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Journal List > Mol Biol Cell > v.18(5); May 2007

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Mol Biol Cell. 2007 May; 18(5): 1554–1569.
doi: 10.1091/mbc.E06-09-0805.
PMCID: PMC1855012
Copyright © 2007 by The American Society for Cell Biology
Sensory Ciliogenesis in Caenorhabditis elegans: Assignment of IFT Components into Distinct Modules Based on Transport and Phenotypic ProfilesAn external file that holds a picture, illustration, etc. Object name is dbox.jpg
Guangshuo Ou,* Makato Koga,†‡ Oliver E. Blacque,‡§‖ Takashi Murayama,† Yasumi Ohshima,† Jenny C. Schafer, Chunmei Li,§ Bradley K. Yoder, Michel R. Leroux,corresponding author§ and Jonathan M. Scholeycorresponding author*
*Center for Genetics and Development, Section of Molecular and Cellular Biology, University of California, Davis, CA 95616;
†Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan;
§Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;
‖School of Biomolecular and Biomedical Science, University College Dublin Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; and
Department of Cell Biology, University of Alabama at Birmingham Medical Center, Birmingham, AL 35294
Erika Holzbaur, Monitoring Editor
corresponding authorCorresponding author.
Address correspondence to: Michel R. Leroux (Email: leroux/at/sfu.ca) or Jonathan M. Scholey (Email: jmscholey/at/ucdavis.edu)
‡ These authors contributed equally to this work.
Received September 11, 2006; Revised February 2, 2007; Accepted February 7, 2007.
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Abstract
Sensory cilium biogenesis within Caenorhabditis elegans neurons depends on the kinesin-2–dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs (Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as “modules.” We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegans IFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis.
INTRODUCTION
Projecting from most eukaryotic cells, the cilium is an important microtubule (MT)-based apparatus that acts as a motile organelle and/or sensory antenna for proper cellular physiology; loss of ciliary function causes various human diseases including polycystic kidney disease, primary ciliary dyskinesia, Bardet-Biedl syndrome (BBS) and Meckel syndrome (Rosenbaum and Witman, 2002 blue right-pointing triangle; Scholey, 2003 blue right-pointing triangle; Pan et al., 2005 blue right-pointing triangle; Badano et al., 2006 blue right-pointing triangle; Marshall and Nonaka, 2006 blue right-pointing triangle; Singla and Reiter, 2006 blue right-pointing triangle). Ciliary proteins include those that are part of the axoneme, the ciliary membrane, and the matrix, as well as those required for building and maintaining the integrity of the microtubule-based organelle. Many of the latter proteins are involved in intraflagellar transport (IFT), a kinesin-2–dependent motility process in which macromolecular complexes called IFT particles deliver ciliary precursors to their site of incorporation into cilia (Kozminski et al., 1993 blue right-pointing triangle; Rosenbaum and Witman, 2002 blue right-pointing triangle; Scholey, 2003 blue right-pointing triangle). The nature of the components, as well as the organization and specific functions of the IFT machinery, is incompletely understood. Biochemically and genetically, IFT components are shown to include one or more kinesin-2 motors that drive anterograde transport in motility assays performed in vivo and in vitro (Cole et al., 1993 blue right-pointing triangle; Snow et al., 2004 blue right-pointing triangle; Imanishi et al., 2006 blue right-pointing triangle; Pan et al., 2006 blue right-pointing triangle), a presumptive retrograde IFT-dynein motor (Piperno et al., 1998 blue right-pointing triangle; Pazour et al., 1999 blue right-pointing triangle; Porter et al., 1999 blue right-pointing triangle; Signor et al., 1999 blue right-pointing triangle; Wicks et al., 2000 blue right-pointing triangle), and the IFT particle subcomplexes A and B (which together contain at least 6 and 11 proteins, respectively; Cole et al., 1998 blue right-pointing triangle; Baker et al., 2003 blue right-pointing triangle; Lucker et al., 2005 blue right-pointing triangle). Any one of these components could conceivably be used as an attachment point for ciliary cargo (Rosenbaum and Witman, 2002 blue right-pointing triangle; Scholey, 2003 blue right-pointing triangle).
To assess the role of such ciliary proteins in IFT and cilium biogenesis, we previously developed a time-lapse fluorescence microscopy assay in C. elegans that allows us to monitor the motility of tagged components of the IFT machinery in live animals (Orozco et al., 1999 blue right-pointing triangle; Signor et al., 1999 blue right-pointing triangle; Snow et al., 2004 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle). Time-lapse fluorescence motility assays and analyses of mutants have subsequently revealed that additional proteins participate in IFT. For example, DYF-1, DYF-3, DYF-13, and IFTA-1 are also important for ensuring the proper assembly and structural integrity of the cilium and at least in the case of DYF-1 and DYF-13, are specifically required for building the distal segment of the cilium (Blacque et al., 2005 blue right-pointing triangle; Murayama et al., 2005 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle,b blue right-pointing triangle; Blacque et al., 2006 blue right-pointing triangle). This observation is of significant interest because many cilia, including those present in vertebrate retinal cells and olfactory neurons, those of C. elegans amphid channel neurons, as well as those of Chlamydomonas engaged in mating, possess a bipartite structure (Reese, 1965 blue right-pointing triangle; Mesland et al., 1980 blue right-pointing triangle; Perkins et al., 1986 blue right-pointing triangle). The ciliary axoneme nucleates from a transitional zone containing a basal body (modified centriole) and begins with a so-called “initial” or “middle” segment, built of doublet microtubules, and can terminate with a “distal” segment composed of singlet microtubules (see Figure 1Figure 1., A and B; Perkins et al., 1986 blue right-pointing triangle). In C. elegans, the formation of the middle segment depends on the concerted action of two distinct kinesin-2 motors (heterotrimeric kinesin-II and homodimeric OSM-3-kinesin), whereas the assembly of the distal segment is independent of kinesin-II, requiring only the OSM-3-kinesin and associated regulators, such as DYF-1 (Snow et al., 2004 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle; Evans et al., 2006 blue right-pointing triangle; Pan et al., 2006 blue right-pointing triangle). The function of distal segments is unclear, but in the channel cilia they appear to be required for sensory signaling. The six known BBS proteins from C. elegans are bona fide IFT components that appear to mediate the interaction between the two motors and the subcomplexes A and B, but their organization within motor-IFT particle complexes and mechanism of action is poorly understood (Blacque et al., 2004 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle).
Figure 1.
Figure 1.
Figure 1.
Schematics and fluorescence microscopy of C. elegans neuronal cilia. (A, C, D, I, and L) Cilia structure schematics adapted from Ward et al. (1975) blue right-pointing triangle. (B) The schematic drawing of axonemal MTs in amphid channel cilia which contain 1-μm-long transitional (more ...)
Although this description is satisfactory in outline, two major questions about the mechanisms of cilium biogenesis in C. elegans remain unanswered: 1) how is the IFT-protein machinery organized? 2) what other components are involved in anterograde and retrograde IFT, as well as cilium biogenesis? Here, we used in vivo transport assays in ciliary mutants to obtain insights into the architecture of the IFT machinery and we found that many components can be organized into several functionally specialized modules. In complementary studies, we carried out forward genetic screens to isolate novel components involved in cilium biogenesis and used positional cloning approaches to identify the molecular identity of one of them. Furthermore, based on the transport behavior of IFT-particles and ciliary structure defects in the newly and previously obtained ciliary gene mutants, they were phenotypically categorized, allowing us to also assign the encoded proteins to the IFT modules, or to specific aspects of cilium biogenesis.
MATERIALS AND METHODS
Strains and Genetic Crosses
Worms were grown on an NGM plate seeded with the Escherichia coli strain OP50 at 20°C using standard methods (Brenner, 1974 blue right-pointing triangle). Wild-type (WT) C. elegans strains used in this work were Bristol (N2) and CB4856. Fluorescence-tagged reporters (e.g., bbs::gfp proteins) were crossed from WT worms to various mutant backgrounds and single-worm PCR was used to follow the mutations in each case. Deletions [bbs-1(ok1111) and bbs-8(nx77)] were detected using a single PCR reaction, where primers flanking the deletion can distinguish WT and mutant copies of the gene. Two PCR reactions detected point mutations [osm-12(n1606)], where one reaction preferentially amplifies the WT gene and a second reaction preferentially amplifies the mutated gene. Mutants used in this work are summarized in Tables 1 and 2. The following strains expressing green fluorescent protein (GFP) or RFP (red fluorescent protein) markers were used to mark cilium structures: amphid and phasmid channel cilia: SP2101, osm-6(p811); mnIs17[osm-6::gfp; unc-36(+)]; SL16, Ex[osm-1::gfp + rol-6(su1006)]; PT47, Ex[che-2::gfp + rol-6(su1006)]; OLQ cilia: CX3716, lin-15(n765); kyIs141[osm-9::GFP5 + lin-15(+)]; AWA cilia: CX3344, kyIs53[odr-10::GFP]; AWB cilia: CX3553, lin-15(n765); kyIs104 X [str-1::GFP+ lin-15(+)]; and AWC cilia: CX3695, lin-15(n765); kyIs140[str-2::GFP + lin-15(+)]; PY2417, oyIs44[odr-1::RFP]; AFD cilia: PY1322, oyIs18[gcy-8::GFP].
Table 1.
Table 1.
Summary of amphid channel ciliary defects in novel dye-filling mutants
Table 2.
Table 2.
Summary of amphid channel ciliary defects in previously known mutants
The strain names of each mutant carrying the above marker are listed in Tables 1 and 2.
GFP Expression Analysis in C. elegans
Translational gfp fusion constructs were generated via fusion PCR as previously described (Hobert, 2002 blue right-pointing triangle). For the translational gfp fusion constructs, the entire exonic and intronic sequence, along with a 5′UTR promoter fragment, was fused in-frame with gfp. The translational 5′UTR consisted of 536 base pairs for Y110A7A.20. Transgenic animals expressing translational gfp transgenes as extrachromosomal arrays in dpy-5(e907);Ex[dpy-5(+)] animals were generated as described previously (Ansley et al., 2003 blue right-pointing triangle).
Genetic Screen and Behavioral Assays
N2 adults were treated with EMS, and F2 progeny were screened for defects in chemotaxis assays toward NaAc or E. coli (OP50) lawn assay. Isolated mutants were further examined in dye-filling assays with DiI (Molecular Probes, Eugene, OR). All of the animal behavioral assays were performed as previously described (Uchida et al., 2003 blue right-pointing triangle; Murayama et al., 2005 blue right-pointing triangle). All mutants were backcrossed once with N2 animals.
Genetic Mapping and Germline Rescue
Genetic mapping was performed by the snip-SNP method (Wicks et al., 2001 blue right-pointing triangle). Genomic DNA of F35D11.11 was amplified from dyf-14(ks69) via PCR with a high-accuracy LA Taq polymerase (Takara, Tokyo, Japan) and purified with a QIAquick PCR purification kit (Qiagen, Chatsworth, CA). The PCR fragments were sequenced to identify the molecular lesion. YAC Y74E4 DNA was injected into the germline of dyf-14(ks69) for rescue.
Fluorescence Microscopy
Intraflagellar transport and cilium morphology was assayed as described previously (Snow et al., 2004 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle; Evans et al., 2006 blue right-pointing triangle). The fluorescent transgenic worms were anesthetized with 10 mM levamisole, mounted on agar pads, and maintained at 21°C. Images were collected using an Olympus microscope (Melville, NY) equipped with a 100×, 1.35 NA objective and an Ultraview spinning disk confocal head with excitation by a 488- argon ion laser (Perkin Elmer, Norwalk, CT). Time-lapse images were acquired at 0.3 s/frame using a cooled charge-coupled device camera (ORCA-ER; Hamamatsu, Bridgewater, NJ). Kymographs were created from the resulting stacked tiff images using Metamorph software (Universal Imaging, West Chester, PA), and the rates of fluorescent IFT particle motility along middle and distal segments were measured as described previously (Snow et al., 2004 blue right-pointing triangle).
RESULTS
Strategy for the Comprehensive Analysis of the IFT Machinery and Sensory Ciliogenesis
Our aim for this study was to analyze, as comprehensively as possible, the IFT machinery that builds the ciliated dendritic endings on ciliated neurons in the C. elegans nervous system (Ward et al., 1975 blue right-pointing triangle; Perkins et al., 1986 blue right-pointing triangle; Evans et al., 2006 blue right-pointing triangle) based on assays of IFT and ciliary mutant phenotypes. To begin with, we examined ciliary morphology and IFT in the various neuronal cilia of C. elegans, which have diverse shapes and distinct sensory functions (Figure 1Figure 1.; Ward et al., 1975 blue right-pointing triangle). The cilia of monociliated ASE, ASG, ASH, ASI, ASJ, ASK, and biciliated ADF and ADL neurons together form bundles of amphid channel cilia whose endings are exposed directly to the environment through openings in the cuticle, allowing them to sense water-soluble chemicals (Figure 1Figure 1., A, C, D, E, G, and H; Perkins et al., 1986 blue right-pointing triangle; Perens and Shaham, 2005 blue right-pointing triangle). By contrast, the olfactory neurons that detect volatile odorants, namely AWA, AWB, and AWC, have wing- or fan-shaped ciliated endings that are ensheathed by the adjacent sheath cells (Bargmann, 1997 blue right-pointing triangle). Specifically, the AWA cilia form extensively branched “filaments” (Figure 1Figure 1., I and M); AWB cilia form irregular “forks” with two branches of varying length (Figure 1Figure 1., J and N); and AWC cilia form two flattened, extended and “fan”-like sheets (Figure 1Figure 1., K and O). Finally, the thermotactic AFD neuron has a short cilium and many microvilli-like projections (Figure 1Figure 1., L and P; Bargmann, 1997 blue right-pointing triangle). Among these diverse cilia subtypes, we were only able to reliably detect robust IFT in the amphid and phasmid channel cilia, where IFT particles move in a biphasic manner along the initial and distal segments of the axoneme (Snow et al., 2004 blue right-pointing triangle; Figure 1Figure 1., B and F). For example, using a known fluorescent IFT-particle marker, OSM-1::GFP, IFT particles moved at 0.70 ± 0.09 μm/s (n = 105) in the initial segment and 1.18 ± 0.14 μm/s (n = 110) along the distal segment of all measured phasmid cilia. This latter finding differs from previous data (Qin et al., 2005 blue right-pointing triangle) and indicates that the coordinate action of kinesin-II and OSM-3-kinesin drives biphasic IFT particle transport in the phasmid cilia, as well as in the amphid channel cilia (Snow et al., 2004 blue right-pointing triangle). Accordingly, in the studies described below, we focused on an examination of the transport and distribution of IFT particles in the hydrophilic molecule-sensing amphids and phasmids in various known and novel ciliary mutants.
Previously Identified Mutants Implicated in Cilium Biogenesis
Work done by the C. elegans community has produced a valuable collection of ciliary mutants that can be used to illuminate mechanisms of IFT and ciliogenesis (Scholey, 2003 blue right-pointing triangle; Inglis et al., 2006b blue right-pointing triangle). For example, the existing dye-filling (dyf), chemotaxis-defective (che), osmotic avoidance–defective (osm), and dauer larva formation–defective (daf) mutants [with the exception of osm-8(n1518), osm-10(n1602), and osm-13 (e329)] have defective sensory cilia structure and/or function (Table 2; Starich et al., 1995 blue right-pointing triangle; Scholey et al., 2004 blue right-pointing triangle), and in addition, the mechanosensation defective mutant mec-8 was also reported to display ciliary defects (Perkins et al., 1986 blue right-pointing triangle). Furthermore, it is possible that some of the uncoordinated (unc) mutant genes might also be involved in cilium biogenesis, including, for example, UNC-119, whose ortholog in Chlamydomonas is a POC protein (proteome of centriole; Keller et al., 2005 blue right-pointing triangle) and whose ortholog in Drosophila is a cilia “compartment” protein (Avidor-Reiss et al., 2004 blue right-pointing triangle). Accordingly, we investigated IFT and ciliary structure in five Unc mutants, namely unc-6, unc-33, unc-101, unc-104, and unc-119 mutants. In all five animals we found abnormalities of dye uptake and in unc-101 and unc-119 mutants the distal segments of cilia were not formed (Table 2).
Genetic Screen for Novel Components involved in Cilium Biogenesis
To identify new IFT and ciliogenesis components, we mutagenized N2 worms with ethyl-methanesulfonate (EMS) and used behavioral assays to screen for F2 mutants defective in chemotaxis toward the hydrophilic chemical, sodium acetate (NaAc) or E. coli food (Figure 2Figure 2.A). One hundred twenty-six independent NaAc chemotaxis-defective strains were isolated from 150,000 mutagenized haploid genomes, and we used dye-filling assays to identify 47 of them as being dyf mutants, which are likely defective in ciliary structure. We used single-nucleotide polymorphisms between Bristol N2 strain and Hawaii CB4856 strain to map the Dyf phenotype (Table 1 and Supplementary Table S1), allowing us to position 42 mutations onto specific chromosomes, with 21 of them being positioned between two genetic markers. This snip-SNP mapping data provides a good basis for further positional cloning of the novel Dyf mutations; for example, many of the dyf mutants initially identified and mapped (Starich et al., 1995 blue right-pointing triangle) were subsequently cloned (Blacque et al., 2005 blue right-pointing triangle; Murayama et al., 2005 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle; Bell et al., 2006 blue right-pointing triangle; Efimenko et al., 2006 blue right-pointing triangle).
Figure 2.
Figure 2.
Figure 2.
Identification and characterization of novel ciliary components. (A) Strategy for forward genetic screens to isolate novel ciliary components. N2 worms were mutagenized with EMS. About 150,000 haploid genomes were screened with NaAc chemotaxis assays, (more ...)
Five additional mutant strains were isolated by screening ~100,000 EMS-mutagenized haploid genomes for defects in “food-orientation” E. coli assays. In these mutant strains, 2–8% of animals moved to an area more than 1.5 cm away from a bacterial lawn within 3 h, whereas fewer than 0.1% of WT animals did so. Among such mutants, FK247 (ks68) and FK263 (ks69) mutants displayed a Dyf phenotype, suggesting abnormal ciliary structures. ks68 is allelic to the che-2 gene, which is known to encode an IFT-particle B subunit homologue, IFT80, whereas we now show that ks69 is a novel ciliary mutant that we name dyf-14.
We examined the distribution of the fluorescence-tagged IFT particle protein, OSM-6::GFP, in the amphid channel cilia of dyf-14(ks69) and observed a severe loss of cilia and abnormal IFT particle aggregates in the dendritic endings (Figure 2Figure 2., B and C). Similarly, no ciliary structure was detectable in the phasmid cilia of dyf-14(ks69), and the GFP signal terminates at the posterior region of the cell body (Figure 2Figure 2., D and E). Given that the dyf-14(ks69) mutant exhibited an interesting ciliary phenotype consistent with the gene encoding a protein important for cilium biogenesis, we sought to clone dyf-14(ks69). SNP mapping was first used to place dyf-14(ks69) within a 2.7-map unit region on chromosome II, and the germline transformation of the YAC clone Y74E4 fully rescued its Dyf phenotype. Sequencing of a predicted open reading frame (F35D11.11) of Y74E4 in dyf-14(ks69) animals revealed a G-to-A transition at 2210 nt, which changes 737 R to a premature stop codon. These data suggest that F35D11.11 corresponds to the dyf-14 gene (Figure 2Figure 2.F). To identify its coding region, we amplified the sequences expressed by dyf-14 cDNAs using RT-PCR. The dyf-14 gene encodes three proteins of 1901 (DYF-14C), 1959 (DYF-14A), or 1972 (DYF-14B) amino acid residues, with different C-termini (Figure 2Figure 2., F and G). DYF-14 proteins contain four predicted coiled-coil regions and one myosin tail-like domain. A database search revealed that the DYF-14B protein shows sequence similarity to the conceptual translation product of a human cDNA clone, named ENSP00000357793 (E value 2.6e−61), whose involvement in cilium biogenesis is unknown.
A Modular Architecture for the IFT Protein Machinery in Amphid Channel Cilia
To examine the relative organization of most C. elegans IFT proteins compared with known Chlamydomonas ciliary protein homologues, as well as recently discovered C. elegans IFT-related proteins within the IFT particle, we expressed GFP-tagged versions of these proteins in bbs mutant backgrounds and analyzed their in vivo transport profiles. On the basis of our previous findings (Ou et al., 2005a blue right-pointing triangle), we expected the GFP-tagged proteins to associate with either the kinesin-II/IFT-A subcomplex or the OSM-3/IFT-B subcomplex, which are dissociated in bbs mutants and to travel at their respective velocities of 0.5 μm/s along only the middle segment or 1.3 μm/s along the middle and distal segments. By employing this experimental approach, we have obtained an extensive set of transport profiles, which has now allowed us to present the first comprehensive description of the IFT machinery that builds C. elegans sensory cilia.
In addition, we have also examined the distribution and transport of a fluorescent IFT particle protein marker in the cilia of our collection of ciliary mutants. By careful comparison of the observed cilia/IFT defects with those of known cilia/IFT mutants, we assigned them to the same set of phenotypic classes. In this way, we were able to assign the known and yet-to-be-identified ciliary proteins into the following functionally specialized modules or to steps in the pathway of ciliogenesis (summarized in Figure 7Figure 7.).
Figure 7.
Figure 7.
Figure 7.
Modular architecture of the IFT-protein machine and molecular framework for cilium biogenesis in C. elegans. (A) Model of the modular architecture of the C. elegans IFT-protein machine. Bolded IFT proteins denote those for which data were obtained in (more ...)
IFT-Particle Subcomplex A and B Modules: Conveyors for Anterograde and Retrograde IFT
The IFT particles, which were first identified in Chlamydomonas flagellar extracts, consist of two subcomplexes, A and B (Cole et al., 1998 blue right-pointing triangle; Lucker et al., 2005 blue right-pointing triangle). These subcomplexes are thought to be capable of binding multiple cargo molecules such as ciliary structural precursors and signaling molecules and transporting them along the cilium (Rosenbaum and Witman, 2002 blue right-pointing triangle; Scholey, 2003 blue right-pointing triangle). Previously, we showed that in bbs mutants, CHE-11/IFT140 (IFT complex A) moves independently from both OSM-5/IFT88 and CHE-2/IFT80 (IFT complex B), suggesting that IFT particle A and B subcomplexes are dissociated and moved separately by kinesin-II and OSM-3-kinesin motors, respectively (Ou et al., 2005a blue right-pointing triangle). Here, we rigorously test and extend this model by determining the transport profiles of another three known subcomplex B components (OSM-1/IFT172, CHE-13/IFT57, and Y110A7A.20/IFT20) in bbs mutants. First, we constructed a translational GFP reporter for the nematode homolog of Chlamydomonas IFT20, Y110A7A.20, which has not been studied in C. elegans, and determined that, like other IFT particle components, IFT-20::GFP undergoes biphasic transport along the middle (~0.7 μm/s) and distal segments (~1.3 μm/s) of WT cilia (Figure 3Figure 3.C and Table 3). In agreement with our previous observations (Ou et al., 2005a blue right-pointing triangle), we found that all three GFP-tagged subcomplex B proteins displayed OSM-3-kinesin–associated fast rates in the middle (~1.1 μm/s) and distal segments (~1.3 μm/s) of bbs mutant cilia (Figures 3Figure 3., D, E, G, and H, and and4,4Figure 4., B–D, and H, and Table 3).
Figure 3.
Figure 3.
Figure 3.
Transport of IFT-particle, accessory and cargo proteins in WT and bbs mutants. Shown are representative “still” fluorescence images and corresponding kymographs (M, middle; D, distal) and kymograph schematics (M′ and D′), (more ...)
Table 3.
Table 3.
Transport velocities of GFP-tagged IFT proteins in WT and bbs mutant animals
Figure 4.
Figure 4.
Figure 4.
BBS proteins participate in IFT as functionally interdependent components of the same biological process. Shown in A–H are representative “still” fluorescence images and corresponding kymographs (M and D) and kymograph schematics (more ...)
DYF-3/Qilin likely functions as a subcomplex B component based on its IFT motility and mutant ciliary phenotype (Murayama et al., 2005 blue right-pointing triangle; Ou et al., 2005b blue right-pointing triangle). Interestingly, the C. elegans interactome project (Li et al., 2004b blue right-pointing triangle) uncovered a protein–protein interaction between BBS-7 and DYF-3, indicating that DYF-3 may help dock, or link, subcomplex B to the BBS protein module. As predicted by this hypothetical interaction network, we found that in bbs mutants, DYF-3:: GFP is moved uniquely by OSM-3-kinesin along the middle and a few remaining distal segments, establishing its association with OSM-3-kinesin/subcomplex B (Figure 3Figure 3., J and M, and Table 3). Verification of these interactions and uncovering additional connections between and among BBS and core/peripheral IFT proteins will necessitate a global analysis of these proteins by yeast two-hybrid and coimmunoprecipitation studies. We also note that, although DYF-3 has been found in the Chlamydomonas flagellar proteome, its apparent absence from the biochemically isolated IFT particle B subcomplex suggests that it may possess a more “peripheral” location or loose association within the macro-molecular architecture of the IFT machinery.
Our data suggest that IFT-particle subcomplexes A and B from C. elegans form discrete functional modules, as they do in Chlamydomonas, and they describe the approximate spatial position of these modules within the IFT machinery (see Figure 7Figure 7.A). These findings are also consistent with numerous functional studies conducted in C. elegans and Chlamydomonas, which describe differential roles for subcomplex B in anterograde transport and subcomplex A in retrograde transport (Rosenbaum and Witman, 2002 blue right-pointing triangle; Scholey, 2003 blue right-pointing triangle). Loss of function of components in IFT-particle subcomplex B inhibit anterograde IFT, resulting in ~2-μm-long residual middle segments and posterior aggregation along the dendrites (Rosenbaum and Witman, 2002 blue right-pointing triangle; Scholey, 2003 blue right-pointing triangle). By “phenoBLASTing” the new mutants against previously characterized ciliary mutants, we found that dyf-9, dyf-11, qj8, qj11, aj25, qj26, qj27, qj32, qj38, qj40, qj45, qj49, and qj53 may function along with other components of IFT-particle subcomplex B (Figures 5Figure 5., ,6,6Figure 6., and and7B7Figure 7.B and Tables 1 and 2). On the other hand, loss of function of components in IFT-particle subcomplex A block retrograde IFT driven by CHE-3, the IFT-dynein (Signor et al., 1999 blue right-pointing triangle; Wicks et al., 2000 blue right-pointing triangle). Mutations of genes encoding known subcomplex A proteins (e.g., che-11, daf-10) and likely subcomplex A–associated components (e.g., dyf-2, ifta-1) are characterized by the formation of aggregates within the remaining cilia and no, or very little IFT is detectable in these mutants (Qin et al., 2001 blue right-pointing triangle; Blacque et al., 2006 blue right-pointing triangle; Efimenko et al., 2006 blue right-pointing triangle). Here, we report that IFT-particle (OSM-6::GFP) aggregates form along sensory cilia in qj9, qj10, qj12, qj16, qj17, qj21, qj22, qj28, qj30, qj31, qj33, qj34, qj37, qj41, and qj48, which indicates that they may function together with IFT-particle A or dynein components in retrograde IFT (Figures 6Figure 6. and and7B7Figure 7.B and Table 1).
Figure 5.
Figure 5.
Figure 5.
Structural defects of amphid channel cilia in previously known dye-filling mutants. Representative images of the ciliary structures of dyf mutants, as visualized with GFP-labeled IFT-particle proteins (OSM-6::GFP or CHE-2::GFP). The first row demonstrates (more ...)
Figure 6.
Figure 6.
Figure 6.
Structural defects of amphid cilia in novel dye-filling mutants. Representative images of ciliary structures of novel dyf mutants, as visualized with a GFP-labeled IFT-particle protein (OSM-6::GFP). The detailed description of genotypes and structural (more ...)
In addition to the molecules that are required for retrograde IFT in general, we uncovered several components whose mutations appear to cause defects in IFT-particle recycling at specific sites, such as the distal segment tip or the junction between the middle and distal segments. For example, DYF-5, an MAPK-related serine/threonine protein kinase (Chen et al., 2006 blue right-pointing triangle) may control IFT-particle recycling because immotile OSM-6::GFP accumulates in the distal segment, whereas IFT along the middle segment persists. The tip of the middle segment is an important site for kinesin-II recycling, and qj14, qj24, and qj51 may be specifically involved in turnaround at this site because OSM-6::GFP accumulates at the junction between the middle and distal segments of these mutants (Figures 5Figure 5., ,6,6Figure 6., and and7B,7Figure 7.B, and Tables 1 and 2). Further work will be required to determine the localization of the corresponding products of these candidate “turnaround” genes.
The BBS Protein Module: Stabilization of IFT Particles
BBS is a pleiotropic disorder related to defects in 12 human genes that affect basal bodies and ciliary axonemes (Ansley et al., 2003 blue right-pointing triangle; Badano et al., 2006 blue right-pointing triangle; Blacque and Leroux, 2006 blue right-pointing triangle; Stoetzel et al., 2006 blue right-pointing triangle, 2007 blue right-pointing triangle). One possible explanation for the oligogenic nature of BBS is that the BBS proteins may be involved in a common genetic pathway and could form a functional hetero-oligomeric complex whose subunits have interdependent functions, i.e., the function of each one depends on the proper function of all the others. The abrogation of any subunit might be predicted to disrupt the formation and conformation of the whole module, thereby preventing it from stabilizing the motor-IFT particle assembly. The availability of live cell imaging and genetics in C. elegans neuronal cilia makes it an appealing system to test this hypothesis.
We first compared the ciliary structural and IFT defects of a novel C. elegans bbs-1 allele with the known bbs mutants to examine if all three have similar phenotypes. bbs-1(ok1111) possesses a deletion spanning exons 7–9, and loss of BBS-1 function causes animal behavioral abnormalities in chemotaxis and dye filling, which are known to be related to proper ciliary function (data not shown and Mak et al., 2006 blue right-pointing triangle). As in bbs-7 and bbs-8 mutants (Blacque et al., 2004 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle), abrogating the function of BBS-1 results in the separation of moving kinesin-II (KAP-1)/IFT-A (CHE-11) and OSM-3/IFT-B (CHE-2 and OSM-1) complexes (Figure 4Figure 4., A–H, and Table 3). These data support the hypothesis that the BBS-1, -7, and -8 proteins are all involved in a common process, and that disruption of this process leads to the destabilization of the IFT-particle. Given that the C. elegans BBS-1, -2, -3, -7, and -8 proteins were previously shown to undergo IFT and be bona fide IFT-related components (Blacque et al., 2004 blue right-pointing triangle; Fan et al., 2004 blue right-pointing triangle), we suspect that all BBS proteins share this common function.
To further investigate the close genetic association of the bbs genes, we next examined the ciliary structure and IFT in double and triple bbs mutants. Using IFT motility assays, we demonstrated that the transport phenotypes observed in bbs single mutants are exactly phenocopied in bbs double mutants. Specifically, we found that GFP-tagged CHE-11 (IFT subcomplex A) and IFTA-1 (IFT subcomplex A-like, C54G7.4; Blacque et al., 2006 blue right-pointing triangle) are transported only along the ciliary middle segments of bbs-7;bbs-8 double mutants at kinesin-II's slow rate (~0.5 μm/s) and that GFP-tagged CHE-2, CHE-13, and OSM-5 (IFT subcomplex B) are transported along the bbs-7;bbs-8 middle and distal segments at the OSM-3-kinesin–associated fast rate (~1.3 μm/s; Figure 3Figure 3., A, B, G, and H, and Table 3). These data are essentially identical to those previously found in bbs-7 or bbs-8 single mutants (Ou et al., 2005a blue right-pointing triangle) and demonstrate that, like the bbs single mutants, IFT complex A and B components are delivered separately by kinesin-II and OSM-3-kinesin, respectively, in bbs-7;bbs-8 double mutants.
Next, we used the gcy-5p::gfp reporter to illuminate the ASER cilium of single, double, and triple bbs mutants. Using this scheme, the ASER ciliary axoneme of WT animals is measured to be ~6 μm long (Blacque et al., 2004 blue right-pointing triangle, 2005 blue right-pointing triangle). In all examined bbs mutants, bbs-1 single (4.13 ± 0.94 μm), bbs-7;bbs-8 double (3.92 ± 1.07 μm; Blacque et al., 2004 blue right-pointing triangle) and bbs-1;bbs-7;bbs-8 triple (4.43 ± 0.97 μm) mutants possess comparable shortened cilium phenotypes (Figure 4Figure 4.I). Hence, loss of function of two or even three BBS proteins does not produce any more severe defects in ciliary length than do bbs single mutants.
Analysis of the transport profiles of several GFP-tagged BBS proteins (BBS-1, -5, and -8) in bbs-1, -7, and -8 mutants further support the hypothesis that the C. elegans BBS proteins possess interdependent functions. In WT animals, BBS proteins undergo biphasic IFT (Figure 4Figure 4., J and K, and Table 3), but in bbs mutants, GFP-tagged BBS proteins accumulate at the transition zones (basal bodies) of bbs mutant cilia and fail to enter the ciliary axonemes or undergo IFT (Figure 4Figure 4., L–N, and Table 3). These data indicate that the function of individual BBS proteins depends on the proper function of other BBS proteins. Importantly, this phenotype is strikingly different from that observed with other IFT markers in that subcomplex A and B components can still enter the cilium in all examined bbs mutants (Figure 3Figure 3., A–H; Blacque et al., 2004 blue right-pointing triangle, 2005 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle). This indicates that the BBS proteins likely operate in the same process by forming a functional, hetero-oligomeric module that is associated with, but acts independent of, the IFT particle A and B subcomplexes (Figure 7Figure 7.A). Consequently, removal of a single BBS protein disrupts the assembly/conformation of the entire BBS module, thereby preventing its incorporation into the motor-IFT particle assembly and resulting in the destabilization and dissociation of the two kinesin motors with their associated IFT subcomplexes.
Accessory Motor Module: Ciliary Distal Segment Assembly for Cilium-based Signaling
In certain cilia, such as those found in C. elegans sensory neurons, the canonical pathway appears to be modulated by an accessory anterograde motor, OSM-3-kinesin (the nematode homolog of human KIF17), which cooperates with heterotrimeric kinesin-II to build middle segments, but is solely responsible for building the distal segments of cilia (Snow et al., 2004 blue right-pointing triangle; Evans et al., 2006 blue right-pointing triangle). Recently, we found that DYF-1 is required to dock OSM-3-kinesin onto IFT particles, because loss of DYF-1 function results in the abrogation of OSM-3 motor activity and a loss of distal segment structure (Ou et al., 2005a blue right-pointing triangle). Interestingly, another IFT protein, DYF-13, also appears to function specifically in building the distal segments of cilia (Blacque et al., 2005 blue right-pointing triangle). Taken together, these data suggest that DYF-1 and DYF-13 may form a module that functions in association with OSM-3-kinesin to build distal segments. Accordingly, we predict that DYF-1 and DYF-13 will display the same transport profile as OSM-3-kinesin along bbs mutant cilia. When examined in bbs-7 and bbs-8 mutant animals, we observed that, similar to OSM-3::GFP, GFP-tagged DYF-1 and DYF-13 move along both the middle and distal segments at ~1.3 μm/s, suggesting that these DYF proteins are closely associated with, and may bind to, OSM-3-kinesin (Figure 3Figure 3., I, K, and L, and Table 3).
One intriguing puzzle concerning the accessory motor module is how the DYF-1 protein docks and activates OSM-3-kinesin, given that no physical interaction can be detected between them and that DYF-1 does not directly activate OSM-3-kinesin in a single-molecule motility assay (Ou et al., 2005a blue right-pointing triangle; Imanishi et al., 2006 blue right-pointing triangle). We proposed that DYF-1 might function with additional and unidentified molecules to form a docking complex for OSM-3-kinesin's docking and activation. One obvious implication is that our forward genetic screen may uncover novel mutants defective in this docking complex that would be expected to phenocopy osm-3 or dyf-1 or dyf-13 by displaying a specific loss of the ciliary distal segment. We isolated three such mutants, namely qj23, qj47, and qj55. snip-SNP mapping data suggest that qj23 and qj55 are novel components required for the assembly of the distal segment because neither of these alleles map to the same genetic loci as osm-3 or dyf-1or dyf-13. Accordingly, we have classified qj23 and qj55 as new Dyf alleles, namely dyf-15 and dyf-16, respectively. In contrast, qj47 maps to the same region as osm-3, and a null allele of osm-3(p802) fails to complement qj47, suggesting that qj47 is a new allele of osm-3 (Figures 6Figure 6. and and7B7Figure 7.B and Table 1). Our set of 49 new Dyf mutants also contains partial dyf mutants. For example, ks101 does not develop any distal segments in a small subset of chemosensory neurons. It is therefore difficult to determine if this gene is specifically involved in the distal segment pathway or if it is a hypomorph, for which more severe loss-of-function alleles and molecular cloning may be needed.
We identified several other known mutants that might be involved in ciliary distal segment assembly (Figures 5Figure 5. and and7B7Figure 7.B and Table 2). dyf-6 has been recently cloned and it was reported that it has a very short middle segment without any visible IFT, suggesting that it functions as an IFT-particle B component (Bell et al., 2006 blue right-pointing triangle). However, we found that dyf-6(m175) only loses its distal segment and IFT still persists in the middle segment. Similarly, UNC-101 and UNC-119 are necessary for distal segment assembly, and their mutation causes the complete loss of the distal segment, with IFT still continuing in the residual middle segment. In addition, we noticed that dyf-10, dyf-12, and qj35 have full-length middle segments and shorter distal segments. IFT persists in all of their middle segments, and in the remaining distal segments of dyf-10 and qj35, IFT is detectable, whereas small aggregations form at the distal tip of dyf-12 (Figures 5Figure 5., ,6,6Figure 6., and and7B7Figure 7.B and Tables 1 and 2). Molecular cloning of the genes corresponding to these mutations will provide critical insights into the functions of the corresponding gene products in building the ciliary distal tips.
Finally, our genetic screen suggests that that the proper assembly of ciliary distal segments may depend on molecules other than components of the accessory motor module. The distal segment follows a linear trajectory along the channel to the amphid pore where it contacts the environment. daf-6 and che-14 were previously shown to be required for this linear orientation because in daf-6 and che-14 mutants the distal segment trajectories deviate into the adjacent sheath cells (Perens and Shaham, 2005 blue right-pointing triangle); interestingly, we observe that dyf-4 and qj20 display a similar phenotype (Figures 5Figure 5., ,6,6Figure 6., and and7B7Figure 7.B and Tables 1 and 2). It is notable that IFT persists in the cilia of these mutants allowing the distal segment to assemble. Thus, dyf-4 and qj20 might function together with daf-6 in the sheath cells to control proper distal segment orientation.
IFT Cargo
Arguably the most poorly characterized proteins associated with the IFT machinery are the cargo molecules that are delivered into the cilia to enable cilium biogenesis and function. Candidate cargo molecules likely include structural components of the ciliary axoneme, membrane, and matrix, as well as the signaling and regulatory molecules that underlie cilia function (e.g., sensory reception). With the possible exception of radial spoke proteins and the TRPV Ca2+ channels, OSM-9 and OCR-2 (Qin et al., 2004 blue right-pointing triangle, 2005 blue right-pointing triangle), no bona fide IFT cargo components have been identified and functionally characterized. However, IFTA-2 (T28F3.6), a RAB-like protein that undergoes IFT, may represent another IFT cargo (or cargo-docking) component, because this protein appears to function as a vesicle-associated signaling molecule required for the function of cilia but not for the function of the motor-IFT machinery (Schafer et al., 2006 blue right-pointing triangle). Interestingly, we show here that in WT animals, IFTA-2::GFP undergoes biphasic IFT, exactly like other IFT-particle proteins, but in bbs-8 mutants, it undergoes transport along the middle (~1.1 μm/s) and distal (~1.3 μm/s) ciliary segments at rates characteristic of OSM-3 (Figure 3Figure 3., N and O, and Table 3). This indicates that an IFTA-2–associated cargo module may associate peripherally with IFT subcomplex B and OSM-3-kinesin and may be the first indication of a docking site of cargo protein(s) on the motor-IFT machinery (Figure 7Figure 7.A).
Cilium Initiation
The loss of function of basal body or ciliary axoneme components could inhibit the initiation of sensory cilia, and we found seven mutations that are defective in ciliary initiation (Figure 5Figure 5., ,6,6Figure 6., and and7B7Figure 7.B and Table 1 and 2). For example, there is no detectable OSM-6::GFP in the presumptive ciliary region of qj36, qj42, qj50, whereas in qj46, OSM-6::GFP forms bright dots in the ciliary region. Che-10 (Perkins et al., 1986 blue right-pointing triangle) and dyf-14 appear to have less severe defects in cilium initiation per se, and both of them can form one or two short cilia, whereas other cilia do not develop at all. Interestingly, in the remaining cilia of dyf-14, IFT is detectable, whereas no IFT is visible in the residual cilia of che-10. Unlike the above mutants, mec-8 does not have defects in cilium initiation per se, but the positioning of the transition zones is dispersed rather than being tightly juxtaposed to the cilium base as in WT. This suggests that MEC-8 is involved in the positioning of the transition zone.
DISCUSSION
In the current study we used transport assays and phenotypic profiling to characterize the role of several known and novel genes and their products in IFT and cilium biogenesis and to provide the most comprehensive picture so far available of the components involved in C. elegans IFT and sensory ciliogenesis. The results are consistent with the C. elegans machinery being organized into distinct modules with specialized functions in cilium biogenesis.
Systematic approaches including proteomics and comparative genomics have recently been used to identify ciliary components (Gherman et al., 2006 blue right-pointing triangle; Inglis et al., 2006a blue right-pointing triangle). Human primary culture respiratory epithelial cilia, Chlamydomonas and trypanosome flagella were isolated and their protein composition was analyzed by protein sequencing (Ostrowski et al., 2002 blue right-pointing triangle; Pazour et al., 2005 blue right-pointing triangle; Broadhead et al., 2006 blue right-pointing triangle). Comparative genomics yielded candidate ciliary components by comparisons of the genomic differences between ciliated and nonciliated organisms or the expression pattern change in ciliated and nonciliated cells or during cilium regeneration (Avidor-Reiss et al., 2004 blue right-pointing triangle; Li et al., 2004a blue right-pointing triangle; Blacque et al., 2005 blue right-pointing triangle; Efimenko et al., 2005 blue right-pointing triangle; Keller et al., 2005 blue right-pointing triangle; Kunitomo et al., 2005 blue right-pointing triangle). Forward genetic studies provide a useful complementary method to these powerful systematic tools for identifying ciliogenesis components.
This latter approach is justified by our identification of a new protein required for cilium biogenesis, namely DYF-14. The function of this protein was previously unknown except that the human protein homolog, trichohyalin, has been reported to associate with a hair follicle intermediate filament (Rothnagel and Rogers, 1986 blue right-pointing triangle; Fietz et al., 1993 blue right-pointing triangle), but ciliary functions were not reported previously. Protein domain analysis of DYF-14 uncovered four coiled-coil domains and one “myosin tail-like” domain. Interestingly, several components of the IFT-particle B subcomplex have coiled-coil domains including IFT81, IFT74/72, IFT57/55, and IFT20 (Cole, 2003 blue right-pointing triangle), which may mediate protein–protein interactions (Lucker et al., 2005 blue right-pointing triangle) so DYF-14 might assemble into a protein complex, possibly the IFT-particle, that is required for ciliogenesis. More work on the expression pattern, cellular localization, and dynamics of DYF-14 is needed to determine how it functions in cilium biogenesis in C. elegans.
Our use of transport assays of ciliary proteins provides the most detailed model yet of the modular architecture of the IFT machinery. In the resulting model, IFT depends on a network of dozens of interacting IFT proteins organized into modules that may execute distinct subprocesses in the assembly and maintenance of cilia (Figure 7Figure 7.A). BBS proteins appear to function interdependently in the same IFT-related genetic pathway, consistent with them forming a heteromeric multiprotein complex that stabilizes IFT-particle subcomplexes A and B. Two known IFT subcomplex A proteins studied (CHE-11 and IFTA-1) associate with the heterotrimeric kinesin-II motor, whereas all the known IFT subcomplex B proteins that were examined (CHE-2, CHE-13, OSM-1, OSM-5, OSM-6, IFT-20) are in close proximity to the homodimeric OSM-3-kinesin motor.
Three recently identified but relatively uncharacterized IFT regulators, DYF-1, DYF-3, and DYF-13, appear to be positioned close to OSM-3-kinesin/IFT subcomplex B. Although DYF-1 and DYF-13 associate with OSM-3-kinesin in the same manner as IFT subcomplex B, we propose they form a separate functional module, because their disruption produces overlapping phenotypes that differ from that of IFT subcomplex B mutants. Specifically, dyf-1 and dyf-13 mutants possess intact middle segments and loss of DYF-1 function specifically affects the function of the OSM-3-kinesin motor (Blacque et al., 2005 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle). Further studies are needed to uncover the specific functions of DYF-1 and DYF-13 in relation to their association with OSM-3-kinesin/subcomplex B. It is possible that these two proteins exert their distal segment-specific functions at a position that is peripheral to IFT subcomplex B, yet in close proximity with OSM-3-kinesin (Figure 7Figure 7.A).
In our forward genetic screens, we identified additional components that appear to function in the OSM-3-kinesin–dependent distal segment pathway. For example, dyf-15(qj23) and dyf-16(qj55) display a specific loss of the complete distal segment and their unique phenotypes suggest that the corresponding gene products could be novel components that function with DYF-1 and DYF-13 in OSM-3-kinesin docking and activation. Alternatively, they may be cargo molecules required for distal segment assembly. Careful characterization of the IFT of all available markers and gene cloning will be necessary to distinguish these possibilities. Of note is the fact that both DYF-1 and DYF-13 have homologues in other ciliated organisms, suggesting that their functions are conserved in IFT pathways of different species (Blacque et al., 2005 blue right-pointing triangle; Ou et al., 2005a blue right-pointing triangle).
Although very poorly characterized, it is thought that numerous cargo “modules” may associate peripherally with the IFT machinery. Cargo proteins are likely to display functional properties and dynamics that are distinct from those of the integral IFT machinery (e.g., IFT-particle subunits and BBS proteins) yet even this aspect of IFT-cargo is poorly understood. For example, although the “core” IFT machinery displays persistent movement back and forth between the basal body and the distal tip of the cilium, once a cargo molecule has accumulated in the appropriate ciliary compartment, there may be a significant temporal delay before it gets recycled to the cell body by retrograde transport, and it is possible that the disruption of cargo proteins will affect cilia function without directly affecting IFT motility or the integrity of motor-IFT particle assemblies. Our present work and the findings of Schafer et al. (2006) blue right-pointing triangle on IFTA-2 suggests that it may represent cargo or likely be required for cargo association with the IFT-particle, because it is required for ciliary function but not for ciliary structure or IFT. Clearly, further work is required to identify and better characterize the cargo of the IFT machinery.
Obviously some of the proteins identified in our forward genetic screen may not be components of IFT modules, but instead they may function at different sites to control IFT and ciliogenesis. Examples include proteins involved in turnaround of the IFT machinery at the tips of the middle or distal segments (e.g., qj14), in transition zone positioning (e.g. mec-8), and in distal segment orientation (e.g., dyf-4) (see Figure 7Figure 7.B). Indeed some of the corresponding gene products may function outside of ciliated sensory neurons, for example in the surrounding sheath or socket cells (e.g., daf-6, [Perens et al., 2005]). Further cloning and characterization of these genes, including tagging, localizing and studying transport of their products, may provide novel insights into the mechanisms of sensory ciliogenesis in C. elegans neurons.
Axon outgrowth was previously shown to be defective in ciliary mutants, indicating that sensory activity is required for sensory axon development (Peckol et al., 1999 blue right-pointing triangle). We studied cilium biogenesis in mutants defective in axon guidance (unc-6), axon outgrowth (unc-33), and axon transport (unc-104), and their cilia appear to be properly formed (Figure 5Figure 5. and Table 2), indicating that abnormal axonal structure and function do not affect cilium biogenesis. In addition, we characterized the ciliary phenotypes of another two Unc mutants, unc-101 and unc-119. Both of them have defective dye-filling phenotypes, and loss of their ciliary distal segments (Figures 5Figure 5. and and7B7Figure 7.B and Table 2). UNC-101 is the AP-1 mu1 clathrin adaptor and mediates polarized dendritic transport of odorant receptors to olfactory cilia (Dwyer et al., 2001 blue right-pointing triangle). UNC-119 encodes a highly conserved protein required for proper development of the nervous system, and its Chlamydomonas ortholog is a centriole protein (Maduro and Pilgrim, 1995 blue right-pointing triangle; Maduro et al., 2000 blue right-pointing triangle). Further work on the cellular localization and dynamics of UNC-101 and UNC-119 is necessary to explain how they function in cilium biogenesis.
Although our work provides a useful description of the protein machinery involved in sensory ciliogenesis in C. elegans, further work will be needed to better characterize the architecture and function of the individual modules. For example, efforts must be directed toward extending and improving the identification of cargo and regulatory molecules associated with IFT particles, and in directly testing predictions of the current model (Figure 7Figure 7.) by physical binding assays and structural studies. Nevertheless, the work described here should provide a framework for studying the functional hierarchy of the IFT machinery, which in turn may eventually illuminate the temporal dynamics of its assembly. In addition, by correlating the IFT motility signatures described here with phenotypic profiles of IFT components and biochemical analyses of direct physical interactions between IFT proteins, it should be possible to develop network models of the modular IFT machinery, analogous to those developed for the protein machinery involved in embryogenesis (Gunsalus et al., 2005 blue right-pointing triangle). Taken together, our work on known and novel ciliary mutants, based on in vivo assays of the transport and distribution of functional GFP fusion proteins, complements the pioneering biochemical studies done elsewhere (Cole et al., 1998 blue right-pointing triangle; Piperno et al., 1998 blue right-pointing triangle; Lucker et al., 2005 blue right-pointing triangle) and provides the first comprehensive picture and modular description of the IFT machinery that builds C. elegans sensory cilia.
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ACKNOWLEDGMENTS
We thank T. Stiernagle and the C. elegans gene-knockout consortium for providing strains. Work in the Scholey laboratory is supported by National Institutes of Health Grant GM50718. Support was from Japan Science and Technology Corporation research grant PREST to M.K. M.R.L. holds Michael Smith Foundation for Health Research (MSFHR) and CIHR scholar awards and was supported by the Canadian Institutes of Health Research (CIHR) Grant CBM134736 and the March of Dimes (equal funding from grants to M.R.L.). O.E.B. was supported by a MSFHR fellowship.
Abbreviations used:
IFTintraflagellar transport
BBSBardet-Biedl syndrome.

Footnotes
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-09-0805) on February 21, 2007.
An external file that holds a picture, illustration, etc. Object name is dbox.jpg The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
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