 BBS-9 gk471* Cargo TRPV B0212.5 OSM-9 n1516, Colbert et ai, 1997 channel ok1677, Qin et al., 2005 kv10 TRPV T09A12.3 OCR-2 ak47, Tobin et aI., 2002 channel ok1711

53 CHAPTER 2. CILIARY COMPARATIVE GENOMICS OF THE CHYTRID FUNGUS BATRACHOCHYTRIUM DENDROBATIDIS

Note regarding contributions: The sections of this chapter that address the new comparative genomics study are the result of a collaboration of with Dr. Christina Cuomo (BROAD Institute, Cambridge, MA, USA) as part of her broader genomic sequence analysis of the chytrid fungus Batrachochytrium dendrobatidis. With initial technical support from Dr. Cuomo, I analysed/interpreted all of the raw data into the form presented in this chapter.

54 2.1 Abstract

Batrachochytrium dendrobatidis is a representative organism of the chytrids, an evolutionary ancient class of fungi that are capable of generating ciliary structures in their zoospore stage. The recent sequencing of the B. dendrobatidis genome has presented cilia researchers with the opportunity to examine the organelle in a potentially ancestor-like state, or primordial form, of cilia present in extant metazoan cell types. Here, we present a comparative genomic analysis, in which we identify the B. dendrobatidis genes that have putative orthologues in at least one additional ciliated organism but are not present in non-ciliated organisms. This analysis resulted in the identification of approximately 300 genes, one third of which encode proteins with established cilium-associated functions. We supplement this study by examining the B. dendrobatidis genome for genes that encode known ciliary/basal body components, and discovered an apparent absence of core BBSome components, as well as the presence of genes associated with Meckel-Gruber syndrome and nephronopthisis. The genes identified in the comparative genomics study will assist in the identification of additional, evolutionarily conserved ciliary genes, and the manual BLAST analysis can offer insights into the relative essential or specialised nature of particular ciliary proteins with respect to cilium structure and function.

55 2.2 Introduction

Since 1980, conservation biologists have noted a dramatic decrease in the global amphibian population. Recent estimates suggest that nearly 10% of amphibian species are Critically Endangered and another 10% are deemed to be rapidly declining (Stuart et aI., 2004). Another 30% of total amphibian populations, considered to be data-deficient (lacking sufficient scientific analysis to assess), are also considered to be at risk (Stuart et aI., 2004). Remarkably, in the same time frame (1980 - present), a number of previously unknown diseases affecting amphibians have been connected to the rapid declines, the most notable of which being chytridiomycosis (Dazak et aI., 1999). Chytridiomycosis is a highly lethal epidermal infection affecting a wide range of amphibian species, and histological studies of affected individuals has resulted in the identification of a novel species of chytrid fungus, Batrachochytrium dendrobatidis, as the infective organism associated with the disease (Longcore et aI., 1999).

Chytrids (members of the fungal division Chytridiomycota), are saprobic organisms, feeding off chitin and keratin. They have two distinct stages in their life cycle: an immotile reproductive stage known as the zoosporangium, and the motile, unicellular, ciliated zoospore that travels in water to new hosts or additional sites on the original host (Garner et aI., 2006). In the case of B. dendrobatidis, zoospores chemotax to the keratinized skin of amphibians, where they encyst and form zoosporangia (Berger et aI., 2005).

It has been posited that ancestral fungi were aquatic organisms with flagellated aquatic spores, much like the Chytridiomycota phylum. In fact, the

56 sole extant fungal phylum that is known to possess these basal characteristics is the Chytridiomycota; other fungal phyla, namely the Ascomycota, Basidomycota,

Zygomycota, and the potential fungal relatives Microsporidia, secondarily lost their cilia during evolution (Liu et aI., 2006). The notion of chytrids being the most basal fungi has been supported by recent molecular phylogenetic analyses, strongly suggesting that they may closely resemble the precursor of all fungal and animal cell types (James et aI., 2006).

Structurally, the flagella of B. dendrobatidis zoospores closely resemble those of Chlamydomonas (Longcore et al., 1999). A microtubule triplet basal body (9 + 0 arrangement) nucleates a canonical 9 + 2 axoneme that extends for

-40 IJm (Longcore et aI., 1999). Structures that resemble the machinery required for flagellar motility, including inner/outer dynein arms and the central pair apparatus (discussed in Chapter 1; see Figure 1-2) can be observed, as well as some, but not all, of the more elaborate basal body-associated structures

(Longcore et aI., 1999).

The recent unpublished sequence of the B. dendrobatidis genome

(http://www.broad. mit.edu/annotation/genome/batrachochytrium_dendrobatidis) provides an excellent opportunity for researchers of cilia/flagella to examine, via comparative genomics, the core requirements of cilium structure/function. The comparative genomic study presented in this chapter represents a detailed and more robust survey of the genomes of ciliated organisms, including 15 representative ciliated organism genomes (anchored by B. dendrobatidis), and 5 non-ciliated organism genomes. In order to gain further insight into the ciliary

57 gene complement of B. dendrobatidis we also attempted, using thorough but non-automated bioinformatic analyses, to identify putative orthologues of cilia­ associated genes not likely to be identified in the comparative genomic study due to relatively high false negative rates associated with automated bioinformatics.

Briefly, our results confirm the presence of most genes associated with ciliary motility and intraflagellar transport (1FT). Surprisingly, our searches revealed numerous ciliopathy-associated genes, but not all, suggesting that some of these may have more essential (or less specialised) functions in ciliated organisms.

2.3 Results/Discussion

Using the criteria described in the Materials and Methods section of this chapter, our comparative genomic analysis identified 309 genes (See Appendix

A and Supplementary File 2-1 on attached CD) that are present in B. dendrobatidis but not other fungi, and thus have a probability to be cilium­ associated. By cross-referencing this dataset with existing cilia-related literature, we found that 95 of the genes in our dataset (-31 %) have confirmed ciliary roles

(Table 2-1), strongly implying that it is highly enriched for cilium-associated genes.

For the remaining 214 genes, we found an additional 5 genes that possess WD repeats or TPR domains that are commonly found in cilium associated proteins (Table 2-2), 3 genes associated with activation of small

GTPases (Table 2-3), and 6 genes that encode proteins that likely interact with tubulin (Table 2-4). The remaining 5 genes have human homologues that are associated with the following conditions: ataxia telangiectasia (also known as

58 Louis-Bar syndrome), Parkinson's Disease, Wiskott-Aldrich syndrome, deafness,

as well as an autoimmune disease (Table 2-5). While none of these diseases

have been connected to cilia so far, it should be noted that deafness and ataxia

are symptoms often found in ciliopathies (see Chapter 1). Furthermore, the

disease-associate gene BDEG_00015 encodes a protein homologous to a

transmembrane protein induced by tumor necrosis factor alpha (TNFa). TNFa

also is known to be associated with Traf3ip, a protein that is linked to the

microtubule cytoskeleton via interactions with MIP-T3 (Ling & Goeddel, 2000).

Chapter 2 in this thesis connects MIP-T3 to 1FT, perhaps hinting at a novel role

for MIP-T3, and perhaps 1FT, in mammalian tumorigenesis.

As explained in the introduction, a notable complication associated with

automated comparative genomic analysis is a high false negative rate, especially

in cases where genes encode proteins containing highly conserved domains. For

example, the NIMA-related kinases NEK1 and NEK8 are ciliated organism­

specific (Parker et aI., 2007), yet they would necessarily be omitted from this

comparative genomic analysis because both have strong homologues in yeast.

In order to compensate for this limitation, we manually performed BLAST

analysis on a set of 138 known ciliary genes (See Appendix A and

Supplementary File 2-2 on attached CD). Perhaps not surprisingly, the vast

majority of proteins known to participate in ciliary motility (inner/outer dynein

arms, radial spoke components, etc.) had clear homologues in the B.

dendrobatidis genome, as did all known components of the 1FT machinery. 86 of

59 the 138 control genes were not identified in the comparative genomic analysis, highlighting the need for complementary approaches.

Interestingly, however, when searching the B. dendrobatidis genome for putative orthologues of BBSome-associated genes (as described by Nachury et aI., 2007), we determined that only one, BBS3, was present. This observation is surprising, given that previous genomic analyses have pointed to BBS5 being the most conserved BBSome peptide, based on it being the sole BBS protein present in Toxoplasma gondii (Nachury et aI., 2007). BBS3 (also known as

ARL6) is not a component of the core BBSome (Nachury et aI., 2006); it is a small GTP-binding protein of the ARF superfamily, and thus may playa role in vesicular trafficking at or near the cilium.

The absence of any core BBSome protein-encoding genes in the B. dendrobatidis genome is somewhat surprising, and may hint at a less central role played by the BBSome in ciliogenesis. Notably, B. dendrobatidis does appear to possess homologues of genes associated with Meckel syndrome (including the

B9 domain-containing Meckel syndrome-related genes described in Chapter 4) and nephronophthisis. Extensive genetic analyses in C. elegans have determined that these genes encode proteins (MKS-1, MKSR-1, MKSR-2,

NPHP-1, NPHP-4) that specifically localise to basal bodies/transition zones, and participate in cilium-based signalling (Williams et aI., 2008; Bialas et aI., 2009).

60 2.4 Conclusion

Altogether, our bioinformatic analysis of the newly sequenced genome of

Batrachochytrium dendrobatidis has determined that the gene complement of this ancient fungus is capable of generating a cilium that closely resembles the canonical axonemal structures of Chlamydomonas. We performed comparative genomic analysis that resulted in a list of approximately 300 genes, highly enriched with known ciliary components. The list generated by this study is markedly shorter than previous comparative genomic attempts to produce a cilia/basal body proteome (Li et aI., 2004; Avidor-Reiss et aI., 2004). We gained further insight into the core requirements for cilium structure and function by manually scanning the genome database for known ciliary genes. We determined that B. dendrobatidis did not possess any of the core BBSome components, while encoding likely orthologues of other ciliopathy-associated genes, including those associated with Meckel-Gruber syndrome and nephronophthisis.

2.5 Materials and Methods

2.5.1 Comparative genomic survey and orthologue identification

Orthology with additional eukaryotes was established by the best BLAST hit criteria, requiring at least 1e-10 with 30% identity and 50% coverage. Orthologs were identified in the following species: E. cuniculi, A. locustea, E. histolytica,

Monsosiga brevicollis, C. elegans, D. melanogaster, M. musculus, H. sapiens, P. infestans, P. falciparum, T. theromolytica, G. lambia, D. discoideum, A. thaliana, and C. reinhardtii, as well as the nr protein database. To identify B.

61 dendrobatidis proteins with only ciliary orthologs, we required that the protein not have a hit to a fungal protein database, nor any of the non-ciliated genomes (A. thaliana, D. discoideum, E. cuniculi, and A. 10Gustea). A total of 528 Bd proteins only had a non-fungal, ciliary genome-specific orthologue by these criteria. This set was further filtered to remove transposon-related proteins, genes specific to

B. dendrobatidis, and genes found in only the B. dendrobatidis and P. infestans genomes, which all appear to be involved in a pathway unrelated to cilium structure/function (C. Cuomo, personal communication).

2.6 Tables

Table 2-1. Established ciliary genes found in the comparative genomics study. Human BdGene Homolgue Annotation References Dawe et aI., BDEG 00264 MKS1 Meckel syndrome, type 1 2007 Li et aI., 2004 BDEG 00280 TTC21B IFT139 (CHAPTER 5) BDEG 00443 RP2 Retinitis PiQmentosa 2 Shu et aI., 2007 Inversin (nephronopthisis type II Otto et aI., 2003 BDEG 00463 INVS associated) Flagellar inner arm dynein Kamiya, 2002 BDEG 00476 WDR78 intermediate chain Deane et aI., BDEG 00556 IFT52 IFT52 2001 Pazour et aI., BDEG 00568 n/a WD-40 repeat (FAP187) 2005 Pazour et aI., BDEG 00668 WDR65 WD repeat domain 65 (FAP57) 2005 Lucker et aI., BDEG 00751 IFT81 IFT81 2005 Pazour et aI., BDEG 00883 WDR63 WD repeat domain 63 2005 BDEG 00906 HSPB11 IFT25 Follitetal.,2009 Pazour et aI., BDEG 01018 WDR65 WD repeat domain 65 (FAP57) 2009

62 Pazour et aI., BDEG 01019 WDR65 WD repeat domain 65 (FAP57) 2009 zinc finger, MYND-type containing 12 Pazour et aI., BDEG 01138 ZMYND12 (FAP146) 2009 dynein, axonemal, intermediate Pennarun et aI., BDEG 01316 DNAI2 polypetdie 3 2000 Coiled-coil domain containing 113 Pazour et aI., BDEG 01463 CCDC113 (FAP263) 2005 Pazour et aI., BDEG 01561 WDR65 WD repeat domain 65 (FAP57) 2005 Bartoloni et aI., BDEG 01802 DNAH9 Dynein Heavv Chain 9, axonemal 2001 Retinal cGMP phosphodiesterase Linari et aI., BDEG 01818 PDE6D (RP3 interactor) 1999 Sperm Associated Antigen 6 (Central Smith & BDEG 01859 SPAG6 Pair-associated) Lefebvre, 1997 ADP-ribosylation factor related Blacque et aI., BDEG 01928 ARFRP1 protein 1 2005 Zhang et aI., BDEG 01954 DNAH7 Dynein Heavy Chain 7, axonemal 2002 Coiled-coil flagellar protein, regulates Tam & Lefebvre, BDEG 01977 CCDC146 flaQellar beatinQ 2002 DiBella et aI., BDEG 01988 TCTEX1D2 Inner arm dynein liQht chain 2004 deleted in a mouse model of primary Zariwala et aI., BDEG 02010 DPCD ciliary dyskinesia 2004 Chapelin et aI., BDEG 02050 DNAH12 Dynein Heavy Chain 12, axonemal 1997 Zhang et aI., BDEG 02051 DNAH7 Dynein Heavv Chain 7, axonemal 2002 Norrander et aI., BDEG 02136 RIBC2 flaQellar protofilament ribbon protein 2000 Hildebrandt et BDEG 02269 NPHP1 Nephronophthisis 1 aI., 1997 Dynein Intermediate Chain 1, Zariwala et aI., BDEG 02271 DNAI1 axonemal 2006 Doxsey et aI., BDEG 02304 PCN1 Pericentrin 1 1994 Pazour et aI., BDEG 02624 CCDC147 Coiled-coil protein (FAP189) 2005 Yang et aI., BDEG 02679 TXNDC3 Radial spoke protein 23 2006 coiled-coil domain containing 65 Pazour et aI., BDEG 02772 CCDC65 (FAP250) 2005 Pazour et aI., BDEG 02775 DNAH8 Dynein Heavy Chain 8, axonemal 2006 BDEG 02841 CLUAP1 Clusterin associated protein 1/qilin Sun et aI., 2004 Li et aI., 2008 BDEG 02985 TRAF31P1 MIP-T3/DYF-11 (CHAPTER 3)

63 Bialas et aI., 2009 BDEG 03036 B9D1 B9 protein (CHAPTER 4) BDEG 03041 GAS8 Growth-arrest-specific protein 8 Yeh et aI., 2002 BDEG 03067 IFT140 IFT140 Cole et aI., 1998 Blacque et aI., BDEG 03092 TTC26 DYF-13 2005 Bloodgood, BDEG 03178 CAMK2B calcium-dependent protein kinase 1992 similar to retinitis pigmentosa Arts et aI., 2007 GTPase regulator interacting protein BDEG 03262 RPGRIP1L 1 isoform 3 Williams et aI., BDEG 03461 RSPH3 Radial spoke head Protein 3 1989 IQ motif containing with AAA domain Pazour et aI., BDEG 03497 IQCA1 (FAP82) 2005 Zhang et aI., BDEG 03574 DNAH7 Dynein heavy chain 7, axonemal 2002 Dymek et aI., BDEG 03581 KATNB1 Katanin p80 subunit 2004 Bialas et aI., 2009 BDEG 03725 B9D2 B9 Protein (CHAPTER 4) Coiled-coil domain containing 19 Pazour et aI., BDEG 04006 CCDC19 (FAP45) 2005 BDEG 04194 IFT80 IFT80 Cole et aI., 2008 Kozminski et aI., BDEG 04421 KIFAP3 Kinesin-associated protein (KAP) 1995 Rompolas et aI., BDEG 04432 DNC2L11 dynein 2 light intermediate chain 2007 DC2-related axonemal dynein Kamiya, 2002 BDEG 04434 CCDC151 intermediate chain 4 BDEG 04571 DNAH10 Dynein heavy chain 10, axonemal Maiti et aI., 2000 Norrander et aI., BDEG 04579 EFHC1 EF-hand domain containing protein 1 2000 EF-hand domain-containing family Norrander et aI., BDEG 04651 EFHC2 member C2 2000 Dynein light intermediate chain 1, Kastury et aI., BDEG 04909 DNALl1 axonemal 1997 BDEG 05041 UNC119 UNC-119 HomoloQue Ou et aI., 2007 Pedersen et aI., BDEG 05050 IFT172 IFT172 2005 Blacque et aI., BDEG 05074 WDR35 WD repeat domain 35 (IFTA-1) 2006 Chapelin et aI., BDEG 05139 DNAH1 Dynein heavy chain 1, axonemal 1997 Coiled-coil domain containing protein Pazour et aI., BDEG 05388 CCDC147 147 (FAP189) 2005 BDEG 05430 RSPH10B Radial spoke head 10 homolog B Kamiya, 2002

64 Krock & Perkins, BDEG 05440 IFT57 IFT57 2008 BDEG 05530 TTC25 Outer arm dynein binding protein Kamiya, 2002 Keller et aI., BDEG 05632 TUBE1 Epsilon Tubulin 2006 BDEG 05701 IFT122 IFT122 Cole et aI., 1998 Efimenko et aI., BDEG 05864 WDR19 WD repeat domain 19/DYF-2 2006 BDEG 05930 TTC30B DYF-1 Ou et aI., 2005 Coiled-coil domain containing protein Pazour et aI., BDEG 06120 CCDC37 37 (FAP100) 2005 Coiled-coil domain containing protein Pazour et aI., BDEG 06140 CCDC39 39 (FAP59) 2005 Andersen et aI., BDEG 06278 CEP78 centrosomal protein 78kDa isoform b 2003 BDEG 06333 IFT20 IFT20 Follit et aI., 2006 Leucine rich repeat containing 48 Pazour et aI., BDEG 06423 LRRC48 (FAP134) 2005 Flagellar outer dynein arm docking Kamiya, 2002 BDEG 06718 CCDC63 complex protein ODA1 Pazour et aI., BDEG 06826 WDR16 WD repeat domain 16 (FAP52) 2005 Coiled-coil domain containing protein Pazour et aI., BDEG 06843 CCDC42 42A (FAP73) 2005 Tctex1 domain containing 1 (Outer Kamiya, 2002 BDEG 06904 TCTEX1 arm dynein Iiqht chain) Keller & BDEG 07129 WDR67 WD repeat domain 67 (POC18) Marshall,2008 Andersen et aI., BDEG 07240 CEP76 Centrosomal protein of 76 kDa 2003 Chapelin et aI., BDEG 07384 DNAH2 Dynein heavy chain 2, axonemal 1997 Coiled-coil domain containing protein Pazour et aI., BDEG 07416 CCDC96 94 (FAP94) 2005 Coiled-coil domain containing protein Marshall et aI., BDEG 07530 CCDC61 61 2001 BDEG 07544 DNAH6 Dynein heavy chain 6, axonemal Maiti et aI., 2000 Pazour et aI., BDEG 07580 IFT88 IFT88 2000 BDEG 07589 RSPH4A Radial spokehead-like protein 3 Inaba, 2003 Lechtreck & BDEG 07678 HYDIN Hydin Witman, 2007 Wirschell et aI., BDEG 08122 AK1 Flagellar adenylate kinase 2004 Ikeda et aI., BDEG 08345 PACRG parkin coregulated gene protein 2007 BDEG 08408 IFT46 IFT46 Cole et aI., 1998 BDEG 08433 TUBE1 Epsilon Tubulin Keller et aI.,

65 2006 Bowman et aI., BDEG 08489 DYNLRB2 Roadblock-like Dynein LiQht Chain 1999 Pazour et aI., BDEG 08543 DYNHC2 Cytoplasmic dynein heavy chain 1b 1999

Table 2-2. Genes identified in the comparative genomic study that encode proteins with TPR domains and WD repeats. Human BO Gene Homologue Annotation Notes Strong xbox in C. BDEG 00321 WDR60 WD repeat domain 60 elegans BDEG 02355 WDR34 WD repeat domain 34 tetratricopeptide repeat BDEG 03324 TTC14 domain 14 Tetratricopeptide repeat BDEG 03745 TTC25 protein 25 similar to WD repeat domain BDEG 07355 49

Table 2-3. Genes identified in the comparative genomic study that encode small GTP-binding proteins. Human BO Gene Homologue Annotation Notes BDEG 00172 ARHGAP27 Rho GTPase activating protein 27 BDEG 03046 RAB6B Ras related protein 6B Stromal membrane-associated protein 1 BDEG 06027 SMAP1 (ARF-GAP)

Table 2-4. Genes identified in the comparative genomic study that encode proteins which likely interact with tubulin. Human BO Gene Homologue Annotation Notes Tubulin polyglutamylase complex BDEG 01294 LRRC49 subunit BDEG 01903 TTLL4 Tubulin tyrosine ligase-like Tubulin polymerization-promoting BDEG 06075 TPPP3 protein family Tubulin polyglutamylase complex BDEG 06704 C18orf10 subunit 2 Tubulin polyglutamylase complex BDEG 06738 C18orf10 subunit 2

66 BDEG 07664 TPX2

Table 2-5. Genes identified in the comparative genomic study that have putative human orthologues implicated in disease, but have yet to be associated with ciliary dysfunction. Human BO Gene Homologue Annotation/disease association Transmembrane protein induced by tumor necrosis BDEG 00015 TMEM120B factor alpha (Coeliac disease) BDEG 00210 ATM Ataxia telangiectasia mutated BDEG 02137 PDDC1 Parkinson disease 7 domain containinq 1 Deafness locus associated putative guanine BDEG 04271 SERGEF nucleotide exchanqe factor

67 CHAPTER 3. AN ESSENTIAL ROLE FOR DYF-11/MIP-T3 IN ASSEMBLING FUNCTIONAL INTRAFLAGELLAR TRANSPORT COMPLEXES

Note regarding contributions: The following chapter is a slightly modified version of a manuscript published in PLoS Genetics. The authors of the study are listed below. Li C*, Inglis PN*, Leitch CC, Efimenko E, Zaghoul NA, Mok CA, Davis EE, Bialas NJ, Healey MP, Heon E, Zhen M, Swoboda P, Katsanis N, and Leroux MR. (2007). PLoS Genet. 4: e1000044. (* - equal contributions) As co-first author of this manuscript, I was responsible for much of the C. elegans work presented. Specifically, I performed the chemosensory (Che) and dye-filling (Dyf) assays, as well as all C. elegans microscopy (with one exception noted below). In addition, I co-wrote the manuscript with M. Leroux and N. Katsanis. C. Li performed the genetic crosses and GFP transgene construction, as well as the ciliary length measurements. N. Bialas and M. Healey performed the lifespan and lipid accumulation assays, respectively. C. Mok performed FRAP/time-Iapse microscopy on the bbs-B (DYF-11 ::GFP) strain, of which I performed analysis. E. Efimenko and P. Swoboda sequenced the dyf-11 allele and identified DYF-11 ::GFP localisation in the AWC neuron. C. Leitch, N. Zaghoul, E. Davis and N. Katsanis contributed a complementary zebrafish study to this paper that I have omitted from this chapter.

68 3.1 Abstract

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia

1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. e/egans MIP-T3 ortholog DYF-11 is an intraflagellar transport (1FT) protein that plays a critical role in assembling functional kinesin motor-1FT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the 1FT machinery, including Kinesin-II, as well as 1FT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that

DYF-11 functions as a novel component of 1FT subcomplex B. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis.

3.2 Introduction

Cilia are slender subcellular structures that protrude from the surfaces of most eukaryotic cell types, where they carry out functions associated with sensation and/or motility. Motile cilia are used for the locomotion of spermatozoa or organisms such as the unicellular green alga Chlamydomonas reinhardtii, as well as for generating fluid flow, as is the case in respiratory airways (Pazour &

Rosenbaum, 2002). Non-motile (primary) cilia are nearly ubiquitous in multicellular organisms, and perform a wide range of sensory functions, including

69 chemosensation/olfaction, photoreception, and mechanosensation (Pazour &

Rosenbaum, 2002; Davenport & Yoder, 2005; Takeuchi & Kurahashi, 2005; Satir

& Christensen, 2007; Yoder, 2007). Primary cilia are also associated with several signaling processes critical for development, including Hedgehog signaling,

PDGFRaa signaling, as well as canonical and non-canonical (planar cell polarity)

Wnt signaling pathways (Christensen et aI., 2007; Davis et aI., 2006; Schneider et aI., 2005; Singla & Reiter, 2006; Germino, 2005; Haycraft et aI., 2005; Gerdes et aI., 2007). Hence, defects in ciliary structure or function affect nearly every organ in humans, and are associated with several pleiotropic genetic disorders.

For example, Bardet-Biedl syndrome (BBS), Alstr6m syndrome, Meckel syndrome, Senior-L0ken syndrome, Joubert syndrome, and several cystic kidney diseases are all believed to involve dysfunction of primary cilia and/or basal bodies, the modified centriolar structures that nucleate ciliary axonemes (Ansley et aI., 2003; Pazour, 2004; Badano et aI., 2006a; Blacque & Leroux, 2006;

Hildebrandt & Otto, 2005; Dawe et aI., 2007; Li et aI., 2007; Tobin & Beales,

2007).

Cilia are organelles that require several hundred proteins to support their motility and/or sensory and signaling functions (Inglis et aI., 2006; Gherman et aI., 2006). Of particular relevance to the present study, cilia possess a specialized microtubule-based transport system, termedintraflagellar transport

(1FT), which shuttles 1FT complexes bi-directionally along the axoneme and supports the formation and maintenance of the organelles (Kozminski et aI.,

1995; Rosenbaum & Witman, 2002; Scholey, 2003). The 1FT particles, first

70 observed in Chlamydomonas (Kozminski et aI., 1993) consist of anterograde

Kinesin-2 motor(s) that move cargo into the cilia and a retrograde dynein motor

involved in recycling components back to the base (basal body). The molecular

motors are associated with two biochemically-separable multisubunit assemblies

termed 1FT particle subcomplexes A and B (Piperno & Mead, 1997; Cole, 2003;

Cole et aI., 1998). In Chlamydomonas, 1FT subcomplexes A and B consist of at

least 6 and 11 subunits, respectively.

In recent years, the nematode Caenorhabditis elegans has emerged as a

powerful model organism for the study of cilia and ciliogenesis. The cilia of C.

elegans are non-motile and restricted to a subset of sensory neuronal cells

principally localized in the head and tail of the animal (Perkins et aI., 1986). While

structurally similar to the canonical flagella of Chlamydomonas, C. elegans cilia

emanate from a potentially more degenerate basal body (termed transition zone)

and exhibit, following the doublet microtubule-containing ciliary middle segment,

a pronounced extension of singlet axonemal microtubules in their distal

segments (Perkins et aI., 1986). Despite these differences, most, if not all, of the

core 1FT components identified in Chlamydomonas appear to be conserved in

nematodes (Inglis et aI., 2007). Additionally, several other proteins associated

with and necessary for the function of the 1FT machinery and cilium formation

have been discovered in C. elegans, namely DYF-1 (Ou et aI., 2005a), DYF-2

(Efimenko et aI., 2006), DYF-3 (Murayama et aI., 2005), DYF-13 (Blacque et aI.,

2005), and IFTA-1 (Blacque et aI., 2006). Like known 1FT particle subcomplex

AlB components, orthologs of these C. elegans proteins are enriched within the

71 membrane-plus-matrix fraction of the recently identified Chlamydomonas flagellar proteome (Pazour et aI., 2005), supporting the notion that they represent conserved 1FT components. In addition, research in the worm has shown that

Bardet-Biedl Syndrome (BBS) proteins are themselves associated with 1FT and are required to maintain the integrity of the 1FT particle during transport along the cilium (Satir & Christensen, 2007; Ou et aI., 2005b). The discovery of novel C. elegans 1FT proteins suggests that the 1FT machinery is more complex than suspected originally from biochemical fractionation studies, raising the possibility that additional components critical for 1FT have yet to be identified.

On the basis of several bioinformatic, genomic and proteomic studies, we recently surmised (Inglis et aI., 2006) that the microtubule-associated MIP­

T3/TRAF3IP1 protein likely represents a previously unknown but conserved ciliary protein. Here, we show that the C. elegans dyf-11 mutant harbors a loss of function mutation in the gene encoding the MIP-T3 ortholog. We demonstrate that the dyf-11 gene product, DYF-11, is a novel 1FT-associated protein required for the proper assembly and function of the 1FT machinery, as well as the sensory abilities of cilia. Consistent with these findings, mammalian MIP-T3 localizes to the basal body in a pre-ciliated cell and to the ciliary axoneme in ciliated cells. Our findings therefore demonstrate new roles for the evolutionarily conserved DYF-11/MIP-T3 protein in building and maintaining functional cilia.

72 3.3 Results/Discussion

3.3.1 The C. e/egans MIP-T3 gene ortholog C02H7.1 is disrupted in dyf-11 mutants

Several lines of evidence support the notion that MIP-T3 orthologs have a ciliary function (Inglis et aI., 2006). First, MIP-T3 is found exclusively in ciliated organisms (Avidor-Reiss et aI., 2004; Li et aI., 2004). Second, its expression in C. elegans and Drosophila is restricted to ciliated cells and under the control of an X box motif, which regulates genes required for ciliogenesis (Blacque et aI., 2005;

Avidor-Reiss et aI., 2004; Efimenko et aI., 2005; Chen et aI., 2006). Similarly,

Chlamydomonas MIP-T3 (C_140070) is upregulated during flagellar regeneration, and proteomic analyses uncovered MIP-T3 in the Chlamydomonas flagellar proteome (Pazour et aI., 2005; Stole et aI., 2005). MIP-T3 proteins range in size from 484 to 625 amino acids and have no recognizable domains except for a predicted coiled-coil region near the C-terminus (Figure 3-1A), providing no indication of their specific cellular function(s).

To test the hypothesis that MIP-T3 encodes a ciliary protein and to analyze its in vivo function, we sought to obtain a strain with a disruption in the C. elegans C02H7.1 open reading frame that encodes MIP-T3 (Figure 3-2). We noticed that a previously identified mutant, dyf-11(mn392), whose compromised fluorescent dye uptake is suggestive of cilia dysfunction (Starich et aI., 1995), maps within a genetic interval that contains C02H7.1. Sequencing of C02H7.1 in the dyf-11 (mn392) mutant strain revealed a nonsense mutation predicted to give rise to a null allele (Figure 3-1 B). Importantly, the dye-filling defect of dyf-

11 (mn392) is fully rescued by introducing a transgene expressing the wild-type

73 copy of the C02H7.1 coding region fused to GFP (Figure 3-1 C). This confirms the cloning of dyf-11 and demonstrates the functional nature of the GFP-tagged

C02H7.1 protein, which we use below for in vivo characterization. We henceforth refer to the C. elegans MIP-T3 gene ortholog C02H7.1 as dyf-11, and its gene product as DYF-11.

3.3.2 DYF-11 is required for the formation of structurally intact and functional cilia

To determine if the dye-filling anomaly observed in the dyf-11 strain stems from structural defects in cilia that are normally exposed to the environment, we expressed GFP in the ASER amphid (head) or two PHA/B phasmid (tail) neurons using reporter constructs driven by the gcy-5 or srb-6 gene promoters, respectively; in those neurons, GFP diffuses freely to highlight the entire cell, including the cell body, axon, dendrite, transition zone/basal body and cilium, so as to permit cilium length measurements (Swoboda et aI., 2000). Although no morphological defects with the dendritic processes or transition zone positioning were observed, the cilia of dyf-11 mutants were truncated substantially (2.4±0.3

IJm and 2.6±0.4 IJm for amphids and phasmids, respectively) compared to those of wild-type animals (both 5.7±0.4 IJm) (Figure 3-3A). These findings likely explain the dye-filling defect in dyf-11 animals and directly implicate MIP-T3 in ciliogenesis. Additionally, the observed lengths of dyf-11 mutant cilia are similar to those of 1FT subcomplex B mutants.

We next used established assays (Perkins et aI., 1986; Bargmann, 2006;

Hart, 2006; Vowels & Thomas, 1992) to ask whether dyf-11 animals exhibit

74 phenotypes consistent with cilia dysfunction, including anomalies in chemotaxis, avoidance of high osmolarity, ability to form stress-resistant dauer larvae, and lifespan.

We first compared the ability of wild-type and dyf-11 mutant animals to detect a volatile odorant, isoamyl-alcohol. Although the dyf-11 mutants are not impaired in their movement (data not shown), they show a pronounced inability to chemotax towards the attractant, indicative of an abnormal Chemotaxis (Che) phenotype (Figure 3-38). In addition, compared to their wild-type counterparts, dyf-11 animals show a clear defect in avoiding moving into a solution of high osmolarity (8 M glycerol; Figure 3-3C), an Osmotic avoidance-abnormal (Osm) phenotype frequently encountered in ciliary mutants (Perkins et aI., 1986;

Bargmann, 2006). These dyf-11 mutant phenotypes are consistent with the original observations of Starich ef al. (1995).

We then tested for the ability of dyf-11 mutants to enter the alternative dauer lifestage at two different temperatures (20/25°C). While wild-type larvae become dauers at these temperatures upon starvation, mutants with defects in

1FT (e.g., osm-5) or the cilium-dependent insulin signaling pathway (e.g., daf-16 and daf-2) are either dauer formation-defective (Oaf-d) or constitutively form dauer larvae (Oaf-c) (Vowels & Thomas, 1992) (Figure 3-30). We found that dyf­

11 mutants are Oaf-d at both temperatures, consistent with abrogated ciliary function (Perkins et aI., 1986; Starich et aI., 1995; Vowels & Thomas, 1992)

(Figure 3-30). Lastly, we noted severe male mating defects (data not shown), which can probably be ascribed to the improper mechanosensory/chemosensory

75 functions of cilia in the male tail (Liu & Sternberg, 1999). For all four behavioral phenotypes observed, the defects could be rescued by expression of the GFP­ tagged wild-type OYF-11 protein (Figures 3-38-0 and data not shown for male mating). Finally, although some ciliary mutants are long-lived (Apfeld & Kenyon,

1999), for example the che-11 mutant, we found no statistical difference in lifespan between wild-type and dyf-11 animals (Figure 3-3E).

Intriguingly, dyf-11 mutants also show, compared to the wild-type and rescue strains, a pronounced increase in Nile Red staining within intestinal cells, indicative of an increased lipid accumulation phenotype (Figure 3-3F). This result is remarkable, as past C. elegans screens for lipid accumulation identified only two ciliary proteins, namely the tubby obesity-associated gene ortholog TUB-1

(Ashrafi et aI., 2003) and BBS-1, which is linked to the obesity disorder Bardet­

Biedl syndrome (Mak et aI., 2006). These observations, coupled with our present findings with dyf-11, suggest an evolutionarily-conserved connection between cilia and lipid homeostasis. It should be noted that since the genome-wide lipid accumulation screens were RNAi-based, it is likely that many more ciliary proteins are involved in lipid homeostasis, as RNAi has been found to be considerably less penetrant in ciliated neurons.

Altogether, our cilium length measurements and sensory behavioral analyses (Che, Osm and Oaf) demonstrate that the dyf-11 mutant strain possesses prominent structural and functional ciliary defects, confirming our hypothesis that MIP-T3 plays a role in ciliogenesis and cilia function. The role(s) of OYF-11/MIP-T3 in cilia formation and function appear to be highly specific, as

76 we have not noted any gross morphological, developmental or locomotory defects in dyf-11 mutant animals.

3.3.3 DYF-11/MIP-T3 is a novel intraflagellar transport (1FT) protein

To directly observe whether the C. e/egans DYF-11 protein associates with ciliary structures (transition zones/basal bodies and/or cilia), we generated transgenic lines bearing a translational fusion construct of the complete dyf-11 gene (with its endogenous promoter) and GFP. Fluorescence microscopy observation of the lines revealed that dyf-11 ::gfp is expressed specifically in ciliated (e.g., amphid and phasmid) sensory neurons (data not shown),

consistent with expression patterns obtained using transcriptional GFP-fusion constructs obtained in two large-scale studies (Blacque et aI., 2005; Kunitomo et aI., 2005). Importantly, the DYF-11 ::GFP protein was found to be highly enriched

at transition zones and within ciliary axonemes (Figure 3-4A, Figure 3-5). This

subcellular localization is indistinguishable from that of other C. e/egans proteins

associated with 1FT, including BBS proteins, newly-discovered 1FT proteins, and

1FT particle subcomplex A and B proteins (Ou et aI., 2005; Efimenko et aI., 2006;

Murayama et aI., 2005; Blacque et aI., 2005; Blacque et aI., 2006; Blacque et aI.,

2004). Consistent with an evolutionarily-conserved role for MIP-T3 at basal

bodies and cilia, V5 epitope-tagged human MIP-T3 also localizes to the basal

body in cells that have not yet ciliated, and to the ciliary axonemes of ciliated

IMCD3 kidney cells (Figure 3-48).

Time-lapse microscopy in C. elegans revealed that DYF-11 ::GFP

localization is not static; the GFP-tagged protein moves bi-directionally along the

77 length of amphid and phasmid ciliary axonemes (Figure 3-4A). Kymograph analyses show that DYF-11 ::GFP anterograde movement is bi-phasic, exhibiting a velocity of 0.74±0.08 IJm/sec along doublet microtubules in middle segments and 1.15±0.21 IJm/sec along singlet microtubules in distal segments (Figure 3­

4A). These observations show that DYF-11 is transported cooperatively by

Kinesin-II and OSM-3-kinesin in middle segments and then OSM-3-kinesin alone in distal segments, exactly as with other C. elegans IFT/BBS proteins (Ou et aI.,

2005; Snow et aI., 2004). Importantly, DYF-11 ::GFP motility depends on the 1FT machinery itself, as abrogating the 1FT particle subcomplex A protein CHE­

11/IFT140, which is required for retrograde transport, results in the expected accumulation of DYF-11 ::GFP at distal tips (Figure 3-4C).

Interestingly, DYF-11 ::GFP can be observed in several additional dendritic extensions that are not typically seen with established GFP-tagged 1FT protein markers (see hollow arrowheads in Figure 3-4A and Figure 3-5). At least some of these unexpected structures appear to overlap with the cilia of the wing neuron

AWC in the DYF-11 ::GFP-containing strain (Figure 3-6), which might indicate that the other unaccounted-for structures represent the axonemes of the remaining wing neurons (AWA, AWB). This unexpected observation may reflect stronger accumulation of DYF-11 ::GFP in the wing neuron cilia, or could be indicative of additional roles for DYF-11 in the development of morphologically distinct cilia (wing cilia have elaborate structures) (Perkins et aI., 1986). Another possible explanation for the observed dendritic extensions is that they represent elongated cilia not sufficiently aligned to enter the amphid channel, a phenotype

78 reminiscent of those recently seen in dyf-5 mutants, which have defects in IFT- kinesin-mediated transport (Burghoorn et aI., 2007).

3.3.4 DYF-11 is transported in the cilium in a manner similar to 1FT particle subcomplex B

The molecular architecture of the motor-1FT machinery has been studied in some detail, using mainly Chlamydomonas and C. elegans as model systems

(Scholey, 2003; Cole, 2003; Snow et aI., 2004; Haycraft et aL, 2003; Lucker et aI., 2005; Ou et aI., 2007; Pedersen et aI., 2006; Pan et aI., 2006). In C. elegans, the motor-1FT machinery consists of at least 32 components organized into three main modules (Ou et aI., 2007): a motor module with two kinesin-2-like anterograde motors for anterograde transport (the more canonical heterotrimeric

Kinesin-II, and homodimeric OSM-3) and a dynein motor for retrograde transport; another module containing two 1FT particle multisubunit subcomplexes (A and B) that are separable genetically and biochemically (Piperno & Mead, 1997; Cole,

2003; Cole et aI., 1998;Ou et aI., 2005b; Snow et aL, 2004; Ou et aI., 2007); finally, a BBS protein complex/module that mediates the association between

Kinesin-II/subcomplex A and OSM-3/subcomplex B (Satir & Christensen, 2007;

Ou et aI., 2005; Nachury et aI., 2007). Having identified DYF-11 as a novel 1FT protein, we sought to characterize its spatial relationship (i.e., localization in one of the aforementioned modules) and function with respect to other components of the 1FT machinery.

To test whether DYF-11 may be a component of either the Kinesin-II or

OSM-3 anterograde motor modules, similar to the association of DYF-1 with

79 OSM-3 (Ou et aI., 2005), we queried whether GFP-tagged DYF-11 enters the ciliary axonemes of mutants lacking either motor (compared with Figure 3-SA, which shows DYF-11 ::GFP in wild-type animals). We found that in the klp-11 mutant, which lacks Kinesin-II motor function, DYF-11 ::GFP readily entered the full-length cilia produced by the redundant OSM-3-kinesin (Figure 3-S8).

Likewise, DYF-11 ::GFP could be detected along the entire length of the ciliary middle segments in the osm-3 kinesin mutant, which specifically lacks distal segments (Figure 3-SC). These observations suggest that the assembly of DYF­

11 with the motor-1FT particle machinery is not dependent on either of the kinesin anterograde motors, and leaves the possibility that it is more closely associated with the BBS protein complex or the 1FT subcomplexes A or B.

We therefore analyzed the behavior of DYF-11 ::GFP in the three available bbs mutants (bbs-1, bbs-7/osm-12 and bbs-B). In all bbs mutants, DYF-11 ::GFP was distributed throughout the middle segment and the residual distal segment

(Figures 3-SD-F). This suggests that DYF-11 is not tightly associated with BBS

protein(s), since all examined BBS proteins are unable to enter cilia in animals

lacking bbs-1, bbs-7/osm-12 or bbs-B (Satir & Christensen, 2007; Ou et aI., 2005;

Ou et aI., 2007). This is also consistent with the fact that the dyf-11 mutant cilia

are distinctly shorter (Figure 3-3A) than those of bbs mutant cilia, which possess

part of the distal segment (Ou et aI., 2005; Mak et aI., 2006; Ou et aI., 2007).

Instead, the ability of DYF-11 ::GFP to enter the residual distal segment of bbs

mutants implies that it is associated with the OSM-3-kinesin/lFT subcomplex B;

this conclusion stems from the fact that in bbs mutants, all tested 1FT

80 subcomplex B components enter the distal segment in an OSM-3-dependent manner, whereas subcomplex A components are transported by Kinesin-II, and do not enter the distal segment (Ou et aI., 2005) (see also schematics in Figure

3-5). Furthermore, DYF-11 :GFP travels at a velocity of 1.21 ±0.19 IJm/sec throughout the middle and distal segments of the cilia of osm-12/bbs-7 mutant worms (Figure 3-51), indicative of an OSM-3-dependent transport process typically seen for 1FT subcomplex B components in a bbs mutant background

(Satir & Christensen, 2007; Ou et aI., 2005; Ou et aI., 2007).

When combined with our finding that dyf-11 mutant cilia are truncated to the same extent as those mutants with abrogated 1FT subcomplex B components

such as OSM-5/IFT88, OSM-6/1FT52, and CHE-13/1FT55-57 (Figure 3-3A)

(Burghoorn et aI., 2007; Haycraft et aI., 2003), the above results further support

the notion that DYF-11 associates either directly or peripherally with the 1FT

particle subcomplex B. Previous work has shown that OSM-6 likely anchors 1FT

subcomplex B at the base of the cilium, possibly at transitional fibers in proximity

to the ciliary membrane (Haycraft et aI., 2003; Deane et aI., 2001). For example,

OSM-6 can enter the cilium in che-13 and osm-5 mutants, but CHE-13 and OSM­

5 are excluded from cilia in an osm-6 mutant background (Haycraft et aI., 2003).

Therefore, to further resolve the positioning of DYF-11 within the hierarchy of 1FT

subcomplex B, we examined the localization of DYF-11 ::GFP in che-13 and osm­

6 mutant backgrounds. In both cases, DYF-11 ::GFP enters the truncated cilia,

with little or no observable accumulation in the dendrites (Figures 3-5G,H).

OSM-5::GFP, on the other hand, fails to enter the severely truncated cilia of dyf-

81 11 mutants, showing significant leakage into dendrites (Figure 3-7H). These data suggest that DYF-11 may be more 'centrally' localized (e.g., closer to the OSM-3­ kinesin) than OSM-5, OSM-6 and CHE-13 in the 1FT particle subcomplex B, although additional studies are needed to confirm this potential protein topology.

3.3.5 DYF-11 is required for the integrity of the motor-1FT machinery

To provide additional insight into the function of DYF-11 with regards to the motor-1FT machinery and cilia formation, we analyzed by microscopy the behavior of several GFP-tagged 1FT-associated components in the dyf-11 mutant strain. In addition to the 1FT subcomplex B protein OSM-5 (see above), we tested a Kinesin-II component (the Kinesin-Associated Protein 1, KAP-1), homodimeric kinesin OSM-3, a component of IFT-dynein (the light-intermediate chain XBX-1), an 1FT particle subcomplex A protein (CHE-11/IFT140), and a BBS protein (BBS­

7). In wild-type animals, these proteins always localize prominently at transition zones/basal bodies and clearly undergo bidirectional transport along ciliary axonemes (Figures 3-7A-F) (Inglis et aI., 2007). Remarkably, aside from OSM­

3-kinesin, we reproducibly observed abnormal localization for all of these proteins in the dyf-11 mutant strain. OSM-3::GFP consistently localizes to transition zones and the truncated amphid and phasmid cilia of dyf-11 mutants

(Figure 3-7J). In contrast, CHE-11, XBX-1, and BBS-7 were anchored to transition zones but their signal intensities were significantly reduced compared to wild-type, and none of the proteins were observed to enter the (truncated) amphid and phasmid cilia (Figures 3-7G, I, L). Interestingly, the heterotrimeric kinesin KAP-1 subunit mislocalized consistently along the dendrite, with no

82 apparent anchoring to the transition zones and no observable localization to the ciliary axoneme (Figure 3-7K). All observations are reproducible and are based on the analysis of at least 50-100 animals, with the observer blind to the genotype and GFP-tagged protein under examination. It should be noted that the observed mislocalizations in the dyf-11 mutant animals appear to be more severe than those typically seen in 1FT subcomplex B mutants, perhaps hinting at additional roles for the DYF-11 protein in 1FT. Overall, our findings are consistent with DYF-11 playing a critical role in the assembly and thus functions of various

1FT-associated proteins, including the Kinesin-II motor, into a functional motor­

1FT machinery. Whether DYF-11 operates as an integral 1FT component, or potentially as an associated 'cargo' protein that affects the core machinery, remains to be determined.

3.4 Concluding Remarks

Human MIP-T3 (Microtubule-interacting protein associated with TRAF3), also termed TRAF31P1 (TNF Receptor-Associated Factor 3 Interacting Protein

1), is a poorly-characterized protein previously implicated in TRAF3 function and shown to bind microtubules (Ling & Goeddel, 2000). More recently, Morris et al

(2003) found that MIP-T3 interacts with the DISC1 (Disrupted-in-schizophrenia 1) protein and is required for its localization to microtubules/centrosomes. Yet, given that MIP-T3 protein orthologs are present in organisms lacking both TRAF3 and

DISC1, including B. dendrobatidis, Chlamydomonas and C. elegans (Figure 3­

1A), we hypothesized that MIP-T3 likely also performs a more general, evolutionarily conserved function-one related to cilia formation (Inglis et aI.,

83 2006). Indeed, we now show that C. e/egans MIP-T3 (DYF-11) localizes to transition zones/basal bodies and cilia, as does its mammalian counterpart

(Figures 3-4A, B). Importantly, C. e/egans DYF-11 is an intraflagellar transport protein critical for the formation of full-length, functional sensory cilia (Figures 3­

1 - 3-4). Abrogating DYF-11 function produces ciliary defects consistent with a role for the protein in the 1FT process itself: (i) cilia are approximately the same length as those of mutants lacking 1FT subcomplex B proteins (Haycraft et aI.,

2003; Ou et aI., 2007; Bell et aI., 2006); (ii) four representative classes of IFT­ associated components-OSM-5 (1FT subcomplex B), CHE-11 (subcomplex A),

XBX-1 (dynein motor subunit) and BBS-7 (part of the BBS protein complex)-fail to enter the short, residual cilium in dyf-11 mutant animals (Figures 3-7G, H, I and L); (iii) remarkably, the heterotrimeric Kinesin-II motor subunit KAP-1 also mislocalizes (Figure 3-7K). These observations suggest that DYF-11 is not only involved in maintaining the integrity of the 1FT machinery, but also may help with the assembly of Kinesin-II onto the 1FT complex. Moreover, it is possible that a proportion of the C. e/egans DYF-11/MIP-T3 protein associates directly with microtubules to influence the function of the 1FT motors or components.

Another potentially pertinent discovery by Taya et a/ (2007) is that the

MIP-T3-interacting protein DISC1 regulates the Kinesin-1-dependent transport of a protein complex composed of NUDEL/L1S1/14-3-3£. Consistent with a probable role in transport and neurogenesis, overexpression of a dominant-negative variant of DISC1 results in defective (shortened) neurite outgrowths (Ozeki et aI.,

2003). Hence, one of the apparent functions of DISC1 as a cohesion factor for a

84 multisubunit protein complex (NUDELlLlS1/14-3-3£) has interesting parallels to that of its interacting partner MIP-T3, which we show may function as a subunit of the 1FT subcomplex B that possibly links, either directly or indirectly, a ciliary kinesin to the multiprotein 1FT subcomplexes AlB (Figure 3-5). Whether C. elegans DYF-11 has dendrite-associated functions whose disruption could affect ciliogenesis represents an interesting question that will need to be addressed.

Several genome- and proteome-wide studies are in accord with our finding that MIP-T3 plays a critical role in 1FT. The Chlamydomonas flagellar proteome

uncovered by Pazour et al (2005) identified the MIP-T3 ortholog (FAP116) as an

abundant protein present specifically in the membrane-plus-matrix but not the

axonemal fraction of cilia, precisely like other 1FT proteins. Chlamydomonas MIP­

T3 is upregulated >3.0 fold during reflagellation (Li et aI., 2004; Stole et aI.,

2005), again similar to other 1FT proteins and consistent with a ciliogenic role.

Finally, just as in C. elegans, the Drosophila MIP-T3 ortholog (CG3259) is

expressed exclusively in ciliated sensory neurons (Avidor-Reiss et aI., 2004). It

should also be noted that three independent studies performed similar

characterisations of C02H7.1/DYF-11 in C. elegans, and obtained similar results

(Kunitomo & lino, 2008; Bacaj et aI., 2008; Omori et aI., 2008). Furthermore, a

recent study by Follit et al. (2009) has confirmed, using co-immunoprecipitation

analysis with IFT88, that the murine orthologue of DYF-11, Traf3ip1, is in fact a

component of 1FT subcomplex B, as predicted by the genetic analyses presented

in this chapter.

85 Our morpholino knockdown studies (Li et aI., 2008) in the vertebrate Danio rerio also suggest that MIP-T3 is likely required for basal body/ciliary functions since it is both necessary for gastrulation movements and also demonstrates a potential genetic interaction with Cit least one gene encoding a basal body protein

(BBS4) in the regulation of this process (Davis et aI., 2006; Simons et aI., 2005).

These data support the notion that vertebrate MIP-T3 plays an important role in development, potentially helping to modulate Wnt and/or other signaling pathways (Gerdes et aI., 2007). Intriguingly, the MIP-T3-interacting protein

DISC1 binds FEZ1 (Miyoshi et aI., 2003), a protein that is implicated in neurite outgrowth and co-precipitates with BBS4 (Lee et aI., 2005). One important question raised by these observations that will need to be addressed in future studies is whether vertebrate MIP-T3 performs functions related to trafficking in neurites, possibly in cooperation with DISC1/FEZ1. If so, then such a finding would suggest that MIP-T3 may have been adapted during evolution to perform two distinct roles in protein trafficking, one in cilia as an 1FT protein, and the other in neurites (axons and perhaps also dendrites).

3.5 Materials and Methods

3.5.1 Strain construction and maintenance

All C. elegans strains were maintained at 20°C, and standard genetic crosses were employed to introduce GFP reporter constructs (transcriptional or translational) into wild-type (N2) or mutant animals. PCR or dye-filling assays were used to follow genotypes, as described (Blacque et aI., 2006). The following mutant strains were used in this study: bbs-1(ok1111), bbs-7/osm-12(n1606),

86 bbs-8(nx77), che-3(e1124) che-11(e1810), che-13(e1805), daf-2(e1370), daf­

16(mu86), dyf-11(mn392) klp-11(tm324), osm-3(p802), osm-5(p813), and osm­

6(p811).

3.5.2 Construction of strains harboring a translational DYF-11 ::GFP construct

A translational DYF-11 ::GFP fusion construct was made by fusion PCR as described (Blacque et aI., 2004). The entire genomic coding region of dyf-11

(C02H7.1), along with 528 bp of promoter sequence 5' of the start codon, was fused upstream of, and in frame with, the GFP coding sequence. Aside from

DYF-11 ::GFP, the following strains were used: dpy-5(e907); Ex[gcy-5p::gfp+dpy-

5(+)] dpy-5(e907); nxEx[osm-12::gfp+dpy-5(+)], N2; myEx10[che-11::gfp+rol­

6(su1006)]; N2; Ex[kap-1::gfp+rol-6(su1006)], N2; ejEx1 [osm-3::gfp+rol­

6(su1006)], N2; yhEx2[osm-5::gfp+rol-6(su1006)], N2; Ex[srb-6p::gfp+rol­

6(su1006)] and N2; nxEx[xbx-1::gfp+rol-6(su1006)].

3.5.3 Localization of MIP-T3 in mammalian cells

A tagged expression construct for MIP-T3 was generated by LR clonase II

(Invitrogen) mediated recombination between the pENTR 221-MIP-T3 (Ultimate

ORF clone IOH28851; Invitrogen) and pcDNA6.2/nLumio-DEST (Invitrogen), placing the human MIP-T3 ORF under control of a CMV promoter with an N- terminal V5 epitope tag (pcDNA6.2/nLumio-MIP-T3).

IMCD3 cells were plated on glass coverslips and transfected with the pcDNA6.2/nLumio-MIP-T3 vector when cells reached 60% confluency by using

FuGENE6 (Roche) transfection reagent. Twenty-four hours post-transfection,

87 cells were fixed in methanol and stained using mouse anti-V5 (1 :200, Invitrogen), mouse anti- y-tubulin or mouse anti- acetylated-tubulin (both 1:1000, Sigma), and secondary detection carried out with goat anti-mouse IgG antibody conjugated to

Alexa 488 dye, and goat anti-mouse IgG antibody conjugated to Alexa 594 (both

1: 1000, Invitrogen). Cells were visualized by fluorescence microscopy.

3.5.4 Cloning of dyf-11 (C02H7.1)

The C. elegans MIP-T3 gene homolog, C02H7.1, is physically situated close to the interval defined for the dyf-11 (mn392) mutant allele (X: -18.27±0.244 cM) (Starich et aI., 1995), suggesting that the genetic lesion lies within this gene.

We sequenced the C02H7.1 coding region in the dyf-11 mutant and uncovered a nonsense mutation (TCA-TGA) in the third exon at nt 419 of the coding region.

Primer sets used to detect the mutation were: OPS0320

TGGTCGCAATTTGACCACC and OPS0322 TGATCATTCTCGGGCTCTC

(fragment 1); OPS0321 GACGATCATGAGATTTCTG and OPS0323

CAACATATTGGTGCAACTTC (fragment 2). A second putative dyf-11 allele, ad1303, had no sequence alterations in exons and complemented the dyf­

11 (mn392) mutation, suggesting that it represents a different gene.

3.5.5 C. e/egans phenotypic analyses

Dye-filling assays using the fluorescent dye Oil were performed as described (Blacque et aI., 2004). Chemotaxis assays were carried out for 1 hour essentially as described (Hart, 2006), using isoamyl-alcohol as a chemoattractant. A chemotaxis index was calculated as the number of worms in

88 attractant zone minus worms in control zone, divided by the total number of worms. Osmo-avoidance assays were performed as described (Hart, 2006).

Briefly, -5 worms (for each of at least 20 assays) were placed inside a small ring of 8 M glycerol, and animals found within or beyond the ring after 10 minutes were counted as non-avoiders. An osmo-avoidance index was calculated: (total avoiders-non-avoiders)/total worms.

Lifespan assays were based on the protocol of Apfeld and Kenyon (1999).

Animals were grown for at least one generation at 20°C before eggs were collected. At the L4 molt, worms were transferred to NGM plates containing 16

IJM fluorodeoxyuridine (FUOR) to prevent progeny growth and kept at 20°C throughout the assay. 100 worms were picked for each strain, at 10 worms/plate.

Worms were scored every 1-2 days for viability; those no longer responding to prodding with platinum wire were considered dead, and those that exploded or crawled off the plate were censored.

To test for entry into and exit from the dauer stage, we employed an existing strategy (Malone et aI., 1996). 10 adult worms were allowed to lay eggs on plates with food at 20°C for 4 hours. Adults were then removed and eggs counted. Eggs were allowed to develop for 4 days at 20°C or 3 days at 25°C, after which they were scored as either Oaf-c or Oaf-d as follows. To identify Oaf-c worms, plates were flooded with 1% SOS, where only dauer larvae remained as live thrashing animals after 15 minutes. To identify Oaf-d worms, animals were allowed to grow 4 days following complete consumption of food, after which they were exposed to and unable to survive the 1% SOS treatment. daf-16 (mu86)

89 and daf-2 (e1370) were used as Daf-d and Daf-c controls, respectively. All dauer/lifespan assays were carried out in duplicate or triplicate.

Nile Red staining was performed as previously described (Ashrafi et aI.,

2003). Briefly, Nile Red powder (Molecular Probes) was dissolved in acetone as a 1mg/ml stock solution and kept at room temperature. The stock solution was diluted in 1x PBS to 1 ug/ml and 0.5 ml of diluted solution was applied to NGM plates seeded with E.coli OP50. Plates were allowed to dry for 24 hours. Staged eggs were allowed to develop on Nile Red plates at 20°C. Worms were transferred every 2 days to fresh Nile Red plates and were analyzed two days post-L4 by fluorescence microscopy. Images were captured and processed under identical settings using OpenLab software (Improvision, Inc). Nile Red fluorescence intensity was calculated as the mean pixel intensity after background subtraction.

3.5.6 Analysis of sensory neuron structure and cilia length measurements

ASER amphid and PHAlPHB phasmid sensory neuron structures were visualized by expressing the cell-specific transcriptional reporters, gcy-5p::gfp and srb-6p::gfp (Blacque et aI., 2006), respectively. In these neurons, the GFP diffuses freely throughout the neuron to mark cell bodies, axons, dendrites, transition zones and cilia. Cilium length was measured from the distal end of the transition zone (visible as a 'bulge' of fluorescence) to the distal tip of the cilium.

90 3.5.7 Visualization of 1FT and rate measurements by time-lapse microscopy

Transgenic animals expressing GFP-tagged proteins were mounted on agarose pads and immobilized with 20 mM levamisole. Amphid or phasmid cilia were examined with a 100X, 1.35 NA objective and an ORCA AG CCD camera mounted on an Zeiss Axioskop 2 mot plus microscope. Images and movies were obtained in Openlab version 5.02 beta (Improvision). Kymographs were generated using the MultipleKymograph ImageJ plug-in. Rates from middle and distal segments were obtained essentially as described in Snow et al (2004).

Images for intraflagellar transport were collected using a Zeiss Axiovert

200 equipped with a Hamamatsu Orca AG CCD camera, spinning disk confocal head, Zeiss Plan-neofluar 63X, 1.3 NA, water-immersion objective and a 1.5X magnification lens Improvision Piezo Focus Drive. Images for MX488 bbs-

7(n1606); Ex[dyf-11::GFP+dpy-1(+)] and MX486 N2; Is[dyf-11::GFP+dpy-1(+)] were collected at 7.5 frames/sec and 4.24 frames/sec respectively. Animals were first anaesthetized with 10 mM levamisole, mounted on agar pads and photobleached for 300-1900 ms before images were collected for 2 minutes. The

FRAP module is a Photonic Instruments MOSAIC Digital Diaphragm System with a 488 nm 300 mW laser line. Images were collected using Volocity.

3.6 Acknowledgements

We thank Leon Avery and Joseph Dent for providing us with the ad1303

allele, and T. Stiernagle and the C. elegans Genetics Center for providing strains.

91 We also thank Mathieu Bakhoum, Chrystal Inglis and Harald Hutter for assistance with the project.

92 3.7 Figures

Figure 3-1. The C. e/egans dyf-11 strain contains a mutation in the gene C02H7.1, the MIP-T3 ortholog. (A) Predicted protein structure of C. elegans C02H7.1/DYF-11 as compared to

MIP-T3 orthologs in H. sapiens (human; AAF76984), D. rerio (zebrafish;

Q6PGZ3), C. reinhardtii (green alga; C_140070), D. melanogaster (fruit fly;

CG3259), L. major (a kinetoplastid parasite; CAJ04965), and the ciliated fungus

B. dendrobatidis (BDEG_02985.1). (B) Cloning of dyf-11 (mn392) was accomplished by identifying a nonsense mutation in the third exon of the C. elegans C02H7.1 gene. Note the presence of an X box at nucleotide -182 bp relative to the ATG start codon. Exons are denoted with gray boxes and intervening introns with lines. (C) Dye-filling assays with the red fluorescent dye,

Oil, reveal that compared to wild-type (N2) animals, dyf-11(mn392) mutants do not visibly take up dye (a Dyf phenotype). A transgene expressing wild-type

C02H7.1 fused to GFP in dyf-11 animals rescues this defect. Fluorescence microscopy signals (all 100 msec exposure) are overlaid onto DIC images.

Arrowheads indicate positions of dye-filling neuronal cell bodies. [Note: (B) sequencing performed by E. Efimenko and P. Swoboda]

93 A 100 200 300 400 500 600 a.a. amino a.a. ,I,,, I I,I, I, I,, I,,,,I,, I, I,, II I II acids identity Homo sapiens 625

Danio rerio 629 51% Caenorhabditis elegans 535 24% Chlamydomonas reinhardtii 532 29% Drosophila 26% melanogaster 588 Leishmania major 484 35% Batrachochytrium dendrobatidis 532 42% predicted coiled coil

B 200 bp C02H7.1 (dyf-11) t t X box: mn392 GTCTCC AT GACAAC (-182) nt419 C-'G (Ser140 -. STOP) c

94 Figure 3-2. Amino acid alignment of C. e/egans DYF-11 (C02H7.1) with MIP T3 protein orthologs from C. reinhardtii and H. sapiens. Identical and similar residues are highlighted in black and gray, respectively.

Note that the major difference in size between the human and C. elegans/Chlamydomonas proteins is due to two large insertions found in the human protein sequence. The number of residues is displayed at the ends of the sequences. Caenorhabditis elegans DYF-11/MIP-T3: C02H7.1; Homo sapiens

MIP-T3: MF76984; Chlamydomonas reinhardtii MIP-T3 accession number:

C 140070.

95 ...... '" . _. ~. -= ... :J ...... =

~""'" ~~I"'!~fII'~' ;,~' ~I"~I'1'" .~ ~ ~ ~ , SI-~;I:t'I;I"I:", ..... r r" III" r. 1:.1tJ!,: I .. -I'. ~.r. \ I,FI.. ~. t\rt.. ~ .. I . D."'"RL I' -"11'"i:'_I.', IS' : ) - -, - - •• co·, -r . c·· ,. - : 0 ~_. r c'IT E .. ,- •- F ...... 2~:- '';.;'\".. :::: ,... : _3 .L.I."A._C "".-.~= ,. , '. I.:. "'~ • _ ...... ~ ... _. _ t • •• _ '-'

.... e:".;;..l$ t. L"::-'~

.J' •

1 • • ,~

.. - 3~-;:.i..e::5 !._=_3

',L

96 Figure 3-3. dyf-11 (mn392) mutants display ciliary structure defects, behavioural phenotypes indicative of ciliary anomalies, and a lipid accumulation phenotype. (A) Cilium length measurements using the gcy-5p::gfp and srb-6p::gfp transcriptional markers (which highlight the cell bodies, axons, dendrites, transition zones/basal bodies and cilia of the ASER and phasmid neurons with

GFP, respectively) reveal that the dyf-11(mn392) strain has truncated cilia relative to wild-type (N2) animals. Measurements (in IJm), shown in the graph, are from the transition zone (TZ, arrowhead) to the tip of the cilium. Scale bar, 5

IJm. (8, C) dyf-11 mutants are defective in both chemotaxis and osmo-avoidance, as observed from their significantly lower che or osm indexes compared to wild- type (N2) animals, respectively. The dyf-11 mutant strain defects are comparable to those of other cilia mutants (che-3 and osm-3) and can be rescued by transgenic expression of wild-type OYF-11 ::GFP. (0) dyf-11 mutants are dauer formation defective (Oaf-d) at 20°C and 25°C. The ciliary (osm-5) or insulin signaling (daf-2 and daf-16) mutants are Oaf-d or Oaf-c (constitutively enter the dauer stage) at the indicated temperatures. (+), ability to enter the alternate dauer stage. (E) Lifespan analyses show that dyf-11 mutants have statistically normal longevity when compared to wild-type (N2) animals. che-11 ciliary mutants, on the other hand, are significantly longer lived. Mean lifespan, number of worms assayed, and P value compared to N2 are tabulated. (F) dyf-11 mutants display an increase in lipid content (relative to N2 animals) that is rescued by the wild-type OYF-11 ::GFP transgene. Lipid content is assessed by

Nile Red staining followed by quantitation of relative fluorescence intensities.

97 [Note: (A) performed by C. Li; (D), (E) performed by N. Bialas; (F) performed by M. Healey]

98 A gcy-5p::gfp srb-6p::gfp 6 E N2 3­ ..1::4 ...O'l C ~ E 2 ::J o

dyf-ll N2 dyf-II N2 dyf-ll gcy-5p::gfp srb-6p::gfp (ASER cilium) (PHNB cilia)

B C 0 0.8 20°C 25°C 1.0 N2 (+) (+) 0.6 0.8 x x daf-16 Daf-d Daf-d Q) Q) "0 .!: 0.4 -g 0.6 daf-2 (+) Daf-c Q) .J:: ~ 0.4 Ll 0 osm-5 Daf-d Daf-d 0.2 0.2 dyf-11 Daf-d Daf-d ,-L, ....L. 0 0 dyf-11 (+) (+) N2 che-3 dyf-11 dyf-11 N2 dyf-11 osm-3 dyf-11 rescue rescue rescue F E -0- N2 -. dyf-11(mn392) 0.8 Q) ..... che-11(e1810) .2: ro 0.6 c 0 U 0.4 ~ LL 0.2

0 0 4 8 12 16 20 24 28 32 36 40 44 48 Days

mean P value lifespan n vs. N2 N2 19.69 ± 0.50 85 Fluorescence Relative to N2 o 0.5 1.0 1.5 2.0 2.5 dyf-11 20.95± 0.59 74 0.108 che-11 27.50 ± 0.85 72 <0.0001* N2 + dyf·11 -+ dyf-11 I----~+ rescue I----.....J

99 Figure 3-4. DYF·11 is a component of the intraflagellar (1FT) transport machinery. (A) Kymograph analyses show the bi-phasic anterograde movement of OYF-

11 ::GFP in amphid cilia. Middle segment (MS) motion was determined to be

O.74±O.08 IJm/sec, while the distal segment (OS) velocity was 1.15±O.21 IJm/sec.

The second and fourth panels are actual kymographs, whereas the third and fifth panels are representative traces of moving particles in the indicated middle or distal segments; X axes denote time whereas Y axes denote distance covered by fluorescently-Iabeled particles. In the still fluorescence microscopy image taken from Movie S1 (left panel), solid arrowheads denote the positions of representative transition zones, while hollow arrowheads denote unexpected dendritic endings. (8) Human MIP-T3 tagged with a V5 epitope (V5-MIP-T3) localizes to centrosomes in IMC03 kidney cells that have not yet ciliated, and to the axoneme when cilia are present. Centrosomes/basal bodies are stained with an antibody against y-tubulin (red), V5-MIP-T3 is marked with an antibody against the V5 epitope (green), and the nucleus is stained with OAPI (blue).

Arrowheads and arrows point to centrosomes/basal bodies, and brackets denote ciliary axonemes. Scale bar, 5 IJm. (C) Similar to other well characterized 1FT components, OYF-11: :GFP accumulates in the cilia of che-11 mutants, which displays retrograde transport defects. Arrowheads represent the positions of transition zones, brackets show ciliary axonemes, and stars denote accumulations (compare the localization of OYF-11 ::GFP in head amphid cilia of wild-type (A) and che-11 mutant (C) animals; see also Figure 3·5A for normal

OYF-11 ::GFP localization). Scale bar, 5 IJm. Schematics represent anterograde

100 transport in wild-type animals (no accumulations) and che-11 mutants

(accumulations at the tip of cilia, shown as stars). K, Kinesin-II; 0, OSM-3­ kinesin; A, 1FT subcomplex A; B, 1FT subcomplex B; S, BBS protein complex. TZ, transition zones. [Note: (B) performed by C. Leitch, N. Zaghoul, E. Davis and

N. Katsanis]

101 A Middle Distal c OYF-11 ::GFP Segment Segment OYF-11 ::GFP (Head) (MS) (OS) Head Tail

wild-type che-11

B non-ciliated IMC03 cell ciliated IMC03 cell

y-tubulin

V5-MIP-T3

merge

102 Figure 3-5. C. e/egans DYF-11 ::GFP may be functionally associated with 1FT particle subcomplex B. (A, B) OYF-11 ::GFP localizes along the entire length of amphid/phasmid cilia in wild-type (N2) and klp-11 (a motor component of heterotrimeric Kinesin-2) animals, both of which have full-length cilia. Note the schematics at the bottom of the figure. (C) OYF-11 ::GFP is observed along the length of the middle segments of osm-3 (homodimeric Kinesin-2) mutants, which lack the distal segments (see schematic). (O-F) OYF-11 ::GFP localizes along the entire middle segments and truncated distal segments of bbs mutant worms, consistent with the hypothesis that OYF-11 is associated with OSM-3-kinesinIlFT subcomplex B (Kinesin-

II/subcomplex A does not enter the distal segment; see schematic). Middle segments (MS) and distal segments (OS) are specifically highlighted. (G, H)

OYF-11 ::GFP ciliary localization is not perturbed in the truncated cilia of two subcomplex B mutants (che-13 and osm-6, respectively; see schematic).

Transition zones (TZ) are shown by white arrowheads, unusual dendritic termini are denoted by hollow arrowheads, and brackets designate the observed ciliary axonemes. Note that OYF-11 behaves like an 1FT subcomplex B component (i.e., moves into the cilia of osm-3, klp-11 as well as distal segment of bbs mutants).

Scale bar, 5 IJm. (I) Kymograph analyses show the movement of OYF-11 ::GFP in a bbs-7 mutant animal at a velocity of 1.21 ±O.19 IJm/sec along the middle and distal segments of phasmid cilia, consistent with an association with OSM-3- kinesin. [Note: (I) initial microscopy done by C. Mok, M. Zhen, E. Heon]

103 DYF-11 ::GFP Head Tail Head Tail

~ MiddlelDistal MS os OYF-11 ::GFP Segments (Head) (MS/OS)

5 BBS protein complex

AB 1FT subcomplexes bbs-1, -7,-8 che-13, osm-6

K Kinesin-II

o OSM-3-kinesin klp-11

104 Figure 3-6. Fluorescence images indicating the possible presence of DYF 11::GFP in the distal segments of AWe neuron cilia. Two of the highlighted extensions (hollow arrowheads) from the DYF-11 ::GFP protein overlap with the RFP protein that is most highly expressed in AWe cilia.

Two 'branches' of the AWe cilium are shown with arrows, and the bundle of amphid channel cilia are pointed to. The schematic shows the ultrastructure of the AWe cilium, as visualised in Perkins et al. (1986). Images were acquired in the strain OE3657 dpy-5(e907) I; dyf-11(mn392) X; nxEx[C02H7.1::gfp; dpy-5

(+)}; ofEx457 [odr-3::rtp; elt-2::cherryj. [Note: all information in this figure provided by E. Efimenko and P. Swoboda]

105 Awe cilium ultrastructure axonemes

transition zone dendrite

106 Figure 3-7. C. e/egans DYF-11 is required for the assembly and function of many components of the motor-1FT machinery, including Kinesin-II. (A-F) In wild-type (N2) animals, 6 representative GFP-tagged 1FT-associated proteins (CHE-11, OSM-5, XBX-1, OSM-3, KAP-1, and BBS-7/0SM-12) show the expected prominent localization to transition zones (TZ; arrowheads) and cilia

(brackets). Some overexpressed proteins show some localization along the length of the dendrites, anterior to the transition zones. (G, H, I, K, L) CHE-

11 ::GFP, OSM-5::GFP, XBX-1 ::GFP, KAP-1 ::GFP, and BBS-7::GFP are consistently mislocalized in dyf-11 mutant animals, frequently showing protein accumulations in the dendrites and/or cilia (denoted by asterisks). The anticipated position of ciliary axonemes is noted with a dashed bracket, and transition zones with arrowheads. Question marks represent unclear localization of transition zones and cilia, or of the GFP-tagged proteins. (J) OSM-3::GFP appears to properly enter the severely truncated cilia of dyf-11 mutants. Scale bar,S IJm.

107 N2 dyf-11 Head Tail Head Tail a.. u. (9 ..­ ..- I ill I () a.. u. (9

L[) I ~ oCJ) a.. u. (9 ..- I co>< >< a.. u. (9

(") I ~ CJ) o a.. u. (9

a.. u. (9 l'- I CJ) co co

108 CHAPTER 4. FUNCTIONAL INTERACTIONS BETWEEN THE CILIOPATHY-ASSOCIATED MECKEL SYNDROME 1 (MKS1) PROTEIN AND TWO NOVEL MKS1-RELATED PROTEINS

Note regarding contributions: The following chapter is a slightly modified version of a manuscript published in the Journal of Cell Science. The authors of the study are listed below. Bialas NJ*, Inglis PN*, Li C*, Robinson JF, Parker JDK, Healey MP, Davis EE, Inglis CD, Toivonen T, Cottell DC, Blacque OE, Quarmby LM, Katsanis N, and Leroux MR. (2009). JCS 122: 611-624. (* - equal contributions) As co-first author of this manuscript, I participated in numerous facets of this study. Specifically, I obtained the bioinformatics data used in the phylogenetic analyses (performed by J. Parker), basic behavioural analyses (Che and Osm), and most C. elegans microscopy with related analysis. In addition, I co-wrote the manuscript with M. Leroux. C. Li performed the genetic crosses and GFP transgene construction. N. Bialas performed the lifespan assays and DAF­ 16 localisation studies. M. Healey performed lipid accumulation analysis. C. Inglis aided with the C. elegans genetics. J. Robinson and E. Davis performed the B9 analysis in IMCD3 cells under the supervision of N. Katsanis. 1. Toivonen, D. Cottell and O. Blacque produced the electron micrographs.

109 4.1 Abstract

Meckel syndrome (MKS) is a ciliopathy characterized by encephalocoele, cystic renal disease, liver fibrosis and polydactyly. An identifying feature of

MKS1, one of five MKS-associated proteins, is the presence of a 89 domain of unknown function. Using phylogenetic analyses, we show that this domain occurs exclusively within a family of three proteins distributed widely in ciliated organisms. Consistent with a ciliary role, all C. elegans 89 domain-containing proteins, MKS-1 and MKS-1-related proteins 1 and 2 (MKSR-1, MKSR-2), localize to transition zones/basal bodies of sensory cilia. Their subcellular localization is largely co-dependent, pointing to a functional relationship between the proteins. This localization is evolutionarily conserved, since the human orthologs also localize to basal bodies, as well as cilia. As reported for MKS1, disrupting human MKSR1 or MKSR2 causes ciliogenesis defects. In contrast, single, double and triple C. elegans mks/mksr mutants do not display overt defects in ciliary structure, intraflagellar transport or chemosensation. However, we find genetic interactions between all double mks/mksr mutant combinations, manifesting as an increased lifespan phenotype due to abnormal insulin/lGF-1 signaling. Our findings therefore demonstrate functional interactions between a novel family of basal body/cilia-associated proteins, providing new insights into the molecular etiology of a pleiotropic human disorder.

110 4.2 Introduction

Primary cilia, the hair-like microtubule-based organelles found on the majority of human cell types, have gained attention recently owing to their involvement in a multitude of sensory processes, signaling pathways, and genetic disorders (Davis et aI., 2006; Davenport and Yoder, 2005; Marshall and

Nonaka, 2006; Satir and Christensen, 2007; Singla and Reiter, 2006). These non-motile cilia are now implicated in most physiological sensory modalities, including chemosensation, olfaction, mechanosensation, photoreception, and thermosensation (Davis et aI., 2006; Davenport and Yoder, 2005; Perkins et aI.,

1986; Satir and Christensen, 2007; Tan et aI., 2007). Primary cilia not only capture and transduce environmental stimuli, but also play key roles in the transduction of Wnt, Hedgehog and PDGFRaa signaling pathways (Christensen et aI., 2007; Eggenschwiler and Anderson, 2007; Gerdes et aI., 2007; Breunig et aI., 2008). Moreover, ciliary assembly and disassembly is coordinated intimately with the cell cycle (Pan and Snell, 2007; Quarmby and Parker, 2005). In humans, defects in the sensory and signaling functions of primary cilia lead to numerous developmental disorders that collectively affect the renal, cardiac, hepatic, pancreatic, skeletal, visual, nervous, olfactory, and auditory systems (Badano et aI., 2006; Bisgrove et aI., 2006; Tan et aI., 2007). Numerous cilia-associated disorders (ciliopathies) have been described, including Bardet-Biedl syndrome

(BBS), Meckel syndrome (MKS), and Polycystic Kidney Disease (PKD) (reviewed in Pazour and Rosenbaum, 2002; Badano et aI., 2006; Bisgrove and Yost, 2006;

Blacque and Leroux, 2006; Christensen et aI., 2007; Hildebrandt and OUo, 2005).

111 Meckel syndrome is a rare autosomal recessive disorder characterized by central nervous system malformations (encephalocele), polydactyly, renal cysts and hepatic ductal dysplasia and cysts (Alexiev et aI., 2006; Badano et aI., 2006).

Numerous lines of evidence have defined MKS as a ciliopathy, although the nature of the ciliary defect is unclear. The recently-identified MKS1 protein

(Kyttala et aI., 2006), for example, localizes to basal bodies, which are the centriolar structures required for nucleating eukaryotic cilia. Together with a second identified MKS-associated protein, MKS3/Meckelin (Smith et aI., 2006),

MKS1 is reported to playa role in basal body migration to the apical membrane, and thus, ciliogenesis (Dawe et aI., 2007). A third protein implicated in MKS,

NPHP6/CEP290/MKS4/BBS14, also localizes to basal bodies, but its molecular function is unclear (Baala et aI., 2007a; den Hollander et aI., 2006; Leitch et aI.,

2008; Sayer et aI., 2006; Valente et aI., 2006). RPGRIP1 L (MKS5), also implicated in Joubert syndrome, is a basal body protein that interacts with the nephronophthisis-associated NPHP-4 protein; although it is not required for cilium formation, it plays an important (and presumably cilium-based) role in

Hedgehog signaling (Arts et aI., 2007; Delous et aI., 2007; Vierkotten et aI.,

2007). Most recently, two other proteins, NPHP3 (Bergmann et aI., 2008), and

CC2D2A (MKS6), whose function is unknown but is associated with the formation of cilia, (Tallila et aI., 2008), were found to be disrupted in MKS patients.

The MKS1 protein contains no domains of recognized function.

Nonetheless, it does harbor a predicted so-called 'B9' domain of undetermined

112 utility (Kyttala et aI., 2006). Two additional highly conserved proteins containing

B9 domains, referred to in mammalian systems as B9D1 and B9D2, can also be identified. We previously reported that all three putative B9 protein orthologs in the nematode Caenorhabditis elegans (R148.1/xbx-7, K03E6.4 and Y38F2AL.2) are found solely in ciliated sensory neurons and possess X box sequences in the upstream promoter sequences of their associated genes that are regulated by the ciliogenic transcription factor, DAF-19 (Blacque et al., 2005; Efimenko et al.,

2005). These observations are consistent with comparative genomic analyses of ciliated versus non-ciliated organisms (Avidor-Reiss et aI., 2004; Li et aI., 2004a), as well as the recent discovery that two of the Drosophila melanogaster B9 domain-containing genes are also X box-regulated (Laurencon et aI., 2007), collectively implicating all three proteins in ciliary function(s). This notion is also supported by the recent finding that the murine 8902 gene is abrogated in the stumpy mutant, which is characterized by impaired ciliogenesis, cystic kidneys and hydrocephalus (Town et aI., 2008). Disruption of stumpy in this mouse model was further shown to be required for the proliferation and neurogenesis of astrocyte-like neural precursor (ALNP) cells, likely as a result result of dramatically downregulated cilium-dependent sonic hedgehog signaling (Breunig et aI., 2008).

In C. elegans, defects in proteins implicated in cilia structure/function have been associated with various sensory phenotypes (Bae et aI., 2008; Inglis et aI.,

2007; Perkins et aI., 1986; Scholey, 2003). For example, abrogation of the

C. elegans orthologs of two transition zone/basal body-associated

113 nephronophthisis-linked proteins, NPHP-1 and NPHP-4, leads to chemosensation, male mating and various axonemal/intraflagellar transport (1FT) defects (Jauregui and Barr, 2005; Jauregui et aI., 2008; Winkelbauer et aI., 2005;

Wolf et aI., 1995), while disruption of BBS proteins result in partially truncated cilia as well as chemo- and thermo-sensory phenotypes (Blacque et aI., 2004; Ou et aI., 2005; Tan et aI., 2007). Notably, cilium- and/or basal body-associated sensory inputs are transduced by at least one major signaling pathway in the nematode, namely the insulin/IGF-I pathway, which regulates longevity (Apfeld and Kenyon, 1999). Hence, many basal body/cilium mutants, including nphp-1 and nphp-4, as well as strains with defects in the 1FT process required for building all cilia (e.g., osm-5, che-11, ifta-2, etc.), display increased lifespans indicative of impaired ciliary signaling (Apfeld and Kenyon, 1999; Schafer et aI.,

2006; Winkelbauer et aI., 2005).

Williams et al. (2008) recently reported on the analysis of the three B9 domain-containing proteins found in C. elegans. Using GFP-tagged variants, they observed interdependent localization of the proteins to ciliary transition zones

(akin to basal bodies), and as such, named the proteins encoded by Y38F2AL.2 and K03E6.4 as "ciliary Transition Zone Associate" protein-1 and -2 (TZA-1 and

TZA-2), respectively. Disruption of any of the three respective proteins did not result in overt changes to ciliary morphology nor in any specific behavioral/sensory abnormalities, with the exception of a subtle foraging behavior phenotype. Interestingly, unlike disruption of stumpy in mammals, C. elegans transition zone positioning/ciliogenesis was only observed to be impaired

114 by further disruption of nphp-1 or nphp-4, suggesting a likely genetic redundancy in the system.

In the present study, we shed additional light on the molecular basis of

Meckel syndrome by characterizing in further detail the three C. elegans and human B9 domain-containing proteins. Our phylogenetic analyses demonstrate that B9 domain-containing proteins are invariably absent from non-ciliated organisms but co-occur as a family of three different proteins with orthologs in nearly all ciliated species. We show that the three C. elegans MKS/MKSR proteins localize to transition zones/basal bodies in a largely interdependent manner, and that the subcellular localization to basal bodies is evolutionarily conserved for the human B9 protein counterparts. Knockdown of the human

MKSR1 and MKSR2 genes using RNA interference (RNAi) leads to a ciliogenesis defect, similar to that reported for MKS1 by Dawe et al. (2007). In contrast, rigorous analysis of 1FT and cilia ultrastructure by electron microscopy for each of the single, double and triple C. elegans mks/mksr gene mutants showed no clear defects versus wild-type animals. However, all double mks/mksr gene mutant combinations revealed genetic interactions between the different family members that are manifested by increased lifespan dependent on the

DAF-2 (insulin/IGF-I receptor)-DAF-16 (FOXO transcription factor) pathway.

Together, our data reveal that a highly conserved family of three proteins functionally interacts at basal bodies/cilia to support ciliogenesis in human cells and a cilium-associated signaling process in C. elegans. Based on our present study, we favor a unified nomenclature that captures the evolutionary and

115 functional relatedness of the three proteins, namely Meckel syndrome 1 (MKS-1), and MKS-1-related proteins 1 and 2 (MKSR-1 and MKSR-2).

4.3 Results

4.3.1 An evolutionarily-conserved family of 89 domain-containing proteins in ciliated organisms

The identification of C. elegans xbx-7/R148.1, K03E6.4, and Y38F2AL.2 as genes expressed exclusively in ciliated cells initially provided evidence that they may encode proteins with important ciliary functions (8lacque et aI., 2005;

Efimenko et aI., 2005). Moreover, the presence of a single 89 domain of unknown function in each of these three proteins hinted at the potential significance of this protein motif, a notion that was strongly reinforced following the cloning of the Meckel syndrome-associated MKS1 gene, the human orthologue of xbx-7/R148.1 (Kyttala et aI., 2006). On this basis, we conducted

comprehensive searches of sequence databases to identify all possible proteins

harboring the 89 domain. Without exception, we failed to find 89 domain-

containing proteins in prokaryotes or in eukaryotes lacking cilia, such as

Saccharomyces cerevisiae, Dictyostelium discoideum and Arabidopsis thaliana

(Table 4-1). In the vast majority of fully-sequenced ciliated organisms queried (21

in total), however, we uncovered three different proteins containing a single 89

domain. The only exceptions were the moss Physcomitrella patens, the parasitic

species Giardia lamblia, and the apicomplexan Plasmodium falciparum, which

have no recognizable 89 domain-containing proteins, and the free-living

Tetrahymena thermophila, Paramecium tetraurelia and Chlamydomonas

116 reinhardtii, which contain a fourth family member that likely arose from an independent gene duplication (Table 4-1).

Using amino acid sequence alignments of 89 domains obtained from a comprehensive list of species chosen to represent the major eukaryotic groupings outlined in Keeling et al. (2005), we derived phylogenetic trees of 89 domain-containing proteins. The neighbour-joining algorithm of ClustalW (Higgins et aI., 1994) and the 8ayesian phylogeny inference program Mr8ayes

(Huelsenbeck and Ronquist, 2001) were used independently on the protein alignments to generate the trees. Each method produced essentially identical phylogenies that group the sequences into three clades, which we named MKS­

1, MKSR-1, and MKSR-2 (the 8ayesian tree is shown in Figure 4-1A). These analyses demonstrate that proteins containing 89 domains are evolutionarily ancient; specifically, the diversity of organisms represented in each clade shown in the tree suggests that the gene duplications which led to the three clades preceded the speciation events resulting in the emergence of major eukaryotic lineages, the last common ancestor of which is inferred to be ciliated (Richards and Cavalier-Smith, 2005).

We find that while the MKSR-1 and MKSR-2 family members typically consist of little more than the 89 domain, members of the MKS-1 clade are larger in size, with poorly conserved regions outside of the 89 domain that sometimes sport other domains (for example, in the Drosophila and Chlamydomonas MKS-1 proteins). These observations suggest that the 89 domain is critically important for the function(s) of the proteins. An amino acid sequence alignment of 22 89

117 domains from MKS-1, MKSR-1 and MKSR-2 proteins across 6 different species, shown in Figure 4-1 B, reveals sequence conservation throughout the -115 residue domain, where some residues are invariant in >90% of the sequences.

4.3.2 All three B9 domain-containing proteins localize to basal bodies and/or cilia

Three human proteins associated to date with Meckel syndrome (MKS1,

CEP290/NPHP6/MKS4 and RPGRIP1 L) localize to the basal body at the base of the cilium, and a fourth, MKS3/Meckelin, is distributed along the length of the cilium (Arts et aI., 2007; Oawe et aI., 2007; Oelous et aI., 2007; Keller et aI.,

2005; Sayer et aI., 2006; Valente et aI., 2006; Vierkotten et aI., 2007). These findings, which are consistent with MKS being a ciliopathy, led us to examine the subcellular localization of all three human 89 domain-containing proteins in a ciliated mouse cell line (IMC03) derived from the inner medullary collecting duct of the kidney. We confirmed the localization of a transiently-expressed, V5 epitope-tagged version of MKS1 to the basal body in ciliated cells (Figure 4-2A, top panels), and to centrosomes in non-ciliated IMC03 cells (Figure 4-3) by co- staining with y-tubulin (a centriolar marker) alone or in combination with acetylated a-tubulin (a ciliary marker). The previously uncharacterized human

MKSR1 (8901) and MKSR2 (8902) proteins, also tagged with the V5 epitope and expressed transiently, show the same localization to basal bodies in ciliated

IMC03 cells (Figure 4-2A, middle and bottom panels, respectively), and to centrosomes in non-ciliated IMC03 cells (Figure 4-3). The localization to centrosomes in non-ciliated cells is consistent with the proposed pre-ciliogenic

118 function(s) of MKS1 in centriolar migration (Dawe et aI., 2007), and the recent differential localization of the murine stumpy protein (MKSR2) to basal bodies or

cilia (Town et aI., 2008). Interestingly, in contrast to the transiently-transfected

IMCD3 cells, versions of all three 89 proteins tagged fused with GFP-tagged

produced from stably transfected IMCD3 cells localize to the ciliary axonemes

(Figure 4-28). These results suggest, along with the findings of Town et al.

(2008), that the 89 proteins can localize differentially to the basal body alone or

to the ciliary axoneme.

To investigate whether the co-localization of the three 89 domain­

containing proteins at basal bodies/cilia is evolutionarily conserved, and to initiate

a functional analysis of the three proteins in a genetically tractable system, we

generated transgenic C. elegans strains harboring GFP-tagged versions of the

respective protein orthologs. The three expression constructs included the

endogenous promoter for each gene (encompassing the X box-regulatory

element), as well as the entire coding region fused in-frame to GFP at the

C-terminus (see Figure 4-4A for the gene structures). Similar to our previous

findings using promoter-GFP (transcriptional) fusion constructs (Efimenko et aI.,

2005; 81acque et aI., 2005), the three mks/mksrtransgenes are expressed

specifically in most, if not all ciliated cells, including the amphid and phasmid

sensory neurons. More notable, however, is that the three GFP-tagged proteins

(MKS-1, MKSR-1 and MKSR-2) localize specifically to transition zones at the

base of cilia (Figure 4-2C), which are akin to the basal bodies of other species

(Perkins et aI., 1986). For each amphid bundle, we were able to observe up to

119 -12 transition zones as fluorescent 'spots' near the head of the animal; for the phasmid sensory neurons situated near the tail, two pairs of transition zones can be seen (individual pairs are shown in Figure 4-2C). This localization pattern is equivalent to that observed for the mammalian orthologs (Figure 4-3) except that the latter proteins can also associate with the ciliary axonemes (Figure 4-28;

Town et aI., 2008). Of note, our findings confirm the localization of the C. elegans proteins recently reported by Williams et al. (2008); furthermore, the C. elegans

NPHP-1 and NPHP-4 proteins, whose respective genes interact with the mks/mksr genes, also localize specifically to transition zones (Jauregui et aI.,

2005; Winkelbauer et aI., 2005; Williams et aI., 2008).

Together, our findings establish that all 89 domain-containing proteins associate with, and presumably function at, basal bodies in two highly divergent species (humans and nematodes); in humans, ciliary localization is also observed, suggesting two potentially distinct regions of localization and thus, function. These data, combined with our phylogenetic analysis revealing an essentially strict co-occurrence of the three proteins in ciliated organisms and absence from non-ciliated species (Figure 4-1A and Table 4-1), raise the distinct possibility that all MKS/MKSR proteins may share a common ciliary function at the base of the organelle and within the cilium itself.

4.3.3 Interdependent localization of MKS/MKSR proteins to basal bodies

On the basis that all three C. elegans 89 domain-containing proteins can be observed at transition zones/basal bodies, we hypothesized that the proteins form a functional complex and that their localization might be co-dependent. To

120 test this possibility, we examined the localization of the three individual

C. elegans GFP-tagged MKS/MKSR proteins in each of their two complementary mks/mksr mutant backgrounds.

To perform this study, we first obtained from the National 8ioResource

Project (N8RP, University of Tokyo, Japan) strains with deletions in each of the respective mks-1/xbx-7, mksr-1/tza-2 and mksr-2/tza-1 genes; the three gene models and their lesions, as deduced by RT-PCR analysis of the transcripts, are shown in Figure 4-4A and 4·48, respectively. In the Y38F2AL.2/mksr-2(tm2452) strain, the mutant allele encodes a protein roughly half the size of wild-type, resulting from a significant truncation (approximately 50 amino acids) within the

89 domain. In the case of the xbx-7/mks-1 (tm2705) mutant strain, the deletion results in the splicing of exon 2 with exon 5, ultimately generating a protein lacking approximately 70 residues in the N-terminal region, but does not abrogate any of the 89 domain. Finally, in the K03E6.4/mksr-1(tm3083) strain, mis-splicing results in the inclusion of nucleotides 1032-1057 into the transcript, which then fuse in-frame to nucleotide 1276 of exon 3 (at the start of the 3' flanking sequence of the deletion). The predicted MKSR-1 protein encoded by this allele is one amino acid larger than wild-type, and possesses a distinct 18 amino acid alteration to the 89 domain (the sequences of which are shown in

Figure 4-48). While the possibility exists that all three deletion mutants (tm2452, tm2705, tm3083) are hypomorphs rather than nulls, in two cases (mksr-1 and mksr-2) the 89 domain is disrupted, and we confirm below that all alleles are

121 associated with observable protein mislocalization and/or lifespan/signaling phenotypes.

By carrying out standard genetic crosses, we generated strains carrying all six possible combinations of MKS/MKSR GFP-tagged proteins present in the two complementary mks/mksr gene mutant backgrounds. Visualization of the

MKSR-1 ::GFP and MKSR-2::GFP proteins in the mks-1 mutant background revealed no significant change in localization to the transition zones compared to that of the wild-type strain, and no unusual/prominent accumulations along the dendrites (Figures 4-5C and 4-5E). In contrast, the MKSR-1 ::GFP protein showed consistently reduced or unclear localization to transition zones in the amphid (head) and phasmid (tail) neurons of mksr-2 mutant animals, and pronounced accumulations in the dendrites (Figure 4-50). Similarly,

MKSR-2::GFP showed reduced and less distinct signals at the transition zones of mksr-1 mutants (compared to wild-type animals), although in many cases the protein could be observed at or near the transition zones and thus is likely to be only partly mislocalized (Figure 4-5F). The GFP-tagged MKS-1 protein was similarly mislocalized in the mksr-1 and mksr-2 mutants (Figures 4-5A and 4­

58); specifically, the protein was less apparent at the transition zones and was found consistently along the dendrites in a manner never observed for any of the

MKS/MKSR proteins in wild-type animals (compare Figures 4-5A and 4-58 to

Figure 4-2C). All analyses were performed at least three times using >50 worms/strain and blind to the genotype and the identity of the GFP-tagged protein.

122 Our data are comparable but not identical to that recently reported by

Williams et al. (2008). Unlike the previous study, which employed the ok2092 allele of mksr-1, we were able to observe at least partial mislocalization of

MKSR-2 in the mksr-1(tm30B3) strain. Taken together, these data further demonstrate that the proper localization of the MKS-1, MKSR-1 and MKSR-2 proteins to transition zones in C. elegans is highly co-dependent, suggesting that they interact directly as a hetero-oligomeric complex or indirectly as part of a larger, functional multi-subunit complex.

4.3.4 The C. e/egans MKS/MKSR proteins do not appear to be essential for transition zone positioning, cilium formation, or 1FT function

To date, the majority (but not all) of C. elegans genes regulated by the

X box-binding DAF-19 transcription factor encode ciliogenic proteins, meaning that their disruption causes cilia structure defects; these include Bardet-Biedl syndrome genes (bbs-1, bbs-7 and bbs-B), and genes encoding core intraflagellar transport (1FT) components (e.g., che-2, osm-5, osm-6 and che-11), that are collectively required for proper ciliogenesis in nematodes (Ansley et aI.,

2003; Swoboda et aI., 2000). Because the three C. elegans mks/mksr genes are

X box-regulated (Figure 4-4A; Blacque et aI., 2005; Efimenko et aI., 2005), and

MKS1 was first shown to be required for ciliogenesis in mammalian cells (Dawe et aI., 2006), we hypothesized that all of the B9 proteins may be required for cilium formation in C. elegans, and perhaps more specifically, regulation of the

1FT process. To investigate these possibilities, we first tested each of the mks-1, mksr-1 and mksr-2 single mutants for an inability to take up a fluorescent dye, a

123 dye-filling (Dyf) phenotype that is observed in all bbs and 1FT gene mutants

(Inglis et aI., 2007). None of the mutant strains display a Dyf phenotype

(Figure 4-6A and Figure 4-7A), suggesting that all possess full-length, environmentally-exposed cilia. To examine the structure of the amphid and phasmid cilia in the mutants directly, we introduced into these strains several

GFP-tagged ciliary markers (the anterograde 1FT motor components OSM-3­ kinesin and the Kinesin-Associated Protein KAP-1, the 1FT protein CHE-2/IFT80 and the dynein light chain component XBX-2 which is essential for driving retrograde 1FT). Consistent with the results of the Dyf assays, each of the ciliary proteins localize correctly to the transition zones and cilia, with no overt accumulations seen in dendrites leading to the transition zones or within the cilia; taken together, the data provide strong evidence that the relative positions of the transition zones, and the ciliary structures, are not different from those of wild­ type animals (Figure 4-68 shows the CHE-2::GFP results; data for OSM-3­ kinesin, KAP-1 and XBX-2 are presented in Figure 4-8A). In addition, all GFP­ tagged proteins appear to display normal intraflagellar transport (1FT); this was confirmed by observing using time-lapse microscopy the expected (wild-type) rates of movement of CHE-2::GFP in the middle (-0.7 ~m/sec) and distal

(-1.2 ~m/sec) segments of the cilia in the single mks/mksr mutant animals

(Figure 4-6C; C. elegans 1FT reviewed in Blacque et aI., 2008; Inglis et aI., 2007;

Scholey, 2008). These data are similarly supported using the OSM-3, KAP-1, and XBX-2 markers (Figure 4-88).

124 The presence of a common B9 protein domain for the three proteins raises the possibility that their functions might be at least partially redundant, where one or more mks/mksr genes may mask the effect of a disruption in another (mutated) mks/mksr gene. To address this possibility, we generated all possible double mutant combinations, as well as the triple mutant. Similar to our observations for each individual mutant, the double and triple mks/mksr mutant strains had no observable defects in dye-filling (Figure 4-7A; the representative triple mutant strain is shown in Figure 4-6A), or in transition zone positioning, ciliary structure, or 1FT, as deduced from observing GFP-tagged CHE-2 and

XBX-2 protein localization and transport behavior in live animals (Figures 4-68 and 4-6C; Figure 4-8).

Because of the apparent phenotypic/genotypic overlap between the

Bardet-Biedl and Meckel syndromes (Karmous-Benailly et aI., 2005; Leitch et aI.,

2008), we sought to test for a possible genetic interaction between the three mks/mksr genes and a bbs gene. We therefore generated a quadruple mutant

(triple mks/mksr mutant in a bbs-B mutant genetic background) harboring a CHE­

2::GFP protein ciliary/1FT marker and looked for cilia structure and 1FT defects.

We found the ciliary structures of the quadruple mutant, based on the CHE­

2::GFP marker, to be indistinguishable from those of the bbs-B mutant alone, with nearly full-length cilia and accumulations at the middle/distal segment midpoint and at the tip of the truncated cilia (Figure 4-6C; see also Blacque et aI., 2004).

By itself, the bbs-B mutation has the effect of separating the 1FT machinery into two independently moving sub-assemblies consisting of Kinesin-II/IFT

125 subcomplex A and OSM-3-kinesin/IFT subcomplex B (Ou et aI., 2005; Ou et aI.,

2007). In the mks-1;mksr-1;mksr-2;bbs-B quadruple mutant strain, the localization and behavior of CHE-2::GFP was indistinguishable from that of the bbs-B mutant, wherein it was transported along the length of the proximal and

(partially truncated) distal segments at the fast unitary rate of OSM-3-kinesin

(-1.2 Jim/sec) (Figure 4-6C).

Recently, Jauregui et al. (2008) demonstrated that abrogation of the transition zone-specific protein NPHP-4 results in relatively subtle yet significant changes to the microtubule architecture of ciliary axonemes. We therefore sought to determine if similar ultrastructural changes were present in a representative

(mks-1;mksr-1) double mutant, which we demonstrate (below) has a lifespan/signaling defect. Consistent with the results of our GFP-based analyses of amphid cilia, transmission electron microscopy (TEM) observation of cross­ sections through the heads of the mks-1;mksr-1 mutants revealed no significant differences in the axonemal structures of amphid cilia compared to wild-type worms (Figure 4-9). Specifically, the double mutant exhibited well-formed transition zones, intact doublet microtubules in the middle segments, and intact singlet microtubules in the distal segments.

In contrast, we observed that disruption of the mammalian MKSR1/B9D1 and MKSR2/B9D2 genes by RNA interference in IMCD3 cells causes ciliogenesis defects, namely a smaller proportion of ciliated cells compared to the control cells (Figure 4-60). These results are similar to what was observed for the knockdown of the MKS1 gene (Dawe et aI., 2007). Hence, it appears that in

126 C. elegans, disruption of mks/mksr genes is less detrimental to cilium formation until a 'second-hit' mutation in nphp-4 or nphp-1 occurs, where transition zone/basal body and ciliogenesis defects become clearly apparent (Williams et aI., 2008).

Altogether, our data indicate that in contrast to the mammalian B9 domain- containing proteins, the C. elegans counterparts are unlikely to be directly implicated in the positioning of transition zones/basal bodies, or the biogenesis of sensory cilia, or play an essential role in intraflagellar transport. This raises the possibility that the C. elegans MKS/MKSR proteins participate in other aspect(s) of basal body and/or cilia function that relate not to the building of, but rather, the sensory/signaling roles of the ciliary organelle.

4.3.5 The C. e/egans mks/mksr genes control lifespan via the insulin signalling pathway

To investigate the possibility that the C. elegans MKS/MKSR proteins play roles in the sensory functions of cilia, we first tested the three single mks/mksr mutant strains for defects in their ability to sense and move towards a volatile attractant (isoamyl alcohol) using an established chemotaxis assay. Although bbs and 1FT mutant animals show clear sensory phenotypes in these assays

(Blacque et aI., 2004; Inglis et aI., 2007; Perkins et aI., 1986), no significant differences were observed between wild-type (N2) animals and the single

mks/mksrgene mutants (Figure 4-6E, light gray bars). Similarly, we assayed for the ability of the mks/mksr mutant animals to recognize and avoid a solution of

high osmolarity (8M glycerol). In this assay, wherein bbs and 1FT gene mutants

127 show osmoavoidance defects (Inglis et aI., 2007; Perkins et aI., 1986; unpublished data), the mks/mksr mutant strains exhibited wild-type osmoavoidance (Figure 4-6E, dark gray bars). Given the possibility of functional overlap between the mks/mksr genes, we also tested combinations of the double and triple mks/mksr mutant animals; no defects in chemotaxis or osmoavoidance were observed (Figure 4-6E). Likewise, another assay that can reveal cilia function defects in the nematode, namely Nile Red staining to determine lipid content (Li et aI., 2008; Mak et aI., 2006), revealed no differences between control animals and the mks-1, mksr-2 and mks-1;mksr-2 mutants (Figure 4-78).

These data are reminiscent of other C. elegans transition zone/basal body proteins whose deletion does not overtly/severely affect cilium formation or 1FT, but may instead still be required for specific sensory and/or signaling capacities of cilia. Two examples are the homologs of the nephronophthisis-associated

NPHP-1 and NPHP-4 proteins. Their disruption causes subtle ciliary structure anomalies, as well as male mating defects and an increased lifespan phenotype, the latter two often being associated with cilia dysfunction (Jauregui et aI., 2005;

Winkelbauer et aI., 2005; Wolf et aI., 2005). Similarly, disruption of IFTA-2, a protein associated with the 1FT machinery, does not appear to result in cilia structure or 1FT defects but causes an increased lifespan phenotype (Schafer et al.,2006).

We therefore tested all single and double mks/mksr mutants, as well as the triple mutant, in ageing assays. None of these mutants display alterations to lifespan compared to wild-type animals (Figure 4-10A and 4-108). In contrast,

128 all three double mutant combinations (mks-1;mksr-1, mks-1;mksr-2 and mksr-1;mksr-2) display statistically significant increases in lifespan when compared to the single or triple mutants or wild-type animals (Figure 4-108).

Interestingly, the lifespan increases seen in the mkslmksr double mutants are comparable to those of the ifta-2 (data not shown; see Schafer et aI., 2006) and nphp-1 or nphp-4 (Figure 4-10C; see also Winkelbauer et aI., 2005) ciliary mutants, but are not as pronounced as those exhibited by some 1FT gene mutants, such as che-11 (Figure 4-10C; see also Apfeld and Kenyon, 1999).

Thus, our genetic and functional analyses of the mkslmksr genes suggest that similar to the nephrocystin proteins NPHP-1 and NPHP-4, which localize to the transition zones at the base of cilia, the MKS/MKSR proteins perform function(s) relevant to longevity control, suggesting a role in cilia-associated signaling.

To support this hypothesis, we next tested for an epistatic interaction between a representative double mutant (mksr-1;mksr-2) and daf-2(e1370). The daf-2 gene encodes the lone receptor responsible for the well-established insulin/lGF-1 signaling pathway that regulates longevity in C. elegans (Kenyon et aI., 1993; Kimura et aI., 1997). Lifespan assays demonstrate that the mksr-1;mksr-2;daf-2 triple mutant exhibits the same longevity as that of the extremely long-lived daf-2 mutant (Figure 4-11 A), implying that the 89 proteins function upstream of DAF-2 in the insulin-signaling pathway_ To confirm this result, we tested the epistatic relationships between all three mks/mksr double mutants and daf-16(mu86) , the gene encoding the FOXO transcription factor required for downstream DAF-2 insulin/lGF-1 receptor-mediated signaling (Ogg et

129 aI., 1997). All mks/mksr double gene mutants, when combined by genetic crossing with the daf-16 mutation, display lifespans that are shorter than the mks/mksr double mutants and are indistinguishable from that of the short-lived daf-16 mutant (Figure 4-11 B). These data support the notion that the 89 proteins function upstream of DAF-16 in the insulin/IGF-I signaling pathway.

Consistent with the above observations, we observed a direct effect of disrupting the 89 proteins and the activity of the DAF-16 protein. Within a population of worms at 20°C, DAF-16 is usually phosphorylated in response to

DAF-2 signaling, which leads to its nearly complete exclusion from the nucleus; however, in long-lived mutants such as daf-2(e1370) or cilia mutants such as osm-5, the proportion of animals showing nuclear localization increases significantly as a result of inhibited daf-2 signaling (reviewed in Mukhopadhyay et aI., 2006). We observed that GFP-tagged DAF-16 in control animals normally shows approximately 0.8% localization to the nucleus, similar to that of the mks-1 and mksr-1 single mutant animals, which have a normal lifespan (Figure 4-11C).

In contrast, the mks-1;mksr-1, mks-1;mksr-2 and mksr-1;mksr-2 double mutants all display a statistically significant increase in GFP-tagged DAF-16 nuclear localization (4-4.8%), and a corresponding decrease in cytosolic localization, as expected from their increased lifespan phenotypes (Figure 4-11 C).

In conclusion, our findings strongly support the notion that the C. elegans

MKS/MKSR proteins functionally interact at the base of cilia to support a process that is required upstream of the DAF-2/DAF-16 insulin/IGF-I signaling pathway to specify longevity.

130 4.4 Discussion

A wealth of genomic, transcriptomic and proteomic data have identified numerous basal body and ciliary proteins, several of which are implicated in ciliopathies (Gherman et aI., 2006; Inglis et aI., 2006; Keller et aI., 2005). In this study, we report that 89 domain-containing proteins uncovered in some of these studies belong to three separate phylogenetic c1ades-MKS-1, MKSR-1 and

MKSR-2-that are found exclusively in ciliated species. The three human proteins and C. elegans orthologs localize to basal bodies/transition zones, and, in the case of the mammalian proteins, to the ciliary axoneme as well. We further demonstrate that the C. elegans proteins function cooperatively at the base of cilia to support the proper function of the insulin/IGF-I signal transduction pathway required for the specification of lifespan.

4.4.1 Evolutionary conservation of B9 domain-containing proteins in ciliated organisms

The potential significance of the 89 protein domain was highlighted recently when one of the first two genes linked to Meckel syndrome, MKS1, was identified by Kyttala and colleagues (2006). The 559-amino acid human MKS1 protein lacks known protein motifs(s) or other recognizable features, but harbors an approximately 115-amino acid 89 protein domain of unknown function (Figure

4-1 B). We identified in nearly all ciliated organisms two more members of the

MKS1/89 protein family, which we name MKS1-related proteins 1 and 2 (MKSR1 and MKSR2). The mutual presence of three 89 domain-containing proteins in ciliated organisms, and complete absence from organisms devoid of cilia (Figure

131 4-1A and Table 4-1), suggests that these proteins may perform common cilia­ associated function(s). It is notable that the MKS/MKSR proteins are absent from at least three groups of organisms that possess cilia (the moss Physcomitrella patens, he Diplomonad Giardia lamblia, and the Apicomplexan Plasmodium fa/ciparum). Interestingly, G. lamblia has a much reduced complement of Bardet­

Biedl syndrome proteins compared to most ciliated organisms, and P. falciparum lacks 1FT and BBS proteins altogether. These observations suggest that at least

in some organisms, the MKS/MKSR proteins, similar to the BBS proteins, are not absolutely required to build motile or non-motile cilia. Also suggested is that the

MKS/MKSR proteins may play more specific roles relating to the sensory and/or signaling functions of cilia.

4.4.2 MKS1, MKSR1 and MKSR2 are associated with basal bodies and cilia

Human MKS1 was previously found to localize to basal bodies (Dawe et

al., 2007), as with the Chlamydomonas ortholog POC12 (Proteome of Centriole

protein 12; Keller et aL, 2005). We have confirmed the basal body/centrosomal

localization of a transiently-expressed, V5 epitope-tagged version of MKS1

(Figure 4-2A; Figure 4-3), and further show the same subcellular localization for

the other two human B9 domain-containing proteins (Figure 4-2A and Figure 4­

3). In addit.ion, we found that GFP-tagged variants of all B9 domain-containing

proteins localize; in stably-transfected cells, to the ciliary axoneme (Figure 4-28).

This dual-localization pattern (basal body/cilia) is comparable to that of the

mouse protein stumpy (MKSR-2; Town et aL, 2008; Breunig et aL, 2008).

Importantly, we confirmed that the localization of the MKS/MKSR proteins to

132 basal bodies is evolutionarily conserved by observing-as recently reported by

Williams et al. (2008)-that the C. e/egans MKS-1, MKSR-1 and MKSR-2 proteins reside at ciliary transition zones (Figure 4-2C), which are akin to basal bodies (Perkins et aI., 1986). The MKS/MKSR protein family therefore joins an increasing number of ciliopathy-associated proteins that concentrate at the base of cilia or to the ciliary axoneme, including among others the Meckel syndrome/Joubert syndrome/nephrocystin proteins RPGRIP1 L, CEP290/NPHP6,

NPHP1 and NPHP4.

4.4.3 Function of the MKS/MKSR proteins in ciliogenesis and cilium­ associated signalling

The notion that MKS1 is implicated in cilia function received strong support from a recent study by Dawe et al. (2007), who demonstrated that knockdown of mammalian MKS1, similar to the disruption of MKS3, is associated with basal body positioning and thus ciliogenesis defects. The reason for the basal body position phenotype remains unclear, but the downstream effects on renal tubule formation were found to be pronounced. Similarly, very little is known about the functions of the other two B9 domain-containing proteins, MKSR1 and

MKSR2. Ponsard et al. (2007) showed that knockdown of ICIS-1 (ortholog of

C. elegans mksr-2) by RNA interference in Paramecium tetraurelia results in defects in cilia stability or formation. Another recent study demonstrated that stumpy (MKSR-2) is required for the proper biogenesis or morphology of cilia in a mouse knockout model (Town et aI., 2008), and is essential for a Shh-based signaling pathway in neural stem cells (Breunig et aI., 2008). Another recent

133 study, by the Yoder laboratory (Williams et aI., 2008), revealed genetic interactions between the 89 domain genes and the nphp-4 gene (discussed further below).

Using C. elegans to dissect the relationship between, and functions of, the

MKS/MKSR proteins, we discovered that the proper localization of the proteins was largely co-dependent. Specifically, in mksr-1 mutant animals, the MKS-1 and

MKSR-2 proteins do not reproducibly show clear, wild-type-like localization to the transition zones, and display accumulations in the dendrites (Figures 4-5A and

4-5F). Similarly, in the mksr-2 mutant strain, the MKS-1 and MKSR-1 proteins are also not tightly associated with the transition zones (Figures 4-58 and 4-50).

Nevertheless, some partial localization to, or near, transition zones is often observed, suggesting either that the disrupted genes encode aberrant truncated proteins that retain some functionality or that the proteins can localize independently of each other, but with lower efficiency. This may be particularly true in the case of the mks-1 mutant, where we observe essentially wild-type localization of MKSR-1 and MKSR-2 proteins (Figures 4-5C and 4-5E).

Altogether, these data, which are comparable to that recently reported in

Williams et al. (2008), suggest that the three 89 domain-containing proteins either interact directly, such that the disruption of one can lead to the

mislocalization of the other, or that they are functionally associated via other transition zone-localized proteins. We favour the first possibility, given that a genome-wide C. elegans study uncovered a yeast-2-hybrid interaction between

K03E6.4 (MKSR-1) and Y38F2AL.2 (MKSR-2) (Li et aI., 2004b). The

134 interdependent localization of the C. e/egans MKS/MKSR proteins provides evidence of a functional relationship between this novel family of proteins.

Interestingly, we found that the mks-1, mksr-1 and mksr-2 mutant strains have no obvious defects in transition zone positioning, ciliary structures, intraflagellar transport, chemo- and osmo-sensation, or lipid accumulation

(Figure 4-6A-C, E; Figures 4-7, 4-8, 4-9). Because of the possibility that the mkslmksr gene functions are partly redundant, we generated the three double mutant combinations as well as a triple mutant, and tested them for the same ciliary phenotypes; we could not, however detect any difference from wild-type animals in these assays (Figure 4-6A-C, E; Figures 4-7, 4-8, 4-9). It is unclear whether the gene deletion mutants available for these studies represent hypomorphs instead of nulls, although the conserved B9 domains of the mksr­

1(tm3083) and mksr-2(tm2452) gene mutants are disrupted (Figure 4-4) and likely disrupt the function of the proteins. Nevertheless, all mutant alleles give an observable longevity phenotype when combined (see below), and we contend that the simultaneous disruption of the three mks/mksr genes is highly likely to impair their collective functions. In light of our data, it seems possible or even likely that unlike in mammalian cells (Dawe et aI., 2007; Town et aI., 2008;

Figure 4-60), in C. e/egans, the MKS/MKSR proteins do not perform an essential ciliogenic role (see also Williams et aI., 2008).

However, the possibility remained that the C. elegans mkslmksr genes are specifically implicated in one or more ciliary signaling pathways; we were able to test for this possibility in the absence of potentially confounding major cilia

135 structure defects by analysing the strains for an increased lifespan phenotype that is typical of ciliary gene mutants, including nphp-1, nphp-4, ifta-2, and most core 1FT-associated mutants (Apfeld and Kenyon, 1999; Schafer et aI., 2006;

Winkelbauer et aI., 2005). Although none of the single mks/mksr mutants differed from wild-type animals (Figure 4-10A), all double mutant combinations

(mks-1;mksr-1, mks-1;mksr-2, and mksr-1;mksr-2) displayed statistically significant expansions in lifespan (Figure 4-108). This is reminiscent of the single nphp-1 or nphp-4 mutants, which show no overt cilia-dependent male mating defects, whereas the nphp-1;nphp-4 double mutant animals possess pronounced mating defects (Jauregui and Barr, 2005). Also of note, the enhanced lifespan of mks/mksr double mutants is comparable to that observed when the nphp-1 and nphp-4 genes are individually disrupted, but less than when ciliary structures are severely abrogated (e.g., as in a che-11 mutant; Figure 4­

10C).

It is intriguing that lifespan phenotypes are observed in double but not single mks/mksr mutants given that the abrogation of either MKSR-1 or MKSR-2 causes the improper localization of the others (Figure 4-5). However, our data indicate that in most cases, the co-dependency of localization reflects an effect on the efficiency of localization rather than an essential aspect of correct trafficking (Figure 4-5); thus, at least partial function may be retained in the incorrectly-localized proteins. Nevertheless, it remains perplexing that the mks-1;mksr-1;mksr-2 triple mutant does not exhibit a longevity phenotype.

Although it is unclear why abrogation of the third mks/mksr gene restores normal

136 lifespan, it provides further evidence of a functional (genetic) interaction between all three mks/mksr genes, since the triple mutant phenotype is reproducibly distinct from that of the three double mutant phenotypes. It may also point to a complex regulation-which likely includes NPHP-1 and NPHP-4 proteins

(Williams et aI., 2008)-of the various ciliary signals regulating lifespan at the transition zone.

Longevity in C. elegans is specifically controlled by cilia-dependent sensory inputs and the insulinllGF-1 signaling pathway (Apfeld and Kenyon,

1999). We show by epistasis analyses that this is also the case for the

MKS/MKSR proteins, which appear to function upstream of both the insulin/IGF-I receptor DAF-2 and the FOXO transcription factor (DAF-16), which transduces insulin-like signals to regulate lifespan (Figure 4-11). But what are the function(s) of the MKS/MKSR proteins in this pathway? Given our results, it is possible that components of the insulinllGF-1 signaling cascade might be improperly trafficked in the mks/mksr double mutants; given that the MKS/MKSR proteins localize at the base of cilia in C. elegans, they may cooperate with other basal body proteins

(such as NPHP-1, NPHP-4 or Meckel syndrome-associated RPGRIP1 L, for which there is a C. elegans ortholog, C09G5.8) in vesicle/protein trafficking (or docking at the base of cilia) prior to incorporation into cilia (e.g., by intraflagellar transport).

Although the functions of the MKS/MKSR proteins appear to not be critical for ciliogenesis in C. elegans (and perhaps other organisms), they are essential for establishing important signaling cascade(s). Such a 'non-essential' function

137 may explain why some ciliated organisms, such as Plasmodium and Giardia, lack the MKS/MKSR proteins altogether. In other systems, for example mammalian cells, the MKS/MKSR proteins may carry out similar trafficking/docking roles at basal bodies, but perhaps functional interaction(s) with their cargo or binding partners are absolutely necessary for establishing the positioning of the basal body at the membrane (Dawe et aI., 2007), and thus, ciliogenesis. Once cilia formation has occurred in normal (wild-type) situations, the mammalian

MKS/MKSR proteins may then also be necessary for the targeting/trafficking of ciliary cargo required for the sensory/signaling functions of the primary cilia (e.g., sonic hedgdhog and perhaps other signaling pathways; Breunig et aI., 2008). In

C. elegans, disruption of an MKS/MKSR protein together with the NPHP-1 or

NPHP-4 protein causes essentially the same defects in basal body

positioning/ciliogenesis (Williams et aI., 2008), potentially suggesting greater functional redundancy in the nematode ciliary system. This is particularly

insightful, given how the NPHP-1 and NPHP-4 proteins appear to be important

regulators for the proper localization/function of the IFT/BBS ciliary proteins

(Jauregui et aI., 2008).

4.4.4 MKSR·1 and MKSR·2 as potential Meckel syndrome gene candidates

Our functional data strongly suggest that in addition to the five presently

known Meckel syndrome-associated genes, namely MKS1, MKS3,

Cep290INPHP6IMKS4, RPGRIP1UMKS5, and CC2D2A, the two MKS1-related

genes MKSR1 and MKSR2 are excellent Meckel syndrome, Joubert syndrome

and Leber congenital amaurosis gene candidates. The genetic locations of

138 MKSR1 (8901; 17p11.2) and MKSR2 (8902; 19q13.2d) are clearly outside of the uncloned MKS2 locus, which has been mapped to chromosome 11 q13 by

Roume et al. (1998); thus, MKSR1 and MKSR2 may represent additional disease loci.

4.5 Concluding Remarks

Understanding the roles of basal bodies and ciliary axonemes in various sensory and signaling processes, and their implications in various ciliopathies, will require the identification and analysis of key, conserved components of the basal body-ciliary organelle. Here, we have uncovered and characterized three

89 domain-containing proteins, MKS-1, MKSR-1 and MKSR-2, as a family of basal body/ciliary components whose functional interactions are required for proper ciliary function/signaling in C. elegans, and are needed for ciliogenesis in mammalian cells. Further analyses of these proteins in C. elegans and other model organisms, as well as exploration of their possible involvement in cilia­ associated diseases, will provide important insights into the physiological functions of cilia and the mo.lecular etiologies of ciliopathies such as Meckel syndrome.

4.6 Materials and Methods

4.6.1 C. e/egans strains and genetic crosses

All strains were maintained and cultured at 20°C. Strains carrying deletions in the C. elegans mks-1/xbx-7, mksr-1/tza-2, mksr-2/tza-1 genes,

R148.1(tm2705), K03E6.4(tm3083) and Y38F2AL.2(tm2452) respectively, were

139 obtained from the National Bioresource Project

(http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) and outcrossed to wild-type (N2) at least 5X. Standard mating procedures were employed to introduce GFP- tagged protein constructs into different genetic backgrounds and to make the different combinations of double mutants or the triple mutant. Single-worm PCR reactions were used to genotype the three mks/mksr mutants. The other strains used were bbs-8(nx77), nphp-1(ok500), nphp-4(tm925), che-11(e1810), osm-5(p813), daf-2(e1370), and daf-16(mu86).

4.6.2 Characterisation of the C. e/egans mks-1, mksr-1, and mksr-2 alleles

N2 and mks/mksr cDNAs were initially isolated by RT-PCR. Briefly, following suspension of worms in Trizol Reagent (Invitrogen) and purification with

RNeasy (Qiagen), first-strand cDNAs were generated using the Superscript First-

Strand Synthesis System for RT-PCR (Invitrogen). PCR amplifications specific to each mks/mksr transcript were then performed to isolate appropriate double- stranded cDNA sequences. PCR products were then incorporated into the pGEM-T Easy Vector (Promega) and sequenced.

4.6.3 Subcellular localisation of the human and C. e/egans MKS-1, MKSR-1 and MKSR-2 proteins

To assess the localization of the three human B9 proteins using transient expression ofV5-tagged proteins, the respective cDNAs (MKS1/BC010061.2,

EPPB9/BC002944.2/MKSR1, and LOC80776/NM_030578.2/MKSR2 (Invitrogen

Ultimate ORF clones IOH12254, IOH5726, and IOH4997, respectively) were cloned into the mammalian Gateway pcDNA6.2/c-Lumio vector (Invitrogen),

140 which contains a C-terminal V5 epitope tag. Murine IMCD3 cells were plated on glass coverslips and transfected with both the expression vector and a pUC12­

TAG tRNA suppressor (Invitrogen) at 70% confluency using FuGENE6 (Roche).

48 hours post-transfection, cells were subjected to immunofluorescence analysis

(below). To generate stable IMCD3 cell lines harboring GFP-tagged MKS1,

MKSR1/B9D1 and MKSR2/B9D2, cells were transfected with each GFP-fusion construct, split 1:5 after 24 hours and grown 24 hours later in the presence of geneticin (750 f.lg/ml); cells were kept under selection for 7 days, after which they were split 1:10 and several colonies were picked to produce independent lines.

Cells were grown to confluency and maintained at least 48 hours before immunofluorescence analysis.

For immunofluorescence analysis, cells were fixed in PFA at 4°C for 1

hour, and subsequently fixed in ince-cold methanol at -20°C for 10 min. After

rinsing with PBS, cells were permeabilized using 0.1 % Triton-X (American

Bioanalytical) in PBS (10 min), washed, and blocked with 5.5% FBS (Gemini) for

1 hour. Cells were stained with primary antibodies at 4°C overnight (rabbit anti­

GFP, 1:1000, Invitrogen A11122; mouse anti-y-tubulin, 1:1000, Sigma T6557;

goat anti-acetylated-tubulin, 1:1000, Sigma), washed in PBS, then incubated with

secondary antibodies (goat anti-mouse (A21206) and donkey anti-rabbit IgG

conjugated to Alexa 488 (A11001) or 594 (A11005), 1: 1000, all Invitrogen). Cells

where then incubated with DAPI (1 :5000,500 mg/ml stock) at 25°C for 10 min,

washed, and mounted with VECTASHIELD. Images were recorded using an

epifluorescence microcope at 64X magnification.

141 For the C. elegans MKS-1, MKSR-1 and MKSR-2 proteins, we generated translational constructs containing the natural promoter of each gene and the entire coding region fused in-frame to EGFP, and generated transgenic lines harboring these constructs as reported previously (Blacque et aI., 2004). The subcellular localization of the GFP-tagged proteins was assessed by fluorescence microscopy in either wild-type (N2) animals or in the indicated mks/mksr mutant backgrounds. Mislocalization phenotypes were assayed blind to the genotype and expressed GFP-tagged MKS/MKSR protein, on at least 50 different animals for each strain.

4.6.4 Mammalian RNA interference

Mouse IMC03 cells were grown in a 10 em dish and co-transfected with short-hairpin constructs for either murine MKSR1/B901 or MKSR2/B902, and

GFP as a transfection control using Fugene6 (INVITROGEN) as directed. After

48-72 hours, the cells were fixed in PFA and co-stained with anti-acetylated tubulin and anti-GFP. The cells expressing GFP, which co-expressed the short­ hairpins, were then scored for the percentage of cilia in comparison with cells expressing GFP alone. The MKSR1/B901 short-hairpin construct was purchased from Open BioSystems -The RNAi Consortium (TRC), TRC-Mm1.0 (Mouse), the target sequence for which is not published. The B901 short-hairpin construct is expressed from the p.KLO.1 vector and has the following TRC 10:

TRCN0000198329. The target sequence for MKRSR2/B902 used in the pSuper­

Basic vector (Oligoengine Cat#VEC-PBS-0001/0002) is:

GAACAGTTGGCACGGGCTT. The primers designed for cloning of the short-

142 hairpin into pSuper.basic are: 5'-

GATCCCCGAACAGTTGGCACGGGCTTTTCAAGAGAAAGCCCGTGCCAACTG

TTCTTTTTA-3' and 3'-

GGGCTTGTCAACCGTGCCCGAAAAGTTCTCTTTCGGGCACGGTTGACAAGA

AAAATTCGA-5'.

4.6.5 Phenotypic assays for ciliary structure, chemosensation and lipid content

Chemotaxis assays using isoamyl alcohol as an attractant were performed

as described (Blacque et aI., 2006). Osmoavoidance assays were performed

essential as described (Culotti and Russell, 1978) using a ring of 8 M glycerol as

the source of high osmolarity. The filling of environmentally-exposed ciliated

sensory neurons with Oil was assessed as described in Blacque et al. (2004).

Lipid content measurements were performed essentially as described using Nile

Red (Mak et aI., 2006). Experiments were done blind to the genotype and

repeated at least three times.

4.6.6 Lifespan assays

Lifespan assays were similar to that described in Apfeld and Kenyon

(1999). Animals were grown for one generation at 20°C before eggs were

collected by treatment with sodium hypochlorite. At the L4 molt, worms were

transferred to NGM plates containing 16 Ilm FUOR to prevent progeny growth

and kept at 20°C for the duration of the assay. 100 worms were picked for each

of the indicated strains, with 10 worms on each plate. Plates were scored every

1-2 days for live/dead worms. Individual animals were considered dead when

143 they no longer responded to harsh touch (prodding with platinum wire). Worms

that exploded or crawled off the plate were censored. All assays were performed

at least twice with consistent results.

4.6.7 DAF-16 nuclear localisation analyses

OAF-16 localization assays were performed essentially as described in

Schafer et al. (2006). Briefly, the various mks/mksr single or double mutant

strains were crossed into the integrated OAF-16::GFP strain (TJ356;

N2(zls356)[OAF-16::GFP+pRF4(rol-6)]). To observe the localization, healthy,

.well-fed worms grown at 20°C were mounted on an agar pad containing 20 mM

sodium azide and quantified immediately. To quantify localization, OAF-16::GFP

was determined to be either nuclear, intermediate or cytosolic as reported (Oh et

al. 2005). Control animals (TJ356 strain) were assayed in parallel. For each

strain analyzed, at least 200 worms were assayed in triplicate.

4.6.8 Intraflagellar transport assays

Transgenic animals harboring GFP-tagged proteins were mounted on 1%

agarose pads and immobilized with 100 mM levamisole. Amphid and phasmid

cilia were examined with a 1OOX, 1.35 NA objective and an ORCA AG CCO

camera mounted on an Zeiss Axioskop 2 mot plus microscope, with time-lapse

images being acquired at 300-500 msec/frame, depending on the specific marker

used. Images and movies were obtained in Openlab version 5.02 (Improvision).

Kymographs were generated using the MultipleKymograph ImageJ plug-in

(http://www.embl-heidelberg.de/eamnet/html/body_kymograph.html). Rates from

144 middle and distal segments were obtained essentially as described in Ou et al.

(2007).

4.6.9 Bioinformatic and phylogenetic analyses

B9 domain-containing proteins were identified using the NCBI Conserved

Domain Database (Marchler-Bauer et aI., 2005) with human MKS1 as query.

This dataset was simplified by removal of duplicate sequences from species not of interest. Sequences from species not shown here (but retrieved by the COD) were manually removed. Sequences from additional species were identified and/or confirmed using BLAST (Altschul et aI., 1997) with sequences from species closely-related on the eukarytotic tree (as defined by Keeling et aI.,

2005) as queries at the species' individual genome sites. Sequences were retained only if reciprocal BLAST identified B9 domain-containing proteins as the top hits. Bayesian analysis of phylogenies was carried out using MrBayes 3.1.2

(Huelsenbeck and Ronquist, 2001) as previously described (Parker et aI., 2007), except that whole protein sequences were aligned using the Muscle algorithm

(Edgar, 2004). Trees were visualized with TreeView (Page, 1996) or

Phylodrendrum (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html).An additional analysis (not shown) was carried out on a slightly different protein dataset using the neighbor-joining algorithm of ClustalW (Higgins et aI., 1994).

4.7 Acknowledgements

We would like to thank the C. elegans Genetics Center (CGC) and Shohei

Mitani (National BioResource Project, University of Tokyo, Japan) for providing

145 C. elegans strains used in this study, and the WestGrid computer cluster for phylogenetic analyses. This research is funded by grants from the March of

Dimes (M.R.L.), NSERC (L.M.Q.), grant R01 HD04260 from the National Institute of Child Health and Development, R01 DK072301 and R01 DK075972 from the

National Institute of Diabetes, and Digestive and Kidney Disorders (N.K). M.R.L. holds scholar awards from Canadian Institutes of Health Research and Michael

Smith Foundation for Health Research (MSFHR). E.E.D. acknowledges an

NRSA fellowship (F32 DK079541-01) and a doctoral fellowship from the Visual

Neuroscience Training Program (National Eye Institute). P.N.1. and M.P.H. acknowledge MSFHR scholarships; P.N.1. also holds an NSERC doctoral research award.

146 4.8 Figures

Figure 4-1. Phylogenetic analysis showing that B9-domain-containing proteins from ciliated organisms belong to a family of proteins consisting of three clades or family members, namely Meckel Syndrome 1 protein (MKS-1), MKS-1-related protein 1 (MKSR-1) and MKS-1-related protein 2 (MKSR-2), and sequence comparisons of different B9 domains. (A) Phylogenetic tree of B9-domain-containing proteins from several diverse ciliated eukaryotes. Support for nodes (posterior probabilities) are indicated by blue (1.0), green (>0.9) or red (>0.8) circles. The MKS-1, MKSR-1 and MKSR-2 protein families are shown in blue, green and red, respectively. Scale bar denotes 0.1 substitutions per site. Bd, Batrachochytrium dendrobatidis; Ce,

Caenorhabditis elegans; Cb, Caenorhabditis briggsae; Cr, Chlamydomonas reinhardtii; Om, Drosophila me/anogaster, Dr, Danio rerio; Hs, Homo sapiens;

Mm, Mus musculus; Sp, Strongylocentrotus purpuratus; Tb, Trypanosoma brucei; Thaps, Thalassiosira pseudonana; XI, Xenopus laevis. Table 4-1 provides the full listing of proteins (with accession numbers) considered in the analysis. (B) Multiple amino acid sequence alignment of 22 B9 protein domains from eight different species. MKS-1, MKSR-1 and MKSR-2 protein sequence names are colored blue, green and red, respectively. Dark blue highlights signify sequences that match the consensus sequence. Light blue sequences do not match the consensus sequence, but have a positive Blosum62 score.

Conservation values for each residue are calculated based on identities and conserved physicochemical properties between different amino acid residues in

147 each column. Quality is a measurement of the likelihood of mutations in each column; lower quality signifies greater likelihood of mutations, if any, present between sequences. Species abbreviations are as above except for Tt,

Tetrahymena thermophila. [Note: Phylogenetic analysis (A) performed by J.

Parker & L. Quarmby; sequence analysis (8) by N. Katsanis]

A MKS-l

MKSR-l

CeR148 , : C9/{O::'lE:."64 Cb P06737 Tb 1061 2290":,:":::-=::===::~,-~.-J--'-~r:::""--"------Dm CG1S/JO l)n\ CG14670

0.1 subslitutions/site • 1.0 >0.9 E • o • >0.8 MKSR-2

B 120 ~'S 99{)" fJ~ SSO' .xl MG:'e.J~44 C, B9h.~t Cr MM1.lI.f "'1 E* t.l""'l B9D2 Cr lXol3oo.,r XI '.~GCl1iS86 Cr lli)i4 Cr '8l~J.{ eM CC92:i:7 Ct Y38F~:'L 2 to! MK$1 '.ImMK$l Xl ~.4e".lIeltl Cr MKSl k.t -, X~)J~SJ +f )02'91') Ce K~3E~ol C. ill431 Cf" C:;'S':"3C

Residue does 001 match the consensus resKiue. and the t\'K) reslcues have a negatwe Blosum62 scole. Residue does no! match me consensus residue but the two resMjues ha',e aoosilive BIosum62 score Residue matches the consensus residue

148 Figure 4-2. MKS-1, MKSR-1 and MKSR-2 proteins localize to centrosomes or basal bodies in human cells and transition zones in C. e/egans. (A,B) IMCD3 cells transiently expressing constructs encoding V5 epitope-tagged human MKS1, MKSR1 (EPPB9/B9D1) and MKSR2 (LOC80776/B9D2), are shown in the top, middle and bottom panels, respectively. In ciliated IMCD3 cells

(A), the V5-tagged MKS1, MKSR1 and MKSR2 proteins (red) colocalize with the centrosomal y-tubulin marker (green), as seen in the merged images (yellow denotes overlap in signals). Cilia are labeled with an antibody against acetylated tubulin (also green). In ciliated IMCD3 cells stably expressing GFP-tagged versions of the MKS1, MKSR1 and MKSR2 proteins (green) (B), colocalization is observed with the acetylated tubulin antibody (red), which highlights the ciliary axoneme. Arrows denote centrosomes or basal bodies; brackets show the ciliary axoneme. (C) GFP-tagged C. e/egans MKS-1, MKSR-1 and MKSR-2 localize specifically to transition zones (akin to basal bodies) in ciliated sensory neurons.

Transition zone staining near the tip of the head (left panels) at the base of amphid cilia and near the tail of the animal at the base of phasmid cilia (right panels) are indicated with arrowheads and are shown enlarged in the insets. The relative positions of cilia, transition zones and dendrites are shown in some of the images. Scale bar: 5 IJm (insets magnified x3). [Note: (A), (B) performed by J.

Robinson, E. Davis, and N. Katsanis]

149 A V5-tagged proteins (transient) B ----'G_F_p_--'-'ta-'<9""9'-'-e...;..d...... p_r-'-'ot_e_in-'-'s_('-'s_ta_b_'e----'e--'xp,-,-r_e_s_si-,-,o_n,-) _ merge with acet-tubulin GFP merge ~IiIYII ~

c head tail

150 Figure 4-3. V5-tagged human MKS1, MKSR1 or MKSR2 transiently expressed in non-ciliated IMCD3 cells colocalizes with v-tubulin. V5-tagged human MKS1, MKSR1 or MKSR2 transiently expressed in non- ciliated IMCD3 cells (stained red) colocalizes with y-tubulin, a centriolar and centrosomal marker (green), showing overlap in signals (yellow) in the merged image. Arrows indicate centrosomes. [Note: All data in this figure from J.

Robinson, E. Davis, and N. Katsanis]

V5-tagged proteins (t ansient expresslo,nj

151 Figure 4-4. Mutations in the C. e/egans mks-1, mksr-1 and mksr-2 genes and analysis of the corresponding transcripts. (A) C. elegans strains used in this study harbor the indicated mutations in the mks-1 (xbx-7/R148.1), mksr-1 (tza-2/K03E6.4) and mksr-2 (tza-1/Y38F2AL.2) genes. The positions and sequences of X-box regulatory regions are indicated, exons are shown as gray boxes, and the deleted regions are highlighted by dashed boxes along with their allele designations (tm2705; tm3083; tm2452). (8)

RT-PCR analysis of wild-type (N2) and mks/mksr mutant transcripts by sequencing. Coding regions for the indicated proteins are shown as lines and the respective 89 domains as a blue box. The predicted MKS-1 protein in the mks-

1(tm2705) mutant is missing residues 68-141, which results in a truncated N- terminal region but does not appear to affect the 89 domain. The predicted

MKSR-1 mutant protein (in the tm3083 strain) has residues 78-94 of the wild-type protein replaced by 18 distinct amino acids, resulting from improper splicing of

intron 2 with exon 3 between nucleotides 1032-1057 and 1276. This produces a

89 domain replaced with an entirely different sequence containing an extra

residue. The predicted MKSR-2 mutant protein (in the tm2452 allele) is encoded

by a transcript that results from the fusion of exon 1 with the 5' of the truncated

exon 3, resulting in a peptide that is missing amino acids 30-78, all of which fall within the predicted 89 domain (the deletion in the 89 domain is denoted by a

squiggle).

152 A x box (-68 bp) 500bp GTCACCATAGGAAC••. tm2705 (deletlo ~ R148.1b , ', (mkS-1: lox-7) 5' R exon

X box (-83 bpj: GTICCCTTGGCAAC 200 bp K03E6 4 -~+L.nl------I"I:::=:J (mksr-1: tza-2) ••----f,-"'"' _

X box ({IS bp)· GTTGCCGTGGCAAC • Im2452 (delelton) 200 bp Y38F2AL.2 +------+-- -tII::J..------c> (mksr-2: tza-1) 1 .1

• B9domain

8 MKS-1 tv2

tm2705

MKSR· tv2 -~--- tm3083 ---.. --..~----- N2 WPRLV~N-CFSKDHSGKD tm3083 SSWHRRRRDQRLVYIYIY

MKSR-2

N2 100 amino aCtds tm2452 ~~__----

153 Figure 4-5. Interdependent localization of C. e/egans MKS-1, MKSR-1 and MKSR-2 proteins to transition zones. (A-F) The localization patterns of GFP-tagged MKS-1, MKSR-1 and MKSR-2 proteins in the indicated mks-1, mksr-1 or mksr-2 mutant strains are shown in both amphid (head) and phasmid (tail) sensory neurons. Arrowheads indicate representative (individual) transition zones, and asterisks highlight protein accumulations not normally observed for the GFP-tagged MKS-1, MKSR-1 or

MKSR-2 proteins in wild-type animals (see Fig. 4-2C). The orientations of the animals are the same for all, Le. the head is up and the tail is down. Scale bar: 5

IJm.

154 Head Tail Head

155 Figure 4-6. Single, double and triple C. e/egans mkslmksr mutants exhibit normal transition zone positioning, ciliary axonemal structures, intraflagellar transport and chemosensory behaviors. (A) The mks/mksr single, double and triple mutants display normal filling of amphid and phasmid neurons with the fluorescent dye dil, indicating that the cilium structure is probably intact and that the ciliary endings are properly exposed to the external environment; representative images for N2 (wild-type), bbs-B mutant (dye-filling defective), mks-1;mksr-1;mksr-2 (triple) mutant, and mks-1;mksr-1;mksr-2;bbs-B quadruple mutant animals are shown. Filled and hollow arrowheads indicate amphid and phasmid neurons that took up dye, respectively. (8) The GFP-tagged CHE-2 (IFT80) intraflagellar transport protein localizes normally to the transition zones (TZ; arrowhead in each panel) and ciliary axonemes (labeled cilia) in both the head and tail sensory neurons of N2

(wild-type), and mks/mksr single, double and triple mutants, as indicated. All panels are oriented with the head up and tail down. Scale bar: 5 tJm. (C) The single, double and triple mks/mksr mutants exhibit normal intraflagellar transport.

Kymographs of N2 and mutants (mks-1;mksr-1;mksr-2 triple mutant and mks-

1;mksr-1;mksr-2;bbs-B quadruple mutant) are shown for the ciliary middle segment (MS) and distal segment (OS) in the amphid (head) cilia using CHE-

2::GFP in intraflagellar transport assays. For each strain, fluorescent images of phasmid cilia and the corresponding kymographs (actual kymographs and schematics/traces of particle movement) for the MS and OS are shown; in the first fluorescent image, the transition zones (TZ) as well as MS and OS are labeled. The table shows the measurement of CHE-2::GFP rates (in tJm/second)

156 in the MS and OS for the indicated strains. n, number of 1FT particles measured.

Note that the rates of CHE-2::GFP movement in the mkslmksr mutants do not deviate statistically from N2; in the bbs-B background, CHE-2::GFP moves at the fast unitary rate of OSM-3 kinesin in both the MS and OS (Ou et aI., 2005 ). The asterisks in the image indicate CHE-2::GFP accumulations normally observed in the bbs-B mutant background (Blacque et aI., 2004 ). Scale bar: 5 ~m. (0)

IMC03 cells cotransfected with GFP and short-hairpin RNAi constructs for

MKSR1/89D1 and MKSR2I89D2 each show a significant reduction in the number of cilia, as assessed by staining with an acetylated tubulin antibody, in comparison with those transfected with GFP alone (control). *P<0.05. (E) The mkslmksr single, double and triple mutants show no statistically significant differences compared with wild-type animals with respect to chemotaxis towards a volatile attractant (isoamyl alcohol) or avoidance of a high-osmolarity solution

(8 M glycerol). The osm-5 ciliary mutant, defective in both chemotaxis and osmoavoidance, is included as a positive control. The chemotaxis and osmoavoidance indices are calculated as described in the Materials and

Methods. *P<0.05. [Note: (D) performed by J. Robinson, E. Davis, and N.

Katsanis]

157 A N2 bbs-8 triple mks/mksr bbs-8; tnple I> I> I A J; I

I

B head tail head tail head tail head tail N2 11JKs-1 mksr-1 mksl-2 Cilla[ Q. TZ [ ...... U. Cl N - fTlks-1 n 1 ksr-1 I mks-I mksr-2 I mksr-I mksr-2 I triple I W , I I U I I ..... I ..... I ..... I ..... I c

mks-1: mks-1 :mksr-1 mksr-1; mks-1; mks-1; mksr-1: mksr-2; N2 mks-1 mksr-1 mksr-2 mksr-2 mksr-2 mksr-1 mksr-2 bbs-8 MS 0.69±0.12 0.73±0.19 0.61±015 0.68±0.09 0.77±0.10 0.65±0.09 0.71±0.08 0.62±0.12 1.16±0.29 OS 1.13±021 1.20±0 17 1.18±0.12 1.21±0.19 1.21±0.18 117±0.16 1.18±0.15 119±0.16 1 23±028 n 65 82 101 71 80 70 95 102 60

D 1.01;) c 0.4 x .Q Vl 0.8 ~ 5 :a; 0.3 a,U 0.6 2S 0'0 c a. 20.2 ~ Q) .!!1 * ,...* 0.4 ·~'501 '0 ro...... 0.2 iO Q;O o ~ E oVl

158 Figure 4-7. mkslmksr mutant animals do not display dye-filling defects indicative of abrogated ciliary structures, or Nile Red (lipid content) phenotypes. (A) Wild-type and mks/mksr single, double and triple mutant animals incubated with Dil take up the fluorescent dye equally well, based on the number of sensory neurons that are stained and their relative staining intensity. (8) mks-1, mksr-2 and the mks-1;mksr-2 double mutants diplay equivalent Nile Red staining compared with wild-type (N2) animals based on relative fluorescence intensities of the dye in the intestine. [Note: (B) performed by M. Healey]

159 A

t ~ t

160 Figure 4-8. mkslmksr mutant animals display wild-type localization of intraflagellar transport (1FT) marker proteins and normal 1FT rates along the middle and distal segments of cilia. (A) Fluorescence images of GFP-tagged XBX-2 (dynein component), OSM-3

(homodimeric kinesin) or KAP-1 (component of heterotrimeric Kinesin-II) in the amphid (head) or phasmid (tail) sensory neurons of wild-type (N2) or mutant backgrounds, as indicated. Arrows point to the relative positions of transition zones (TZ) and ciliary axonemes are indicated with brackets. Scale bar: 5 ~m.

(B) 1FT rate measurements of 1FT markers in N2 or mutant animals, as indicated. n, number of particles measured; n/a, not applicable.

161 A Head Tail Head Tail Head Tail

B Middl e segment Dis al segment S ain n velocity (!lm/sec) velocity (!Imlsec) N2 (XBX.2.GFP) 62 0.72 ± C09 58 1.20 ± 0.16 mksr·1 (XBX.2::GFP} 72 O.77±0.11 57 1.08±0.16 mks.1:mksr~1.,mksr·2(X8X.2.:GFP) 83 0.73 ± 0.11 57 1.18 ± 0.13 N2 (OSM-3 GFP) 52 0.7 ± O. 1 39 1.15 ± 0 26 mksr-2 (OS ~-3: G P) 81 0.66 ± 0,08 6/ 1.24±O16 N2 (KAP-1"GFP) 50 0,68 013 nla nla mksr-2 (KAP-1:GFP) 48 0,67 009 nla n/a

162 Figure 4-9. Transmission electron microscopy (TEM) analyses of wild-type (N2) and mks-1;mksr-1 animals cross-sectioned through their amphid sensory neurons, showing their ciliary transition zones, cilia middle segment (doublet microtubules) and cilia distal segment (singlet microtubules), as indicated. No anomalies in ciliary ultrastructure were seen in the sections of the mutants compared to N2, of which only representative samples for the N2 and mks-

1;mksr-2 double mutants are shown. Insets show close-ups of representative

(individual) ciliary axonemes. Scale bars: 200 nm (white) and 500 nm (black).

[Note: electron micrographs provided by T. Toivonen, D. Cottell, and O.

Blacque]

N2 mks-l.mksr·2 double mutant

.e'" ~~------50------, '" b , E '- .

r ,I I I I I I ______JI

-- 1. ", I ~ 1rP!'l"...... ,. I I \ II \ I \ \ \ \ dual rOd winged I 01'<1 alia ,

amphld neuron cilia

163 Figure 4-10. Genetic interactions between the C. elegans mks-1, mksr-1 and mksr-2 genes revealed by an increased lifespan phenotype. (A) The mean lifespans of the individual mks-1, mksr-1 and mksr-2 mutant strains (as indicated) are indistinguishable from that of the wild-type (N2) strain.

(8) All combinations of double mutant animals (mks-1;mksr-1, mks-1;mksr-2, and mksr-1;mksr-2) exhibit statistically significant increases in lifespan relative to N2 animals (indicated by <0.0001*). The lifespan of the triple mutant is not statistically different from that of the N2 strain. (C) mkslmksr double mutant animals have enhanced lifespans comparable to those of nphp-1 and nphp-4 animals (Winkelbauer et aI., 2005) but shorter than that of the long-lived strain with a defect in the CHE-11 intraflagellar transport protein. All graphs show representative experiments, and the inset tables present mean lifespans with standard errors. Experiments were repeated at least twice and the results were found to be reproducible. "*,, indicates statistically different from wild-type. [Note: all data in this figure was generated by N. Bialas]

164 A 1.2

mean P value lifespan n VS. N2 .~ 0.8 -<:rN2 19.71 ± 0.40 75 'iii ..... mks-1 (tm2705) 19.35 ± 0.38 89 0.52 5 0.6 -0- mksr-1(tm3083) 20.63 ± 0.54 82 0.17 is ~ -0- mksr-2(tm2452) 20.36 ± 0.57 67 0.35 u. 0.4

0.2

0 0 4 8 12 16 20 24 28 32 36 40 44 48 Day B mean P value 1.2 lifespan n VS. N2 -<:rN2 19.71 ± 0.40 75 -0-mks-1;mksr-1 23.43 ± 0.53 80 <0.0001' .~ 0.8 -o-mks-1;mksr-2 23.89 ± 0.64 73 <0.0001' 'iii ..... mksr-1;mksr-2 23.36 ± 0.69 88 <0.0001' 6 0.6 ...... mks-1;mksr-1;mksr-2 19.69 ± 0.51 70 0.97 is ~ u. 0.4

0.2

0 0 4 8 12 16 20 24 28 32 36 40 44 48 Day C mean P value 1.2 lifespan n VS. N2 -<:rN2 18.98 ± 0.49 94 -o-nphp-1(ok500) 21.54 ± 0.44 87 <0.0001' .~ 0.8 ..... nphp-4(tm925) 22.13 ± 0.50 77 <0.0001' 'iii -o-che-11(e1810) 27.16 ± 0.86 74 <0.0001' 5 0.6 is l'O u: 0.4

0.2

0 ~'-"...... ,~-~~.-.--~.-,-~ 0 4 8 12 16 20 24 28 32 36 40 44 48 Day

165 Figure 4-11. MKS/MKSR proteins appear to function upstream of DAF-2 and DAF-16 in the insulin signaling/lGF-1 pathway to regulate lifespan. (A) The lifespan of mksr-1;mksr-2;daf-2 triple mutant animals is the same as that of the long-lived daf-2 mutants, supporting the notion that 89 domain-containing proteins function upstream of the DAF-2 insulin/lGF-1 signaling pathway. (8) The lifespan of all mks/mksr double mutants is shortened when introduced into a daf-

16 mutant background, suggesting that the MKS/MKSR proteins function upstream of the DAF-16 FOXO transcription factor in the insulin/lGF-1 signaling pathway. (C) The mks/mksr double mutants have increased levels of DAF-

16: :GFP in the nucleus, consistent with their increased lifespan compared to wild-type (N2) animals and their corresponding single mutants (mks-1 and mksr-

1). The osm-5 ciliary mutant positive control is impaired in DAF-2 signaling and has an increased level of nuclear DAF-16::GFP. Animals were categorized as having mainly cytoplasmic DAF-16::GFP (white bars), intermediate localization between the and nucleus (light gray bars), or mainly nuclear localization (dark gray bars); examples of these three localization patterns are shown on the right. A star (*) indicates statistically different from N2. [Note: all data in this figure was provided by N. Bialas]

166 c c: 100 ..- r- r- III ~ III ..g 90 •nuclear III f- f- ~ f- III 80 f- f- ~ 70 I-- e: 50 t!> 50 f- (C) ~ 40 C§ 30 'E 20 Q) ~ 10 c.. 0 DAF-15::GFP

167 4.9 Tables

Table 4-1. Distribution of 89 domain-containing proteins in ciliated organisms and absence from non-ciliated species. Individual proteins are grouped into their respective MKS/MKSR clades based on results of neighbor-joining ClustalW algorithm (not shown) and Bayesian analysis

(Figure 4-1A). Classification scheme according to Keeling et al. (2005). [Note: information in this table was assembled in collaboration with J. Parker]

B9 Family Members SUDeraroup Subaroup Species Cil NCBI Accession Accession Clade Plantae 8ryophyta P. patens yes No 89 proteins Chlorophyta C. reinhardtii yes XP_001693128.1 137074 MKSR-2 XP_001695895.1 130473 MKSR-1 XP_001698587.1 187742 MKSR-2 XP_001700551 POC12 MKS-1 Prasinophyta O. lucimarinus no No 89 proteins

Tracheophyta A. thaliana no No 89 proteins

Excavates Kinetoplastids T.brucei yes XP_827924.1 10.61.2290 MKSR-1 XP_828130.1 11.03.0750 MKSR-2 XP_847174 927.8.3430 MKS-1 L. major yes XP_001683785 n/a MKSR-2 XP_001685341 n/a MKSR-1 XP_001683495 F23.1710 MKS-1 Oiplomonads G./amblia yes No 89 proteins

Opis- Ascomycetes S. cerevisiae no No 89 proteins thokonts 8asidomycetes P. no No 89 proteins chrysosporium Zygomycetes R. oryzae no No 89 proteins Chytrids B. yes n/a 03036 MKSR-1 dendobatidis n/a 03725 MKSR-2 n/a 00264 MKS-1 Animals H. sapiens yes NP_056496.1 8901 MKSR-1 NP_085055.1 8902 MKSR-2 AAH10061.1 MKS1 MKS-1 M. musculus yes NP_038745.1 8901 MKSR-1 NP_742160.1 8902 MKSR-2 EOL15835.1 MKS1 MKS-1 Klaevis yes NP_001085984.1 82986 MKSR-2 NP_001090590.1 Meckelst1 MKS-1 NP_001086557.1 83744 MKSR-1 D. rerio yes NP_001002394.1 436667 MKSR-2 NP_001019544.1 89like MKSR-1 CAM56561.1 Mks11ike MKS-1 D. yes NP_608998.1 CG9227 MKSR-2 melanogaster NP_650470.1 CG14870 MKSR-1

168 NP_572804.1 CG15730 MKS-1 A. mellifera yes XP_393962.2 66524364 MKSR-1 XP_001120316.1 110748825 MKS-1 XP_001120495.1 110750832 MKSR-2 C. yes NP_500186.1 Y38F2AL.2 MKSR-2 elegans NP_497669.1 R148.1 MKS-1 NP_508203.1 K03E6.4 MKSR-1 C. briggsae yes XP_001677167 CBP10354 MKSR-2 CAE65900 CBP08737 MKSR-1 XP_001664663 CBP20416 MKS-1 S. purpuratus yes XP_795184.2 B9-like MKSR-1 XP_787637 FABBlike MKS-1 XP_001175727 B9 MKSR-2 N. vectensis yes XP_001618299 225297 MKSR-2 XP_001631460 209121 MKS-1 XP_001636548 241009 MKSR-1 Amoebozoa Dictyostelids D. discoideum no No B9 proteins

Chrom- Apicomplexans P. falciparum yes No B9 proteins alveolates C. parvum no No B9 proteins Ciliates T. thermophila yes XP_001022773.1 630490 MKS-1 XP_001020583.2 00219110 MKSR-1 XP_001030337.2 01099210 MKSR-2 XP_001010073.1 00633350 MKSR-2 P. tetraurelia yes XP_001426697.1 hypB9a MKSR-1 XP_001432714.1 hypB9c MKSR-2 XP_001441845.1 hypB9d MKSR-2 XP_001443728.1 hypBge MKS-1 Diatom T. yes n/a 38153 MKSR-2 pseudonana n/a 5008 MKS-1 n/a 264691 MKSR-1

169 CHAPTER 5. ANALYSIS OF INTRAFLAGELLAR TRANSPORT SUBCOMPLEX A (1FT-A) IN C. ELEGANS

170 5.1 Abstract

The vast majority of research examining the process of intraflagellar transport (1FT) in Caenorhabditis elegans has focused on the anterograde (tip­ direct) component of the process, specifically with respect to the interactions between the motors Kinesin-2, OSM-3, and the BBSome. This chapter examines the process of retrograde 1FT by screening a mutant library enriched for ciliogenesis-defective C. elegans strains for impairment specifically in retrograde

1FT. Of over forty mutants examined, nine were apparently defective in retrograde 1FT, with one particular mutant carrying an allele that maps to a small genetic region that contains a component of IFT-A not previously characterised in

C. elegans.

5.2 Introduction

Intraflagellar transport subcomplex A (1FT-A) is one of the two, highly

conserved multi-protein complexes that are deemed to be the core structural

components of 1FT rafts (Cole et aI., 1998; Cole, 2003). The original 1FT

biochemical studies in Chlamydomonas identified at least 5 IFT-A proteins,

named after their respective predicted molecular masses: IFT122A, IFT122B,

IFT139, IFT140, IFT144, and a putative sixth, IFT43 (Cole, 2003). Four of the

proteins (IFT122A, IFT122B, 1FT 140, IFT144) possess WD repeats. IFT139 is

predicted to possess at least 16 TPR motifs; 3 tandem TPR motifs are posited to

created a binding pockets for proteins, indicating that IFT139 is potentially

capable of binding at least 5 polypeptides (Cole, 2003), hinting at it being a more

"core" component of 1FT-A.

171 Genetic analysis in C. elegans appears to have identified a protein likely to be an additional IFT-A component, termed IFTA-1 (Blacque et aI., 2006). The gene encoding IFTA-1, C54G7.4, was uncovered as a likely ciliary candidate based upon the ciliomic studies described in Chapter 1 of this thesis, and is thus highly conserved amongst ciliated organisms. The 1FT defects seen in ifta-1 mutants strongly resemble those observed in other IFT-A components, and IFTA­

1 transports along with IFT-A/Kinesin-2 in ciliary middle segments of bbs mutants. The IFTA-1 protein itself appears to be a more peripheral component of the overall IFT-A structure, as it fails to enter cilia in che-11 (IFT140) mutants.

Beyond this observation, however, no detailed genetic analysis has been done in

C. elegans that focuses on determining the hiearchy of IFT-A components.

Haycraft et al. (2003) demonstrated a hierarchy of several proteins found within

1FT-B.

Studies of paraquat (also termed methyl viogen) (Fujii et aI., 2004) and ivermectin (Dent et aI., 2000) resistance in C. elegans have revealed that the neuronal rearrangements observed in dye-filling defective mutants conferred a degree of resistance, presumably through decreased cuticle permeability. All of the Dyf alleles isolated by Starich et al. (1995) have now been shown to be paraquat resistant, as have all 1FT-associated mutants, with the exception of worms defective in ciliogenic transcription factor mutant DAF-19 (Fujii et aI.,

2004). In the same study, an ethylmethane sulfonate (EMS) mutagenesis screen identified a new allele of che-11 (Fujii et aI., 2004), which encodes the IFT-A component IFT140.

172 In the present study, we examine an existing C. e/egans paraquat mutant library specifically for defects associated with components of IFT-A and IFT­ dynein. Alleles defective in these two 1FT modules have distinctive ciliary structure defects in the nematode, characterised by large, bulbous accumulations of GFP-tagged 1FT markers (see Figures 1-7 and 1-8 for examples). We

identified several mutants defective in the gene encoding the IFT-dynein heavy chain (CHE-3), as well as a mutant that, based on complementation analysis, appears to be a previously unidentified/uncharacterised component of the C.

e/egans 1FT machinery. A mapping strategy employing the snip-SNP method

(Wicks et aI., 2001) determined that the gene abrogated in this mutant is

positioned in a region containing a putative orthologue of the Chlamydomonas

IFT-A component, IFT139 (Li et aI., 2004).

5.3 Results/Discussion

5.3.1 Dye-filling analysis of paraquat-resistant mutant library

A library of 45 paraquat-resistant nematodes generated by EMS

mutagenesis (generously supplied by M. Fujii, Kihara Institute for Biological

Research, Yokohama City University, Japan) was subjected to dye-filling (Oyf)

analysis to determine individual lines likely to boast ciliary defects. 17 strains

(38%) appeared wild-type with respect to dye-uptake, and thus likely represent

abrogation of genes altering the metabolism of paraquat; these are therefore not

pertinent to this study (Table 5-1). 20 (44%) were completely Oyf-defective, that

is, they showed no uptake of Oil under the conditions outlined in the Materials

and Methods (Table 5-1). This phenotype is typically associated with mutations

173 affecting components of the 1FT machinery, although this requires independent confirmation, as some mutations could in principle affect the nature of the sheath and/or socket cells, or affect dendritic development, and hence ciliogenesis

(reviewed in Inglis et aI., 2007; Bae & Barr, 2008). Interestingly, the remaining 8 paraquat resistant strains displayed unusual partial dye-filling phenotypes, in which dye-filling defective and wild-type individuals were observed within the same population. We postulated that wild-type individuals were in fact heterozygotes; attempts to homozygose wild-type and dyf individuals did not alter the variable Dyf phenotype. Such variation has not been described previously in the literature, and may represent the first documented temperature-sensitive alleles of 1FT genes in the nematode, although this possibility has yet to be explored. A summary of the dye-filling analysis of the paraquat resistant alleles is shown in Table 5-1.

5.3.2 Ciliary defects in dye-filling defective strains

In order to determine the nature of the ciliary defect in each of the 28 dye­ filling defective strains, we introduced various GFP tagged 1FT markers (CHE­

11 ::GFP, OSM-5::GFP, and XBX-1 ::GFP) into each line. All lines successfully generated are listed in Supplementary File 1 (see Appendix A and attached

CD-ROM), along with their current physical location. As described in Chapter 1, the behaviour of 1FT markers in certain mutant backgrounds can allow

researchers to generally map the defect to a specific 1FT module. For example,

mutations in IFT-B-associated genes will likely show extremely truncated

axonemes, while mutants generating defective IFT-A or IFT-dynein components

174 generally result in large, bulbous accumulations in lieu of the normal filamentous axonemal structures. BBSome mutants typically have slightly shortened cilia, with subtle protein accumulations seen at the middle segment/distal segment transition and tips distal segments. Worms defective in OSM-3-based anterograde 1FT are characterised by significantly shortened axonemes, corresponding to a loss of ciliary distal segments.

Nine paraquat resistant strains were uncovered that displayed the large ciliary protein accumulation phenotype representative of defects in IFT-A or IFT­ dynein (see Figure 5-2. Complementation analysis was then performed with all alleles known to have ciliary protein accumulations: che-3 (e1224), xbx-1

(ok279), che-11 (e1810), and daf-10 (e1387). The alleles qa5007, qa5021, qa5032, qa5033, qa5040, qa5041, qa5048, and qa5060 all failed to complement with the che-3 allele, indicating that we obtained 8 distinct strains defective in the heavy chain of the IFT-dynein motor. The final putative retrograde 1FT-deficient allele, found in qa5054, complemented all of the alleles tested, and therefore likely represents a previously unidentified mutation affecting the retrograde 1FT machinery.

5.3.3 Progress in the cloning and characterisation of the qa5054 allele.

In order to determine the gene mutated in qa5054 of the paraquat- resistant mutant library, we performed the snip-SNP mapping technique employed by Wicks et al. (2001) to map the dyf-5 allele. First, using the recommended snip-SNPs for determining linkage to I, II, III, and

V, we determined that the allele was linked to chromosome III (data not shown).

175 Next, using the same bulked Iysates, we further resolved the genetic position of the allele to a region right of the centre of Chromosome III (Figure 5-1). Because fewer than recommended Chromosome III snip-SNPs were employed, we viewed this predicted region with considerable scepticism, and thus considered the mapping range of the qa5054 allele to be significantly wider than that

predicted by binomial analysis. By cross-referencing the 825 genes that fell in

between genetic positions -5 and 10 of Chromosome III with the ciliomic meta­

analysis described in Chapter 1 (as well as the comparative genomic study of

Chapter 2), we found 7 genes as likely candidates to be mutated in qa5054:

C27F2.1, C27F2.10, R01H10.6 (BBS-5), R07E5.3, ZC395.10, ZC395.3, and

ZK328.7. R01H10.6/BBS-5 is a known component of the BBSome (Nachury et

aI., 2007), and has been shown in the worms to traffic in a manner similar to

other BBS proteins (Ou et aI., 2007). It is therefore likely that bbs-5 mutants

would exhibit similar defects to other bbs mutants in the nematode, namely

slightly shortened cilia and mild middle/distal segment accumulation (Blacque et

aI., 2004). As a result, bbs-5 is not a likely candidate to be the gene mutated in

the qa5054 allele. C27F2.1 0, R07E5.3, ZC395.1 0, and ZK395.3 all exhibit severe

RNAi phenotypes, including embryonic lethality and sterility (e.g., Kamath &

Ahringer, 2003), none of which are associated with the qa5054 allele, although

the unlikely possibility exists that it could be a hypomorphic, rather than null,

mutation. Of the two remaining candidates, ZK328.7 represents the most likely

candidate gene, as knockout of its murine orthologue, THM1, results in

significant 1FT protein accumulation (Tran et aI., 2008), and has been identified

176 as a core component of 1FT-A, namely IFT139 (Li et aI., 2004; Cole & Snell,

2009).

5.4 Concluding Remarks

The next step in this line of research will necessarily involve an attempt to

rescue the dye-filling defects observed in the strain carrying the qa5054 allele

with a fosmid containing the ZK328.7 gene (as well as its upstream/downstream

promoter elements). Given previous work in Chlamydomonas (Cole et aI., 1998)

and mice (Tran et aI., 2008) have identified that genes orthologous with ZK328.7

encode a core component of 1FT-A, and the predicted mapping region of the

genetic lesions in qa5054 encompass ZK328.7, it is highly likely that ZK328.7 is

the gene whose mutation has resulted in the retrograde 1FT phenotypes

observed in Dyf strain we have identified.

If the ZK328.7-containing fosmid indeed rescues the dye-filling defects of

qa5054, it will be important to generate a translational GFP fusion construct of

the ZK328.7 gene. This construct will be used in a definitive rescue experiment

(i.e., qa5054 harbouring ZK328.7::GFP will be wild-type), but will also allow for

the further characterisation of the ZK328.7 protein. Based on the association of

ZK328.7 with 1FT-A, it is highly likely that the ZK328.7::GFP fusion protein will

traffic in a manner similar to other established IFT-A components (for example,

IFTA-1; Blacque et aI., 2006). Specifically, we would expect ZK328.7::GFP to

travel at an approximate velocity of 0.5 IJm/s, solely in the middle segments of

bbs mutant cilia. Attempts to generate ZK328.7::GFP using methods similar to

those described for DYF-11 ::GFP in Chapter 3 (the fusion PCR method with

177 genomic DNA) have been unsuccessful, perhaps due to the size of the gene

(-4000bp coding sequence, -7000bp genomic, not including upstream promoter sequences). It appears that different methodologies will need to be employed, including cloning into the GFP-containing pPD95.75 vector, or via the recently developed method of recombineering (Zhang et aI., 2008; Dolphin & Hope,

2006).

The possibility also exists that the defects observed in the qa5054 allele are not due to defects in ZK328.7. If this were to be the case, it would be an exciting finding, as only one of the remaining IFT-A or IFT-dynein associated genes (che-11, daf-10, ifta-1, che-3, xbx-1, xbx-2, dyci-1) is found on

Chromosome III. The exception, dyf-2, encodes the C. elegans orthologue of the

Chlamydomonas IFT-A component, IFT144 (Snell & Cole, 2009), but is positioned a considerable distance from the predicted mapping region of qa5054

(-20 map units). The existing allele of dyf-2 (m160) has already been thoroughly characterised (Efimenko et aI., 2006), and is available from the C. elegans

Genetics Center; complementation analysis could easily be performed to rule out qa5054 being an additional mutant allele of the dyf-2 gene. In order to rule out the other primary candidate genes, fosmid rescue will need to be conducted, with the exception of bbs-5, which already has an allele (gk507), that appears to disrupt gene function; a complementation test could performed to examine bbs-5 function in qa5054.

In the event that the aforementioned experiments failed to identify the gene mutated in the qa5054 allele, additional cloning techniques will need to be

178 employed. As previously indicated, fewer snip-SNPs were analysed than recommended by Wicks et al. (2001) for Chromosome III; greater resolution of the mapping region could be obtained via inclusion of additional snip-SNP analysis. Certainly, standard 3-point mapping strategies could also be employed

(reviewed in Fay, 2006), as well as more recently developed techniques,

including whole genome sequencing (Shen et aI., 2008), comparative genomic

hybridisations (CGH; Maydan et aI., 2009), and deficiency mapping (reviewed in

Edgley et aI., 2006).

5.5 Materials and Methods

5.5.1 Strains and genetic crosses

All C. elegans strains were maintained and cultured using standard

techniques. Mutations were tracked during crosses using PCR or dye-filling

assays. 45 paraquat resistant strains isolated in an EMS screen performed by M.

Fujii (Kihara Institute for Biological Research, Yokohama City University, Japan)

were numbered based on the order of isolation, and given a tentative gene class,

Lmev (Leroux-Methyl-Viologen), to ease tracking of strains: qa5005, qa5006,

qa500~ qa5021, qa5022, qa5023, qa5024, qa5025, qa5026, qa5032, qa5033,

qa5034, qa5037, qa5038, qa5039, qa5040,qa5041, qa5042, qa5043, qa5044,

qa5045, qa5046, qa5047, qa5048, qa5049, qa5051, qa5052, qa5053, qa5054,

qa5055, qa5056, qa505~ qa505a qa5059, qa506Q qa5061, qa5062, qa5063,

qa5064, qa5065, qa5066, qa5067, qa5068, qa5069, qa5070. Mutants that were

determined to be dye-filling defective (dyf; see below) were mated with N2 males.

The male, and hence heterozygous, progeny (F1 ) generated by these crosses

179 were then crossed with wild-type strains harbouring one of three GFP translational reporter constructs: N2 [che-11 ::gfp + rol-6J, N2 [osm-5::gfp + rol-6J,

N2 [xbx-1::gfp + rol-6]. Resulting progeny (F2) were allowed to grow to L4 and then were individually placed on NGM plates to lay eggs (F3). A dye-filling analysis of each plate was made (see below), and dye-filling defective individuals carrying the roller phenotype were selected.

5.5.2 Dye-filling (Dyf) assays

The 45 paraquat resistant mutant strains were obtained from M. Fujii

(Kihara Institute for Biological Research, Yokohama City University, Japan), and were examined for dye-filling defects as previously described (Starich et aI.,

1995). Briefly, populations were exposed to 200 IJL of Oil solution (Oi1C18,

Vybrant Oil cell-labelling solution, Molecular Probes; diluted 1:200 in C. elegans

buffer M9). Worms were incubated in the Oil solution for approximately 1 hour in a dark area, and agitated slightly every 15-20 minutes to ensure even distribution. Following incubation, exposed worms were placed on unseeded

NGM plates for 30 minutes to minimize gut fluorescence. Uptake of Oil into the

amphid and phasmid neurons was then determined using the Texas Red filter.

5.5.3 Microscopy

Transgenic animals expressing GFP-tagged proteins were mounted on

agarose pads and immobilized with 20 mM levamisole. Amphid or phasmid cilia

were examined with a 100X, 1.35 NA objective and an ORCA AG CCO camera

mounted on an Zeiss Axioskop 2 mot plus microscope. Images and movies were

180 obtained in Openlab version 5.02 beta (Improvision). Kymographs were generated using the MultipleKymograph ImageJ plug-in

(http://www.embl.de/eamnet/html/body_kymograph.html). Rates from middle and distal segments were obtained essentially as described in Snow et al. (2004).

Still images presented in figures were obtained using a Zeiss LSM 410

Axiovert confocal microscope. Stacks were obtained using the Piezo-z objective focus drive, and were then deconvolved using the Volocity version 4.40 deconvolution module (Improvision). Figures were then cropped and subject to colour modification using Adobe Photoshop version CS3.

5.5.4 Mapping

Mapping was performed essentially as described by Wicks et al. (2001).

Briefly, two bulked Iysates were fromed from groups of wild-type or dye-filling defective (Dyf) F2 adults derived from a cross of the Imev-54 allele (outcrossed

10 times with the N2 strain) and CB4856. Established PCR reactions were then carried out to generate a product specific containing an individual SNP (PCR information provided generously by Harald Hutter, Simon Fraser University).

These products were then digested with a specific enzyme (see Wicks et aI.,

2001 for a non-comprehensive list of enzymes used for individual SNPs) that cuts differently in the N2 and CB4856 strains. Digest products for N2, CB4856, and bulk Iysates of Dyf (+) and Dyf (-) F2s were then run out on an agarose gel.

The relative amounts of snip-SNP products in Dyf(+) and Dyf (-) Iysates were then compared using the ImageJ GelAnalyzer Tool. An arbitrary linkage

181 measure, referred to as the map ratio, was determined using the following equation,

where Dm[CB4856] and Dm[N2] represent the intensities of the CB4856 and N2 bands, respectively, in the Dyf (-) lysate. Dw[CB4856] and Dw[N2] represent the intensities of the CB4856 and N2 bands, respectively, in the Dyf (+) lysate (Wicks et aI., 2001). Markers unlinked to the mutation were expected to have Map

Ratios close to 1.0. The closer the Map Ratio is to zero, the greater the linkage, and thus the shorter the genetic distance between the snip-SNP and the gene mutated.

5.6 Figures

Figure 5-1. Mapping of qa5054 allele to the centre of C. e/egans Chromosome III. Height of bars indicates the map ratio calculated based on batch segregant analysis (BSA) for each snip-SNP as described in the materials and methods section of this chapter. Corresponding to each bar is the gel showing the banding pattern for each specific SNP. In all cases, the left lane represents the Dyf (+) F2 progeny BSA, and the right lane shows the results from the Dyf (-)/mutant F2s. A second order polynomial best fit curve determined by Microsoft Excel (2007) was

182 defined by the equation y =0.0009>1 - 0.0068x. The arrow represents the predicted genetic location of the qa5054 allele based on this equation.

1 2 y =0.0009x - 0.0068x

.2 ro cr: 0.5 Q. ro ::2

0 -30 -20 -10 0 10 20 Map Units • I• --SNP1 SNP9 SNP15 SNP16

183 Figure 5-2. Dye filling-defective (Dyf) paraquat-resistant strains with defects in retrograde intraflagellar transport. OSM-5::GFP labelled amphid (head) and phasmid (tail) cilia in putative retrograde 1FT-deficient strains and wild-type (N2). Scale bar, 10 ~m.

184 Amphid Phasmid Amphid Phasmid

N2 qa5007

qa5021 qa5032

qa5033 qa5040

0­ u. C)

L{) I ~ oen

qa5041 qa5048

qa5054 qa5060

- 185 Table 5-1. Dye-filling results from paraquat-resistant mutant library. Dye-filling results Wild-type Dye-filling defective Variable dye-filling (Dyf) defective qa5037 qa5005 qa5006 qa5038 qa5007 qa5042 qa5039 qa5021 qa5043 qa5045 qa5022 qa5044 qa5046 qa5023 qa5052 qa5047 qa5024 qa5055 qa5049 qa5025 qa5067 qa5051 qa5026 qa5069 qa5053 qa5032 qa5056 qa5033 qa5058 qa5034 qa5059 qa5040 qa5061 qa5041 qa5063 qa5048 qa5065 qa5054 qa5066 qa5057 qa5068 qa5060 qa5062 qa5064 qa5070

186 CHAPTER 6. CONTROL OF C. ELEGANS THERMOSENSATION AND LOCOMOTION BY BARDET-BIEDL SYNDROME PROTEINS

Note regarding contributions: A portion of the material presented in this chapter relating to C. elegans thermosensation (both thermotaxis and thermal avoidance) was published in the following article: Tan PL, Barr T*, Inglis PN*, Mitsuma N*, Huang SM, et al. (2007). Loss of Bardet-Biedl syndrome proteins causes defects in peripheral sensory innervations and function. PNAS 104: 17524-17529. (* - equal contributions) All research presented in this chapter was performed by me or under my direct supervision. Preliminary C. elegans thermotaxis studies were aided by Van Xue (SFU). OSM-9::GFP deconvolution microscopy was performed by Brian Bradley (SFU). Allan Mah (SFU) performed construct microinjection. Anthony Breemo (SFU) conducted the C. elegans motility studies.

187 6.1 Abstract

The ability of an organism to effectively recognise and respond to a wide range of thermal stimuli is essential for survival. Here, we demonstrate that the sensory cilia of Caenorhabditis elegans playa role in the sensation of both physiological and noxious temperature exposures. Specifically, bbs mutant worms that are known to be deficient in ciliogenesis and cilium-based signalling fail to effectively recognise slight physiological temperature differences, and are defective in their response to noxious thermal stimuli. This defect may be due to the improper intraflagellar transport (IFT)-based trafficking of cilium-based thermosensors, as OSM-9, the orthologue of the human heat receptor TRPV4, fails to efficiently localise to the OlQ cilia in bbs mutants. We determine that the thermotactic phenotype correlates with a marked decrease in roaming ability of bbs mutants under our assay conditions. This result is likely not due to decreased individual motility, as bbs mutant worms show no apparent deficiency in body bend rates. Taken together, our data implicates cilia in metazoan thermosensation for the first time.

6.2 Introduction

The behavioural responses of Caenorhabditis elegans to thermal stimuli represent an excellent model for metazoan temperature-responsiveness and neural plasticity. Nematodes will respond to various, non-noxious thermal cues by migrating towards the temperature at which they were reared (Hedgecock &

Russell, 1975; Mori & Ohshima, 1995). This behaviour is advantageous to the

188 reproductive success of individual nematodes, as their natural environment features a diverse range of fluctuating temperatures, and various aspects of worm physiology, including reproduction, growth, food sources and motility, can be sustained only within a small range of temperatures (Ramot et aI., 2008).

The thermotaxis assay (also known as the isothermal tracking assay), first performed by Hedgecock and Russell (1975), represents the standard means of testing the temperature-sensing abilities of C. elegans strains. Briefly, individual worms are placed on a steep thermal gradient (shown in Figure 6-1A) and their movements are traced (Hedgecock and Russell, 1975). Wild-type worms display two distinct behaviours when placed on the gradient. First, they immediately move towards the location of their rearing temperature on the gradient, in a process known as thermotaxis. For the remainder of the assay, individuals will move around the plate in an isothermal manner; that is, they circle the plate, staying within 1 °C - 2 °C of their rearing temperature (Hedgecock & Russell,

1975; Mori & Ohshima, 1995). Mutants that fail to behave as wild-type in this assay are referred to generally as thermotaxis-defective (ttx), and are classified as being athermotactic, cryophilic (migrates towards colder-than-rearing temperatures), or thermophilic (migrates towards warmer-than-rearing temperatures).

Laser ablation studies have identified the amphid AFD neuron as the cell responsible for receiving thermal stimuli (Mori & Ohshima, 1995). The two interneurons, AIY and AIZ, are required specifically for thermophilic and cryophilic responses, respectively (Mori & Ohshima, 1995). A second ciliated cell,

189 AWC, has recently been identified as a secondary thermosensory neuron (Biron et aI., 2008), but is not specifically addressed in this chapter. The portion of the

AFD neuron most likely to be receiving thermal cues from the environment, namely the dendritic ending, possesses a unique structure comprised of a shortened (-1.5 IJm) cilium surrounded by at least 50 villi, each -2 IJm in length

(see diagram in Figure 6-1 B; Perkins et aI., 1986). The unusually short cilium of the AFD neuron has been described as degenerate (Mori, 1999) due to the comparative lack of complexity in its proximal segments, when compared to the cilia of other amphids (Perkins et aI., 1986). These observations potentially explain the wild-type behaviour of ciliogenic mutants in prior thermotaxis studies

(Hedgecock & Russell, 1975; Perkins et aI., 1986).

Genetic studies have identified two channel proteins as being associated

with thermotaxis and isothermal tracking, namely TAX-2 and TAX-4. These

proteins, which together form a heterodimeric cyclic nucleotide gated channel,

appear to be responsible for transducing a large number of sensory signals

within the amphid neurons (Coburn & Bargmann, 1996; Komatsu et aI., 1996).

The genes encoding these proteins are expressed in a subset of amphid

neurons, namely AFD, ASE, AWB and AWC (Coburn & Bargmann, 1996;

Komatsu et aI., 1996). GFP-tagged TAX-2 (Coburn & Bargmann, 1996) and TAX­

4 (Komatsu et aI., 1996) were observed at the distal tips of amphid neurons,

presumably at cilia.

A second putative thermal-responsive channel, OSM-9, is a strong

homolog of the vertebrate capsaicin (the spicy ingredient in chilli peppers)

190 receptor, TRPV4 (Mori, 1999). osm-9 is expressed in a specific subset of ciliated neurons, namely AWA, ASH, and OlQ, and its corresponding protein localises to cilia (Colbert et aI., 1997) and, at least in certain circumstances, undergoes 1FT

(Qin et aI., 2005). However, it should be noted that the expression pattern of osm-9 shares no overlap with that of tax-2/tax-4, suggesting there is no overlapping function on the cellular level (Mori, 1999). Additionally, while osm-9 mutants are defective in olfaction and mechanosensation, they appear to be wild­ type for isothermal tracking and thermotaxis (Colbert et aI., 1997). Similarly, no links between OSM-9 and thermosensation and sensitivity to capsaicin in C. elegans can be seen in thermal avoidance (TAV) studies, although other central ciliary/1FT proteins do (Wittenburg & Baumeister, 1999).

Briefly, the thermal avoidance (TAV) assay, first performed by Wittenburg and Baumeister (1999), scores the ability of individual nematodes to escape exposure to a localised, noxious (>33°C) heat source. Worm responses are grouped into distinct classes based on the responsiveness of the individual to the

noxious signal (shown in Figure 6-10). Mutants isolated in isothermal tracking/thermotaxis mutagenesis screens displayed wild-type TAV responses, consistent with the notion that TAV is distinct from previously studied thermal

behaviours (Wittenburg & Baumeister, 1999). Furthermore, nematodes defective

in ciliogenesis, specifically che-13, osm-1, osm-5 and osm-6, are TAV defective,

strongly implicating cilia in the sensation of noxious temperatures (Wittenburg &

Baumeister, 1999). Finally, worms exposed to between 1 IJM and 100 IJM of

capsaicin for -30 minutes were found to be desensitized to noxious

191 temperatures. osm-9 mutations (which were found to be wild-type in non-treated

TAV assays) were not observed to rescue the TAV phenotype in hyperalgetic

(capsaicin-exposed) animals (Wittenburg & Baumeister, 1999), indicating that another channel is responsible for capsaicin-based responses.

This chapter describes the analysis of motility and thermosensation of C. elegans Bardet-Biedl syndrome (bbs) mutants. Firstly, TAV analysis of bbs mutants show a decrease in response similar to those observed with ciliogenic mutant strains reported by Wittenburg and Baumeister (1999), consistent with the notion that cilia playa role in this behaviour. Next, using a novel, shallow­ gradient thermotaxis assay (Figure 6-1 C), we observed that bbs and other ciliary mutants were less efficient in reaching their rearing temperatures than wild-type controls. This result is surprising, given that ciliogenic defective strains were wild­ type in the original published isothermal tracking assays (Hedgecock & Russell,

1975). In an attempt to reconcile this discrepancy, we examined the behaviour of bbs worms in the isothermal tracking assays and discovered that they were covering a significantly shorter area of the plate than wild-type strains in equivalent conditions. This defect is not the result of impaired motility of mutants, as they were found to be similar to wild-type with regards to body-bends per minute. Overall, the data presented offers the first evidence that cilia are involved in transducing subtle, non-noxious thermal cues in the nematode, and hints at the previously undiscovered role played by the highly-conserved BBS proteins in metazoan temperature sensation.

192 6.3 Results

6.3.1 C. e/egans bbs mutants are thermosensory defective

To test the possible role of C. elegans BBS proteins in thermosensation, we first compared the behaviour of two previously described bbs mutants

(Blacque et aI., 2004), bbs-7 (also known as osm-12) and bbs-B, with that of their

WT counterparts in a thermotaxis assay (similar to that performed in Ito et aI.,

2006; Figure 6-1 C). When placed on a shallow linear thermal gradient ranging from 18.5°C to 21.5°C (across 8 cm), WT animals moved effectively to their rearing temperature of 20°C over a period of 1 h (Figure 6-2A). By contrast, bbs­

7 and bbs-B mutants displayed statistically significant defects (P < 0.00001) in their thermotaxis to the 20°C zone (Figure 6-2A). An equal number of thermotaxis assays were performed by placing the animals on the hot or cold side of the agar slab, and the results implied that ciliary mutants were defective in general thermosensation, showing no preference for warm or cold temperatures.

This phenotype cannot be ascribed to locomotory defects, because the mutant

animals display movement (i.e., bends per minute) indistinguishable from WT

(see Section 6.3.2). In addition, bbs-7 and bbs-B mutant strains rescued with

their respective WT genes showed restored thermotaxis behaviours (Figure 6­

2A; bbs-7 rescue =0.5307 ± 0.073; bbs-B rescue =0.4925 ± 0.063). Importantly,

these defects were not restricted to the bbs mutants, because other ciliary

mutants, including osm-5, which encodes the ortholog of IFT52 (Qin et aI., 2001),

and osm-3, which encodes the homodimeric kinesin required for building the

distal segment of C. elegans cilia (Snow et aI., 2004), also displayed thermotaxis

193 defects (P < 0.00001) that were comparable with those of bbs mutants (Figure 6­

2A). Also of interest, the general morphology of the microvilli-decorated AFD thermosensory neuron cilium was superficially intact, as visualised by examining the AFD-specific gcy-Bp::GFP construct at high magnification in bbs-B mutants, suggesting that defects in the underlying cilium are responsible for the phenotype.

Next, to test whether thermosensory defects extend to noxious temperatures, we performed a thermal avoidance assay, in which the response of individual worms to a nociceptive temperature (-50°C) placed directly in the path of their movement near their head was measured (Wittenburg &

Baumeister, 1999). WT animals (n > 400) stop their forward movement and initiate a distinct, reproducible reflexive reversal in 94.9% of the cases (Figure 6­

28; a typical reversal is demonstrated in Figure 6-1 D) (see also Wittenburg &

Baumeister, 1999). By contrast, bbs-7 and bbs-B mutants (n > 400) both showed poor avoidance phenotypes (P < 0.00001), with proper avoidance responses observed in only 73.1 % and 73.3% of the cases, respectively. Similarly, the osm­

3 and osm-5 mutants displayed avoidance in 78.3% and 73.4% of the cases (P <

0.00001), respectively (Figure 6-28). To ensure that the thermal deficient phenotypes are specifically due to mutations in bbs, we confirmed that bbs-7 and bbs-B mutants expressing their WT transgenes had thermal avoidance responses comparable with that of WT animals (bbs-7 rescue = 95.0% ±3.2%; bbs-B rescue = 94.5% ±0.4%; Figure 6-28).

194 We next sought to probe further the cellular basis of this phenotype. One possibility was that defective ciliary function might perturb the movement of effector molecules, of which TRP receptors would represent ideal candidates.

We therefore examined the movement of OSM-9 in our mutants. Because no C. elegans TRP channel has yet been linked specifically to thermosensation, we focused on OSM-9, a known axonemal protein that is required for chemotaxis and represents a potential functional homolog of mammalian TRPV1 or TRPV4

(Colbert et aI., 1997; Kahn-Kirby & Bargmann, 2006). We found that a GFP­ tagged version of OSM-9, which localizes properly to the ciliary axoneme in WT animals, shows variable but consistent mislocalization in a bbs mutant background. Specifically, bbs-B animals display accumulations/mislocalization not seen in the WT animals or in bbs-B rescue animals, along the dendrite, within or in proximity to the basal body, and along the ciliary axoneme (Figure 6-2C).

Although OSM-9 is not the C. elegans thermosensory receptor per se, our findings suggest that at least one molecular explanation for the thermosensory phenotype is that a cilium-based thermosensory apparatus (e.g., TRP-type receptor) mislocalizes or is not functional when BBS protein function is disrupted.

Interestingly, this phenotype is very similar to that observed in the dyf-11 mutant, where much of the OSM-9 protein still localizes to the ciliary axoneme but some accumulations are observed at the base (Omori et aI., 2008)

195 6.3.2 C. elegans bbs mutants demonstrate reduced roaming on isothermal gradients in a manner unrelated to rate body bends

In an attempt to explain the differing results of the shallow gradient thermotaxis assays in the previous section and the steep thermal gradient analyses of ciliogenesis-defective C. elegans strains (Hedgecock & Russell,

1975; Perkins et aI., 1986), we performed isothermal tracking assays

(Hedgecock & Russell, 1975; Mori & Ohshima, 1995; Figure 6-1A) on bbs-B

(nx77) mutants. Typical results are shown in Figure 6-3A, including those for wild-type (N2) and cryophilic (ttx-3) controls. In all cases (n=15), bbs-B mutant worms appeared to cover a considerably smaller area of the assay surface than either of the control strains. This result could explain the inefficiency of bbs mutant worms in migrating towards the 'target zones' (location of rearing temperature) in the shallow gradient assays (Figure 6-2A).

The decreased roaming of bbs mutants in the isothermal tracking assays could be attributed to impaired mobility. To test this possibility, we performed body-bend assays (Sawin et aI., 2000) on bbs-1, bbs-7 and bbs-B individuals.

Over a range of experimental temperatures (15 ec, 20 ec, and 23 ec), we could observe no significant difference between the rate of total body bends for bbs

mutants and wild-type (N2) controls (Figure 6-38).

6.4 Discussion/Conclusion

BBS proteins have been shown to play key roles in both the process of

ciliogenesis and cilium-based sensation in animals ranging from C. elegans to

humans (Blacque & Leroux, 2006). Here, we present data that, for the first time,

196 links the BBS complex (known as the BBSome) to thermal sensation in the nematode. For the studies relating to thermal avoidance (TAV), this result is far from surprising, as C. elegans mutants deficient in the process of ciliogenesis have already been shown to respond poorly to noxious heat signals (Wittenburg

& Baumeister, 1999). More striking, however, is our observation that bbs (and other ciliary) mutants showed impaired ability to identify and migrate to rearing temperatures when placed on a shallow linear thermal gradient. Several large scale studies that employed steep thermal gradients (isothermal tracking assays) were unable to distinguish ciliary mutants from wild-type with respect to thermal migratory behaviour (Hedgecock & Russell, 1975; Perkins et aI., 1986; Mori &

Ohshima, 1995).

There are two reasonable explanations for the discrepancies observed between the behaviours of bbs mutant worms on steep and shallow gradient thermotaxis assays. First, it is possible cilium of the AFD neuron acts as a 'tuning fork' for thermal recognition; in other words, while the complex arrangement of dendritic villi surrounding the dendritic terminus of the AFD are responsible for recognizing large differences in temperature (e.g., 1 °C - 2 °C variation), the AFD cilium is involved in identifying far more precise deviations (e.g., < 0.5 °C). If this were to be the case, there would likely be an additional thermosensory channel

(i.e., other than TAX-2/TAX-4) or signal, specifically associated with the AFD cilium. While there are no clear candidates at this moment, there is a wealth of functional genomic data available that could expedite the search for such a channel, such as AFD-specific SAGE and microarray studies. A second

197 possibility is that bbs mutant worms have defective locomotion that stem from their inherent sensory impairment.

The latter explanation was given significant support following our placement of bbs mutants on original isothermal tracking assays (Figure 6-3A). bbs-B individuals were found to cover a far smaller region of the assay plate than any other strain analyzed. In light of recent findings (published after the research in this chapter was performed), this result is not surprising, as a number of ciliogenic mutants have been shown to have defective roaming behaviours in assays employing no thermal gradients (Williams et aI., 2008; Kobayashi et aI.,

2007).

The most obvious explanation for the decreased roaming of cilium structure/function mutants is that they simply do not move as much as their wild­ type counterparts. To test this hypothesis, we performed body bend assays

(Figure 6-38) on bbs mutant and control worms. Remarkably, our data suggests that there is no significant difference between wild-type and bbs mutant strains with regards to overall locomotion. Indeed, bbs-1, bbs-7, and bbs-B all showed wild-type mobility in both the forward and reverse directions. Although by no means definitive, these data imply that the roaming defects of ciliary mutants are not due to specific defects in locomotion, but rather some sort of sensory process that regulates locomotion.

Given that bbs and other cilium-defective strains appear to roam less than wild-type yet move at comparable rates, it is likely that they turn more than N2 strains. This will need to be tested experimentally. Studies of chemotaxis in C.

198 elegans have uncovered that worms tend to migrate up and down gradients in a predictable manner, referred to as the pirouette model (Pierce-Shimomura et aI.,

1999; Pierce-Shimomura et aI., 2005; Bargmann, 2006; Figure 6-1 E) chemotaxis. Essentially, when moving down a chemical gradient (Le., away from the attractant), nematodes tend to turn more, increasing the likelihood that the move in the direction of the attracting chemical. When moving towards an attractant, individuals make significantly fewer turns.

While it remains to be seen whether C. elegans thermotax according to the pirouette model, increased turning would be sufficient to explain the lack of roaming by bbs mutants. The pirouette model requires worms to recognize gradients in order for the rate of turning to be modified. Given the results of our shallow gradient thermotaxis assays vis-a-vis the steep gradient isothermal tracking assays, it certainly appears that bbs mutant worms are less likely to recognise the subtle temperatures needed to recognise the thermal gradients as whole. Without being to identify and follow thermal gradients, worms would be predicted to turn with no particular directional bias. In other words, individuals would tend to move in circles, covering very little area.

If this hypothesis were to hold true, a fascinating implication would be that researchers could phenocopy the defective roaming behaviour of bbs or cilium structure/function mutants in wild-type strains by removing all gradients of sensory stimuli from the surrounding environment. Such conditions may prove difficult to generate, as they would necessarily require the creation of a tightly controlled environment in which the roaming assays were to be performed. An

199 exciting approach to this problem could involve the exploitation of emerging lab­ on-a-chip technologies, similar to the recent C. elegans learning assays performed on microfluidic surfaces (Qin & Keever, 2007).

6.5 Materials and Methods

6.5.1 Shallow gradient thermotaxis assay

To analyze the response of C. elegans over a shallow, linear temperature gradient, similar to that described in Ito et al. (2006) , we manufactured an aluminum slab (1.5 cm x22 cm x64 cm) with an aluminum leg (1.5 cm x22 cm x15 cm) attached at each end. A stable thermal gradient was generated by immersing 10 cm of one aluminum leg in an ice water bath, with the other leg immersed 10 cm in a 50 0 e hot water bath. An 8 cm long (4 cm wide) agar slab

(2% agar, 0.005 M potassium phosphate, 0.001 M calcium chloride, 0.001 M magnesium sulfate) was placed 20.8 cm away from the hot aluminum leg. The temperature over this 8 cm slab ranged from 18.5°e to 21.5°e. Worms were placed 1.5 cm from the edge of the agar (either on the hot or cold side), and motility was assessed after 1 h. Worms were scored based on their presence in one of three zones: the "hot" zone, which consists of the first 3 cm of the agar slab, the "ideal" zone, which is the 2-cm region centred around 20 o e, and the

"cold" zone, which comprises the final 3 cm of the agar slab. A thermotaxis index was calculated by examining the ratio of worms found in the ideal zone over the total number of worms counted.

200 6.5.2 Thermal avoidance assay

The response of C. elegans exposed to noxious heat was performed essentially as described (Wittenburg & Baumeister, 1999), with a minimum of

400 worms examined per strain. The heat source was a Dremel Versatip woodburning pen (Dremel Service Center, Racine, WI). Briefly, forward-moving individual worms were exposed to temperatures ranging between 37°C - 45 °C; temperatures within this range have been reported to be noxious to C. elegans

(Wittenburg & Baumeister, 1999). The immediate response of each worm was scored. Individuals that immediately withdrew from the heat source by reversing for at least 1 body length were described as responsive (i.e., thermal avoiders).

Those worms that failed to effectively respond to the heat source were scored as non responsive. Representative examples of both groups are shown in Figure 6­

10.

6.5.3 Analysis of OSM-9 localization

The extrachromosomal array Osm-9::gfp3 (generously donated by Dr.

Cornelia Bargmann, The Rockefeller University, New York, NY) was analyzed in both WT (N2) and bbs-B backgrounds. Because OSM-9 localizes to the cilia outer labial quadrant (OlQ) neurons, it is impossible to view all four cilia simultaneously by using standard microscopy techniques. To optimally analyze

OSM-9 localization patterns, we examined lines, immobilized lines using 30 mM levamisole and mounted on 4% agarose pads, using an Olympus IX70 epifluorescence inverted microscope (100X objective) in a DeltaVision imaging station (Applied Precision, Issaquah, WA). Removal of out-of-focus fluorescence

201 and restoration of optical Z-sections was achieved through deconvolution with the constrained iterative algorithm and point-spread functions supplied with the

DeltaVision system. Images shown represent merged stacks of the deconvolved sections.

6.5.4 C. e/egans body bend assay

The procedure used for the scoring C. elegans locomotion is referred to as the body bend assay, and was performed essentially as described by Sawin et al. (2000). Briefly, late-L4 worms were plated on nematode growth media (NGM) plates, and given sufficient time to lay a significant number of eggs. They were then removed from the plate. Each genotype was grown at three distinct temperatures: 15°C, 20 °c and 23°C. Each population was grown to L4. 50 worms were then picked off of each plate and plated individually on unseeded

NGM plates and left for 10 minutes. Each worm was then picked onto a fresh unseeded NGM plate and allowed to rest for further 3 minutes.

The forward and reverse body bends for each individual worm were scored over a 20 second interval. A body bend was scored when the pharyngeal region of the animal had progressed from on maximum bend position to another

(see Figure 6-1 E for an illustration). To clarify, standard worm locomotion resembles a typical sin wave. When the position of the pharynx has moved from a minimum of the wave to a maximum, one body bend is scored. Conversely, worms that did the opposite, that is, showed pharyngeal movement in the reverse direction, were scored as a reverse body bend. Worms that roamed to within 1 em of the edge of the assay plate were not included in the final analyses.

202 6.6 Figures

Figure 6-1. Experimental approaches used in Chapter 6. (A) 9cm plate setup for the standard isothermal tracking assay as performed by

Ohshima & Mori (1995). A vial of frozen glacial acetic acid is placed in the centre of a 9cm petri dish sitting at 25°C. An individual worm is placed at point X, denoted in figure. Thermal gradient is shown at bottom. (B) Schematic representation of the distal tip of the AFO neuron (both AFOL and AFOR). A single cilium (red), emanating from a transition zone (blue), is surrounded by villi emanating from the dendrite (green). Based on electron micrograph analyses performed by Perkins et al. (1986). (C) Novel thermotaxis assay (Tan et aI.,

2007) employing a shallow gradient along an 8cm x 4cm slab agar pad.

Populations of worms (-25) are placed in the centre of the "warm" or "cold" zones, as shown. Thermal gradient is shown at bottom. (0) Time-lapse images, spanning a total time of 1 second, of typical responsive (top panel) and non- responsive (bottom panel) thermal avoidance (TAV) behaviours. The dark object seen at the bottom of many of the images is the probe supplying the noxious heat. In all pictures, the anterior of the worm is facing the bottom of the page. (E)

Time-Iaspe microscopy to demonstrate the scoring of the C. elegans body bend assay. The range of movement for the single worm depicted in this figure undergoes precisely one body bend according the experimental approach described in the materials and methods. P == Pharynx.

203 A C

8cm

Cold Target Warm Zone Zone Zone

2cm 3cm

• 18,5°C 21,5°C • 25°C 17"C o

~ 'en c o a.

Q) 'en> c o a.

E

204 Figure 6-2. C. e/egans ciliary mutants show thermotaxis and thermal avoidance defects as well as mislocalized OSM-9. (A) Thermotaxis and thermal avoidance of WT (N2), bbs mutants, and osm-3 kinesin mutants. Thermotaxis relates to how well the worms move to their rearing temperature zone (20°C). (B) Nonthermal avoidance is the proportion of worms that do not avoid a noxious temperature presented near the head of the animal.

For A and B, *, P < 0.00001. (C) GFP-tagged OSM-9 mislocalizes in bbs-B mutant animals. TZ, transition zone/basal body; *, abnormal accumulations not seen in wild-type animals.

205 A 0.7 ~ 0.6 '"J -::l c 0.5 ~ ~ 0.4 .~ ;.< :'0 OJ C E 0.2 * * 'J ** * -= 0.1 0 X"~, ~".- ,,;'\ .,'t> "'-', s''I _,'t> 'O'(j. '0'0'" 0o:,'V- Os""~ \\. 'O'(j. C '0'0'" C -Co\): ~C?·c\." ~c'" Strain

Bi 0.3 'J * :.J * * t: * :'0 ~ 0.2 ;c :'0 ...E 0.1 'J ...c:, £= C 0 ~ ~1. .,'1 :i>''t> ~,~ .;1 '0'0'" 'O'(j 0o:,'V- 'O'Os'~\, 00:,':\\,' '0'0'" \.,c ·-c\: ~csc <,.c'" Strain

206 Figure 6-3. C. e/egans bbs mutants show defective roaming in isothermal tracking assays but appear to have normal body movement. (A) Representative isothermal tracking assays of wild-type (N2), cryophilic (ttx-3), and bbs-B mutant worms. The large outer circles represent the petri dish the assay was performed on. The smaller blue circles denote the approximate location and relative size of the cold source (a vial of glacial acetic acid). 'x' denotes the location on the plate where the worm was placed, 'y' denotes the position of the worm at the end of the assay. The lines connecting them are traces of the worm tracks. (B) Body bend motility assays performed on wild-type

(N2), slow moving (unc-43), fast-moving (goa-1), and bbs (bbs-1, bbs-7, bbs-B) mutant worms at three different temperatures. * denotes significant difference from wild-type at the same temperature (P < 0.05).

207 A

N2 ttx-3 bbs-B

B 16 [] 15°C 14 (J) [] 20°C 0 N.... 12 Q) 23°C a. • ~ 10 c:: Q) .0 >- 8 "0 0 .0 Q) 6 OJ ~ Q) 4 «> 2

0 g08-1 ep275 Strain

208 CHAPTER 7. CONCLUSION

209 Overall, this thesis describes a survey of the mechanisms and components underlying cilium biogenesis and function, with a primary focus on the nematode Caenorhabditis elegans. Ultimately the work presented in each chapter can be grouped into two distinct categories: (1) the identification and characterisation of novel proteins associated with the process of intraflagellar transport (1FT); (2) genetic analysis of C. elegans cilium-based signalling pathways, with a specific focus on the role played by 1FT-associated proteins.

7.1 Identifying strong candidate ciliary genes using existing ciliomic data

Central to each of the chapters is the concept of the ciliome, which is the core complement of proteins required for proper cilium biogenesis or function.

Chapter 1.4 provides a more-or-Iess comprehensive description of the many genomic, bioinformatic, and proteomic approaches to defining the ciliome.

Chapter 2 describes a new cilia/basal body-centric comparative genomic study, taking advantage of the recent sequencing of the Batrachochytrium dendrobatidis genome. Each ciliomic approach is similar in that they ultimately result in a long list of candidates. Ultimately, researchers wanting to analyze these genes/proteins need to prioritise candidate genes based on the existing ciliomic data.

The first and perhaps most reliable means of identifying top ciliary gene candidates is the cross-referencing of ciliomic data with existing literature. For example, Chapter 4 describes the characterisation of MKS1 in C. elegans; this protein has been identified in several ciliomic studies, and is also implicated in

210 Meckel-Gruber syndrome, which is characterised by many symptoms typical of ciliopathies. Similarly, the currently uncharacterised MKS6, also implicated in

Meckel-Gruber syndrome, is found in a number of bioinformatic and comparative genomic ciliomic analyses, and therefore represents an excellent focus for future studies.

Another excellent means of identifying novel ciliary proteins is the meta­ analysis of the various ciliomic studies. This approach is described in Chapter

1.4.6, and involves direct comparison of each ciliomic study. A candidate that appears in two or more ciliomic approaches (i.e., proteomics, bioinformatics, and functional genomics) is likely to be a bona fide ciliary protein, as it is unlikely that each approach would have overlapping false positives. Chapter 3 describes the characterisation of such a protein, namely DYF-11, which was recognized as a candidate gene in C. e/egans X-box searches (Blacque et aI., 2005; Efimenko et aI., 2005), the Chlamydomonas ciliary proteome (Pazour et aI., 2005), and was shown to have up-regulated expression during ciliary regeneration in

Chlamydomonas (Stoic et aI., 2005).

Ultimately, both meta-analysis and cross-referencing of ciliomic data with existing literature are extremely limiting approaches, as only a handful of genes fit either criteria, and for the most part, have all been studied. A non-exhaustive

list of uncharacterised C. elegans ciliary gene candidates that fit the above

criteria can be seen in Table 7-1.

In order to identify less obvious candidates, more organism-specific

approaches are needed. Of particular relevance to this thesis is the efficient

211 identification of ciliary gene candidates in the organism C. elegans. There are a wealth of resources available to the nematode cilia researcher, the most useful of which being the bioinformatic searches for x-box sequences in upstream promoter sequences (as described by Blacque et aI., 2005 and Efimenko et aI.,

2005). Depending on the stringency by which candidates are identified, this approach can be extremely accurate or subject to an enormous false-positive rate. For example, genes that contain x-boxes highly similar to a consensus sequence (an updated Hidden Markov Model score of greater than 16 as defined by Blacque et aI., 2005) within the first 200bp of their upstream promoter regions almost certainly encode cilia-associated proteins. Of the 45 genes that meet this criteria (Blacque et aI., 2005), 14 were known ciliary genes, including components of the 1FT machinery and the BBSome, while an additional 10 have been shown subsequently to playa role in cilium structure/function. The remaining 21 genes should necessarily be a focus of future studies, and are shown in Table 7-2. Lowering the stringency of X-box quality will undoubtedly lead to the identification of additional novel components, albeit with a higher false positive rate.

7.2 Future directions of 1FT research in C. e/egans

7.2.1 The role of protein modifiers in 1FT

Table 7-2 lists a number of largely uncharacterised C. elegans genes that likely encode proteins with ciliary functions. Two of the fourteen proteins listed are involved in phosphate transfer, specifically the kinase H01 G02.2 and the phosphatase T12B3.1. By decreasing the stringency of X-box position

212 requirements (from 300bp upstream to 1000bp), a significant number of kinases and phosphatases with canonical X-box sequences can be identified. A classic example is the M04C9.5/dyf-5 gene, which encodes a kinase that has been shown to regulate ciliary length in the worm, specifically by restricting the movement of heterotrimeric Kinesin-2 to ciliary middle segments (Burghoorn et al.,2007).

As seen in the model for nematode 1FT (see Figures 1-6, 1-7, and 1-8), there are a number of key transition points that occur during the ciliary transport process. At the middle-to-distal segment midpoint, the anterograde 1FT motor heterotrimeric Kinesin-2 separates itself from the 1FT complex, and likely returns via a dynein-mediated transport mechanism to the basal body/transition zones.

At the tips of the distal segments, the OSM-3 anterograde motor needs to become deactivated, while, concurrently, the IFT-dynein complex must become' active. While the 1FT motors undergo these alterations, they must also change their respective associations with the two 1FT subcomplexes. Finally, at the base of the cilium, the returning, retrograde-driven 1FT complex likely disassembles and ultimately reassembles into a functional tip-directed raft. In each of the aforementioned steps, there are almost certainly proteins that are specifically involved in facilitating the re-assembly of the 1FT complex. Based on previous observations in Chlamydomonas and C. elegans, it appears that kinases/phosphatases provide some of this functionality. In addition to DYF­

5/LF4, a second kinase, GSK3, has been shown to inhibit Kinesin-light chains and disrupt anterograde 1FT in Chlamydomonas (Wilson & Lefebvre, 2004).

213 Because of the added complexity of 1FT in C. elegans, there may be additional

protein modifiers that play roles in 1FT particle rearrangement, and may in fact be worm-specific.

The study of kinases, phosphatases, and other protein modifiers (such as

phosphodiesterases, deactylases, etc.) with respect to signalling changes that

result in 1FT particle rearrangement is ideal for future study in C. elegans.

Because of the detailed protein transport profiling techniques developed

specifically for 1FT research, it is possible to identify very minute changes in 1FT

trafficking. Subtle phenotypes, such as presence of Kinesin-2 transport profiles in

ciliary distal segments (as seen in dyf-5 mutants), or slightly altered 1FT-particle

velocities and localisation patterns (such as those observed in nph-1 and nph-4

mutants; Jauregui et aI., 2008), require more comprehensive analysis than

previous studies examining more core 1FT components, but are still reasonably

efficient, and have the potential to offer tremendous insight into the regulation of

1FT.

7.2.2 The basal body-transition zone module

Chapter 4 of this thesis describes a complex of B9 domain-containing

proteins (MKS-1, MKSR-1, MKSR-2) that specifically localise to the ciliary basal

body-transition zone region, and likely regulate the addition of ciliary sensory

proteins to the 1FT complex (Bialas et aI., 2009; Williams et aI., 2008). The

aforementioned NPH-1 and NPH-4 proteins also specifically localise to the basal

body-transition zone region, and play subtle roles in the coordination of

anterograde 1FT motors (Jauregui et aI., 2008). Interestingly, combining any of

214 the mks/mksr mutants with any of the nph mutants results in severe ciliogenic defects (Williams et aI., 2008). This data strongly hints at the presence of a multisubunit complex of basal body-transition zone proteins that is involved in the assembly of the 1FT complex at the base of cilia. The specific role played by each protein in 1FT complex assembly is likely varied; some participate in 1FT motor coordination (e.g., the NPH proteins), while others participate in the loading of specific cargo (e.g., the MKS/MKSR proteins).

Connected with the loading of 1FT cargo, such as ciliary membrane proteins, onto the 1FT raft is the process of cilium-directed vesicular transport.

Reports from Trypanosoma cruzi have demonstrated that specific ciliary components are trafficked via post-golgi vesicles to a docking site near the transition zones, and that this process is essential for ciliary localisation (Godsel

& Engman, 1999). Furthermore, observation of rhodopsin transport to the (ciliary) outer segments of frog photoreceptor cells determined that a similar transition zone vesicular docking mechanism was required (Deretic & Papermaster, 1991).

It has been posited that 1FT particles assemble near the transitional fibers of the basal body, the presumptive location of vesicle fusion would take place in a region proximal to this (Deane et aI., 2001).

Recent research has hinted at a cilium-specific golgi-based vesicular trafficking pathway. IFT20, in addition to its ciliary localisation, is also found on the golgi-apparatus (Follit et aI., 2006). Subsequent research has determined that IFT20 interacts with the Golgin GMAP-21 0/TRIP11, which itself appears to perform a subtle ciliogenic function (Follit et aI., 2008). Golgins are a family of

215 proteins that localise to Golgi and Golgi-based vesicles, and are capable of tethering vesicles to membranes and the cytoskeleton (reviewed in Barr, 2009).

The specific targeting function of Golgins is incumbent on interactions with a number of GTPases of the ARF, ARL, and RAB families (Barr, 2009).

Interestingly, a number of established ciliary proteins, as well as many putative ciliary candidates (as seen in Tables 7-1 and 7-2) belong to these families. Such proteins include Rabin8, an interactor of the BBSome (Nachury et aI., 2007) and

BBS3/ARL6. As previously indicated, the incorporation of vesicles carrying ciliary proteins into the 1FT complex is most likely to occur proximally to the transition zones. One might expect, therefore, genetic interactions between members of the ciliary GTPases and the established transition zone localised proteins (MKS-

1, MKSR-1, MKSR-2, NPH-1, NPH-4). Many of the reagents needed to carry out a detailed analysis of the interactions occurring at the transition zone, including

mutants defective in many of the genes, as well as strains expressing GFP- tagged variants of each component, are already available in the worm, and therefore this organism represents an excellent system for studying this exciting and complex story.

7.3 Placing the novel C. elegans 1FT proteins into the core 1FT complex

Due to certain limitations of the C. e/egans model system, it is prohibitively

difficult to perform significant biochemical analyses on the 1FT complex in the worm. The model system Chlamydomonas reinhardtii has consistently

demonstrated its effectiveness with regard to this sort of study, as seen by the

216 original isolation of many of the known IFT-A and IFT-B components (Cole et aI.,

1998), as well as a further resolution of some of the specific physical interactions that occur within each subcomplex (Lucker et aI., 2005). This data is invaluable in terms of piecing together the 1FT complex, as it can ultimately demonstrate the role(s) played by each core 1FT protein in the 1FT complex, and thus, 1FT function.

Over the past five years, a number of highly conserved 1FT components, not originally identified in the original biochemical fractionation studies of

Chlamydomonas, have been identified in C. elegans. These proteins include

BBS-1, BBS-2, BBS-3, BBS-5, BBS-7, and BBS-8 (Ou et aI., 2007; Blacque et aI., 2004; Mak et aI., 2006), DYF-1 (Ou et aI., 2007), DYF-2 (Efimenko et aI.,

2006), DYF-3 (Murayama et aI., 2005; Ou et aI., 2007), DYF-11 (Chapter 3),

DYF-13 (Blacque et aI., 2005), IFTA-1 (Blacque et aI., 2006), IFTA-2 (Schafer et

aI., 2006), and OSM-3 (Ou et aI., 2005). These proteins, for the most part,

appear to play more peripheral roles in their specific 1FT subcomplexes, which

may explain why they were not picked up in the Chlamydomonas 1FT

biochemical studies.

Nonetheless, an excellent next step in characterising the roles and

interactions of these specific proteins would be a more detailed biochemical

analysis, studying the interactions with established 1FT proteins and examining

their subcellular localisation patterns in more canonical ciliary model systems.

This research, to some extent, is already starting to be performed, as evidenced

by a number of recent studies. Follit et al. (2009) recently published an excellent

217 study examining the murine orthologues of DYF-1, DYF-6, and DYF-11. They found that all three, as predicted by the genetic analyses in C. elegans, were biochemically linked to 1FT-B. Furthermore, overexpression of DYF-11, renamed

IFT54 in Chlamydomonas, was found to result in mislocalisation of IFT20, a component of the core 1FT machinery that also localises to golgi bodies and interacts with GMAP21 0 (Follit et aI., 2008), hinting at a dominant negative relationship between the two proteins.

Another powerful model system that can be used to validate the model of

1FT posited by C. elegans researchers are the photoreceptor cells of zebrafish. It has been demonstrated that the D. rerio OSM-3 orthologue, KIF17, is required to build photoreceptor outer segments, which are elaborate ciliary structures

(Insinna et aI., 2008). Loss of Kinesin-2 in the same system appears to affect the non-ciliary inner segments (Insinna et aI., 2009). One of the more unexpected results from the C. elegans 1FT studies was the observation that two anterograde

1FT motors (OSM-3 and Kinesin-2) were acting cooperatively through an interaction with the BBSome. It would be a remarkable discovery to observe a similar interaction occurring in zebrafish photoreceptor cells, as it would strongly imply that the model described in C. elegans is, in fact, an appropriate model for cells that possess cilia that are structurally similar to those of the nematode (i.e.,

possess marked A-tubule extensions).

7.4 General conclusions

Ultimately, the results of this thesis highlight the strengths of C. elegans as

a ciliary model organism. Because of the excellent resources available to

218 nematode researchers, such as high-throughput mutant generation facilities, bioinformatic tools available (especially with respect to X-box sequence identification), genomic tools that include cell expression data, and relative ease of GFP tagging peptides, comprehensive characterisation of putative novel ciliary gene candidates is significantly more rapid than in other model systems.

Ultimately, because of the limitations of the nematode model, especially with respect to biochemical studies in ciliated neurons, it is important to use the knowledge gained in nematode research to focus related studies in other systems, particularly mammalian cell research. As described above, this work has already begun, and is resulting in impactful observations. This is not to say that ciliary research in the worm is losing its usefulness. Quite the contrary; it seems that nematode cilia research is slightly transitioning its focus to examining more subtle alterations to the 1FT machinery and cilium-based signalling, as seen in the nphp and dyf-5 mutants (Jauregui et aI., 2008; Burghoorn et aI., 2005).

Transmission electron microscopy (TEM) will become an invaluable tool with

regards to such analyses, as it can identify extremely subtle axonemal defects in various mutant backgrounds. As seen in Chapter 4 with the B9 domain containing proteins, C. elegans is also a powerful model system with regards to the study of cilium-based signalling. Furthermore, because of the relative

simplicity of the C. elegans neuronal system, the role played by cilia in

behavioural responses to stimuli such as changes in temperature (Chapter 6)

can be more easily deduced.

219 7.5 Tables

Table 7-1. Currently uncharacterised top ciliary candidate genes based on various ciliomic analyses. Shown are the putative C. elegans (Worm) and H. sapiens (Human) gene orthologues. 'x' denotes presence in ciliomic study. Avidor-Reiss: Avidor-Reiss et al. (2004); Li: Li et al. (2004); Bla: Blacque et al. (2005) C. elegans X-box search;

Efi: Efimenko et al. (2005) C. elegans X-box search; Ost: Ostrowski et al. (2002) proteomics of respiratory cilia; Paz: Pazour et al. (2005) proteomics of

Chlamydomonas cilia; Smith: Smith et aI., 2005: proteomics of Tetrahymena cilia.

Comparative Blo- Gene genomics informatics Proteomlcs Avidor- Worm Human Reiss LI Bla. Efi. Ost. Paz. Smith Annotation tubulin-polymerization C32E8.3 TPPP3 x x x x promoting protein C54C6.6 C16orf80 x x x x putative transcription factor qst-1 GSTP2 x x x x qlutathione-S-transferase organic solute transport R10F2.5 OSCP1 x x x protein pde-1 PDE1C x x Phosphodiesterase K07G5.3 CC2D2A x x MKS6 cAMP-dependent protein kin-1 PRKACA x x kinase hydroxyphenyl pyruvate hpd-1 HPD x x x deoxyqenase F13H8.2 WDR3 x x WD repeat-containinq

220 Table 7-2. Putative ciliary genes based that fit stringent X-box criteria. Genes identified by Blacque et al. (2005) that are currently uncharacterised in C. elegans (worm) and possess highly canonical X-boxes (HMM score> 16.0) within 300bp upstream of the start of the gene. Human homologues are shown

(when applicable), as are available C. elegans mutants.

Worm Gene Mutant? Human Annotation gk564, K08D12.2 ok1863 XRP2 Human orthologue implicated in retinitis pigmentosa F56H9.4 gpa-9 GNAT2 GTPase ok582, F40F9.1 xbx-6 ak221 FAIM2 NMDA-receptor bindina Nematode-specific protein implicated in centrosome F56A3.4 spd-5 or213 function ABC transporter; multidrug resistance protein family Y43F8C.12 mrp-7 ABCC3 member

T12B3.1 tm3270 PTPDC1 Phosphatase C27F2.1 WDR60 WD-repeat containina (possiblv nematode-specific) gk1001, C47E8.6 tm3429 Uncharacterised nematode-specific protein tm1745, Y37E3.5 arl-13 tm2322 ARL13B GTPase implicated in Joubert syndrome C06A12.4 gcy-27 Guanylyl cyclase

F19H8.3 arl-3 tm1703 ARL3 GTPase ok698, K07G5.3 gk674 CC2D2A C2 Domain protein implicated in Meckel-Gruber syndrome Nematode-specific Patched-related protein implicated in C53C11.3 gk472 sterol-sensing

H01G02.2 ok200 CDK7 CDK-activating kinase

T19A5.4 nhr-44 tm1834 Nematode-specific nuclear hormone receptor tm776, C15F1.7 sod-1 tm783 SOD1 Superoxide dismutase F41E7.9 Uncharacterised nematode-specific protein F21D5.2 OTUD6B OTU-domain containing

T06G6.3 gk546 Uncharacterised nematode-specific protein F53A2.4 nUd-1 NUDC Nuclear migration protein

221 APPENDICES

Appendix A. CD-ROM Data

The CD-ROM attached forms a part of this work, specifically related to the comparative genomic analyses depicted in Chapter 2. All Data files can be opened with MSExcel, and are saved in the Excel 97-2003 format.

Data Files:

• Supplementary File 1. Location of strains used in thesis 44 KB • Supplementary File 2-1. Results of comparative genomic analysis 145 KB • Supplementary File 2-2. Putative B. dendrobatidis ciliary proteins 73 KB

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