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1 Molecular Genetics Research Laboratory, and
2 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Ciliated sensory neurons (CSNs) are the sole cell type that develops cilium structure in C. elegans hermaphrodites. Dendritic endings of these cells terminate in cilia, which play critical roles in perception of environmental stimuli. Morphological and functional abnormality of cilia can lead to mutant phenotypes such as chemotaxis and odorant response defects (Che and Odr), osmotic avoidance defects (Osm), dauer formation defects (Daf) and fluorescent dye-filling defects (Dyf) (Culotti & Russell 1978; Bargmann et al. 1993; Malone & Thomas 1994; Starich et al. 1995). In wild-type animals, six classes of amphid and phasmid sensory neurons absorb lipophilic fluorescent dye such as DiQ through sensory cilia that are directly exposed to environment (Hedgecock et al. 1985; Starich et al. 1995). At least 14 dyf loci have been genetically identified so far (Starich et al. 1995; Ou et al. 2007). Consistent with the defects in cilium structure, many genes of this class that have been characterized so far are associated with IFT (Murayama et al. 2005; Ou et al. 2005a, 2007; Bell et al. 2006; Efimenko et al. 2006).
The sensory cilia of C. elegans, whose structure is well defined in the amphid and phasmid channel neurons, consist of three parts: the transition zone, which is equivalent to the basal body of motile cilia; the middle; and the distal segments, which contain doublet and singlet axonemal microtubules, respectively (Perkins et al. 1986). Two different kinesin-2 motors, heterotrimeric kinesin-II and homodimeric kinesin, OSM-3, cooperatively work during anterograde IFT in this organism (Snow et al. 2004). They redundantly function to build the middle segment of the sensory cilia. Transport in the distal segment is, however, carried out solely by OSM-3 (Snow et al. 2004).
It has been shown that IFT particles of Chlamydomonas can be isolated biochemically and separated into two complexes, A and B, which collectively contain at least 16 different polypeptides (Piperno & Mead 1997; Cole et al. 1998; Piperno et al. 1998). In C. elegans, mutations in the components of IFT complexes A (IFT-A) and B (IFT-B) cause distinct ciliary phenotypes. That is, mutants of IFT-A (CHE-11 and DAF-10) have slightly short cilia with accumulation of ciliary components along the axoneme (Perkins et al. 1986; Schafer et al. 2003), a very similar phenotype to the mutants of retrograde motor, dynein (Signor et al. 1999; Schafer et al. 2003). In contrast, mutants of IFT-B (CHE-2, CHE-13, OSM-1, OSM-5 and OSM-6) typically show a drastic reduction of cilia length (Perkins et al. 1986; Fujiwara et al. 1999; Haycraft et al. 2001, 2003). These results as well as the observations in complex A mutants in Chlamydomonas collectively suggested that the components of IFT-A are functionally important for retrograde transport, whereas that of IFT-B are important for anterograde transport (Piperno et al. 1998; Cole 2003; Scholey 2003). Although previous studies have substantially clarified the mechanisms of IFT, it is not yet known how the IFT complexes are organized and regulated. Furthermore, the full complement of this process has not been identified and characterized so far.
In this study, we describe C. elegans DYF-11, which shares a significant homology to mammalian Traf3ip1 (tumor necrosis factor receptor-associated factor 3 interacting protein 1). The dyf-11 gene is predominantly expressed in CSNs, and its expression is regulated by DAF-19. We defined the gene structure of dyf-11 and characterized an available mutant allele, mn392, along with a newly obtained deletion allele, pe554. We observed severe morphological and functional defects in cilia of dyf-11 mutants, which is consistent with a ciliogenic role of dyf-11. Using cell biological approaches, we show that DYF-11 is likely to be associated with IFT complex B. Protein domain analysis revealed that the coiled-coil domain is required for proper localization and function of DYF-11. We further show that Traf3ip1 localizes to cilia in renal epithelial cells.
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Results |
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Based on a neuron-specific mRNA pull-down experiment, C. elegans C02H7.1 was identified as a CSN-expressed gene (Kunitomo et al. 2005). Through our analysis, C02H7.1 was identified as the genetically defined gene dyf-11 (see below) and we hereafter refer to it as dyf-11. To reexamine the cellular expression pattern of dyf-11, we newly generated two transcriptional and one full-length translational fluorescent protein fusions (see Experimental procedures). The transcriptional fusions, pGdyf11p(–4.1) :: venus and pGdyf11p(–2.0) :: venus that carry 4.1- and 2.0-kb promoter, respectively, showed essentially the same expression patterns (Fig. 1A). In wild-type background, fluorescence was first detected at the 1.5-fold stage of embryogenesis and persisted throughout the lifespan. At larval and adult stages, most of the ciliated neurons, namely, all 12 classes of amphid sensory neurons, phasmid neurons PHA and PHB, the inner and outer labial neurons (IL1, IL2 and OLQ), AQR, URX, FLP, PDE, PQR and several other neurons (probably OLL, ADE and BAG) were found to express the reporter. In addition to CSNs, fluorescence was often seen in gut cells at larval stages (Fig. 1A); this intestinal expression decreased as development proceeded (see below). It was difficult to determine precise cellular expression pattern of the translational fusion, pGdyf11p(–4.1) :: venus :: dyf11, because of ciliary localization of the fusion protein VENUS :: DYF-11 expressed from this transgene (Fig. 1B and see below).
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In C. elegans, DAF-19, an RFX-type transcription factor that recognizes a DNA sequence called X-box, is known to regulate the expression of many ciliary genes (Swoboda et al. 2000; Haycraft et al. 2003; Schafer et al. 2003; Li et al. 2004; Blacque et al. 2005; Efimenko et al. 2005, 2006; Murayama et al. 2005; Ou et al. 2005b). The promoter region of dyf-11 contains a canonical X-box (Fig. 2A), suggesting that its expression may be regulated by DAF-19 (Avidor-Reiss et al. 2004; Blacque et al. 2005; Efimenko et al. 2005; Kunitomo et al. 2005). To test this possibility, the extrachromosomal array of pGdyf11p(–4.1) :: venus was transferred from daf-19(+) to daf-19(m86) mutant background and the expression level of the reporter was examined in the adult worms. Loss of DAF-19 resulted in severe reduction of the reporter expression in CSNs, whereas the expression in digestive tract still remained (Fig. 1C and D).
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To elucidate the gene structure and biological function of C02H7.1, a full-length cDNA of this gene was isolated. The nucleotide sequence of this clone revealed an ORF of 535 amino acids consisting of seven exons (Fig. 2A). We then isolated a deletion allele of C02H7.1, pe554. Nucleotide sequence of the mutant genome revealed that pe554 carries a 667-bp deletion in C02H7.1 starting in the third exon and ending in the fourth intron, which was replaced by a 7-bp consecutive thymine insertion (Fig. 2A). This lesion is expected to truncate the 535-amino acid gene product at position 145. pe554 homozygotes are fully viable and display no obvious locomotory or fertility phenotypes (data not shown). However, the mutants are slightly shorter than wild-type animals and exhibit fluorescent dye-filling defective (Dyf) phenotype, the characteristics of ciliary abnormalities. The Dyf phenotype of the mutant animals is fully penetrant; CSNs that take up dye in wild-type animals are never stained in pe554 mutants (Fig. 2C, compare with Fig. 2B). Expression of the VENUS :: DYF-11 by its authentic promoter rescued the defects, demonstrating that the deletion in C02H7.1 is responsible for the phenotype and this fusion protein is fully functional (Fig. 2D).
Given these phenotypes of C02H7.1(pe554) and the fact that this gene maps close to the genetic position of dyf-11 (–18.27 ± 0.244) on linkage group X (Starich et al. 1995), we hypothesized that these two genes are identical. dyf-11 was an uncloned locus that shows defects in dye-filling, chemotaxis, dauer formation and body length (Starich et al. 1995). Several lines of experimental evidence were obtained to confirm this hypothesis. First, PCR-amplified DNA fragments that cover the C02H7.1 region of wild-type genome rescued the Dyf phenotype of dyf-11(mn392) mutants. However, the same genome region prepared from dyf-11(mn392) mutants failed to do so (data not shown). Second, C02H7.1(pe554) phenocopied dyf-11(mn392) mutants in dye-filling, morphological and behavioral phenotypes (Fig. 2C, F and see below). Third, C02H7.1(pe554) failed to complement the Dyf phenotype of dyf-11(mn392) (see Experimental procedures). Fourth, dyf-11(mn392) genome carries a C to G substitution converting S140 of the predicted gene product of C02H7.1 to a stop codon (Fig. 2A). And finally, the Dyf phenotype of dyf-11(mn392) animals was completely rescued by the expression of cDNA for C02H7.1 (Fig. 2G). Together, we concluded that C02H7.1 is identical to dyf-11.
dyf-11 encodes a putative microtubule binding protein conserved in ciliated organisms
In addition to the CSN-specific expression, recent studies raise a strong possibility that DYF-11 and its orthologues are involved in ciliogenesis: identification as candidate cilium genes in comparative genomics approaches (Avidor-Reiss et al. 2004; Li et al. 2004), identification of Chlamydomonas orthologue, C_140070, in flagellar regeneration transcriptome analysis (Stolc et al. 2005) and in flagellar proteomics (Pazour et al. 2005), and finding X-boxes in both C. elegans C02H7.1 and its Drosophila orthologue in their promoter region (Avidor-Reiss et al. 2004; Blacque et al. 2005; Efimenko et al. 2005; Kunitomo et al. 2005). The predicted gene product of dyf-11 showed a significant similarity to the gene products evolutionarily conserved from human to Chlamydomonas (Fig. 3A). Among the orthologues, human TRAF3 (tumor necrosis factor receptor-associated factor 3)-interacting protein 1 (TRAF3IP1, also called MIP-T3) is the sole member whose physiological function was previously described. TRAF3IP1 is a microtubule- and β-tubulin-binding protein that recruits TRAF3 to microtubules when co-expressed in HeLa cells (Ling & Goeddel 2000). Interestingly, TRAF3IP1 also binds to the 
1 subunit of interleukin-13 (IL-13) receptor and modulates inflammatory signal transduction mediated by STAT6 (see Discussion, Niu et al. 2003; Low et al. 2006).
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Defects in dye-filling suggest that dyf-11 mutants have abnormalities in cilium structure (Starich et al. 1995). To visualize the morphology of sensory cilia, we utilized fluorescent protein markers that are specifically expressed in CSNs. Gross cilium morphology of the amphid and phasmid CSNs was observed by OSM-6 :: GFP (Fig. 4A–D). OSM-6 is a previously characterized IFT-B protein that plays a critical role in formation of IFT-B complex (Collet et al. 1998; Haycraft et al. 2003). To characterize the morphology of cilia in detail, the ASH and ASER chemosensory neurons were visualized by sra-6 :: venus (Fig. 4E and F) and gcy-5 :: gfp (Fig. 4G and H), respectively. We found that the cilia of dyf-11(pe554) mutants are indeed malformed. Specifically, the length of the ciliary axoneme in dyf-11(pe554) mutants are short (Fig. 4B, D, F and H); the average total length of the middle and distal segments of ASER cilia was 3.1 ± 0.4 µm (n = 25) in comparison to 5.3 ± 0.5 µm in wild-type animals (n = 27). In addition to short cilium length, some dyf-11(pe554) cilia showed abnormal branching (Fig. 4F). Furthermore, aberrant posterior projection was observed in some, but not all, dendrites (Fig. 4H). These abnormalities were also observed in dyf-11(mn392) animals (data not shown). The phenotypes observed in dyf-11 cilia are similar to those found in the IFT-B mutants (Perkins et al. 1986; Fujiwara et al. 1999; Haycraft et al. 2001, 2003), suggesting that DYF-11 is associated with this function (see below).
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DYF-11 acts cell-autonomously for formation and maintenance of sensory cilia
To examine whether dyf-11 acts cell-autonomously in cilium biogenesis, we rescued dyf-11(pe554) mutants by VENUS :: DYF-11 in tissue- or cell-specific manner. As mentioned above, the dyf-11(pe554) mutants show a severe dye filling-defective phenotype (Fig. 2C). Mutant animals expressing the fusion protein by the authentic 4.1-kb promoter restored dye-uptake in all competent neurons (Figs 2D and 5A). However, animals that express VENUS :: DYF-11 in muscle cells by the myo-3 promoter did not (Fig. 5A). When VENUS :: DYF-11 was specifically expressed in ADL neurons using the sro-1 promoter (Troemel et al. 1995), ADL, but not other neurons restored dye-uptake (Figs 2E and 5A). These results indicate that dyf-11 acts cell-autonomously at least for fluorescent dye-uptake.
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DYF-11 is likely associated with IFT
To elucidate how DYF-11 is involved in ciliogenesis, we observed the localization and movement of the functional VENUS :: DYF-11 fusion proteins within cilia. Consistent with a role in cilia, the fusion protein was enriched at the transition zone and along the axoneme of ciliated neurons (Fig. 1B). Time-lapse fluorescence imaging of phasmid cilia revealed that VENUS :: DYF-11 fluorescent particles are moving within the cilia in both anterograde (Fig. 6A) and retrograde (Fig. 6B) directions. Moreover, VENUS :: DYF-11 particles moved in different velocities along the middle (0.87 ± 0.08 µm/s) and distal (1.42 ± 0.41 µm/s) segment in the anterograde direction (Table 1). These velocities of particle movement are comparable to those for IFT proteins (Snow et al. 2004; Ou et al. 2005a; Blacque et al. 2006; Efimenko et al. 2006), suggesting that DYF-11 is moving with IFT particles carried by cooperative anterograde movement of kinesin-II and OSM-3 motors. Dependency of DYF-11 movement on IFT was further supported by the observation that VENUS :: DYF-11 accumulated at the tip of cilia in mutants of the IFT-A gene, daf-10, which is required for retrograde transport (Fig. 6C).
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The coiled-coil region but not the highly conserved putative microtubule-binding region is required for the ciliary function of DYF-11
The orthologues of DYF-11 show the highest similarity with each other in their N-terminal regions; for example, the N-terminal 106 amino acids of DYF-11 show 41.1% identity to the first 112 amino acids of human TRAF3IP1 (Supplementary Fig. S2). This region was shown to be a microtubule- and β-tubulin-binding domain in TRAF3IP1 (Ling & Goeddel 2000). On the other hand, approximately 110 amino acids at the C-terminus of DYF-11 form a coiled-coil structure (Supplementary Fig. S2, aa 416–531), which is known as a protein–protein interaction domain. The equivalent region of TRAF3IP1 is also proposed to form a coiled-coil structure, which contributes to association with TRAF3 (Ling & Goeddel 2000). The amino acid identity between DYF-11 and TRAF3IP1 within the coiled-coil regions is slightly higher than that of central regions (25.0% for the coiled coil regions, and 17.7% for the central regions). To determine which regions of DYF-11 contribute to ciliary biogenesis, several DYF-11 deletion constructs were generated and tested for rescue of the Dyf phenotype of dyf-11 worms (Fig. 3B). Interestingly, DYF-11 (aa 106–535), which lacks the N-terminal conserved region, was fully functional. Furthermore, C-terminal half of DYF-11 (aa 307–535) was sufficient for both rescue of the Dyf phenotype and localization to cilia (Supplementary Fig. S3A). In contrast, a variant that lacked the coiled-coil region (aa 1–447) failed to rescue the Dyf phenotype (Fig. 3B and Supplementary Fig. S3B). Under the wild-type background, this form of DYF-11 (aa 1–447) was uniformly distributed within the cells and hardly incorporated into IFT (Supplementary Fig. S3C). These results indicate that the coiled-coil region but not the putative microtubule-binding region is required for proper intracellular localization and function of DYF-11.
Traf3ip1 localizes to cilia in MDCK cells
Considering that DYF-11 is required for ciliogenesis in C. elegans, we hypothesized that mammalian Traf3ip1 may also be targeted to cilia. To examine this possibility, we determined the subcellular localization of Traf3ip1 in MDCK renal epithelial cells using anti-Traf3ip1/MIP-T3 antibodies. Cells were cultured on permeable filter supports for 7 days, during which time they polarize and develop apical primary cilia. The affinity-purified antibodies recognized a protein of approximately 90 kDa in MDCK cell lysate, which is similar to the molecular mass previously reported for Traf3ip1 (Ling & Goeddel 2000) (Fig. 7A). Immunofluorescence staining of the cells showed that Traf3ip1 co-localized with acetylated tubulin, indicating that it localizes to the basal body, from which ciliary axoneme extends, and to the ciliary shaft (Fig. 7B). Preincubation of the antibodies with excess competing peptide blocked the immunofluorescence signal in cilia, verifying that the ciliary localization pattern was specific for Traf3ip1 antigen (Fig. 7C). These results indicate that ciliary localization of the orthologues of DYF-11 is conserved in higher eukaryotes.
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Discussion |
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The expression pattern of dyf-11 fits the model in which ciliary genes are classified into two groups based on the property of the X-box motifs (Efimenko et al. 2005). In this model, genes that carry symmetric X-boxes (group 1) are expressed in most or all CSNs and required for general aspects of cilia formation, whereas genes that carry asymmetric X-boxes (group 2) are required in more specialized subsets of ciliated neurons (Efimenko et al. 2005). The promoter region of dyf-11 contains a symmetric X-box sequence motif (GTCTCCATGACAAC, in which the underlined residues are critical for symmetrical X-box), which meets the signature of group 1 genes. In accordance with this, dyf-11 mutants showed highly penetrant dye-filling defects. Moreover, they showed various behavioral abnormalities such as osmotic avoidance defects and chemotaxis defects to NaCl and odorants, demonstrating that at least several classes of sensory neurons are affected. Together, these results strongly indicate that dyf-11 is required for general aspects of ciliogenesis and ciliary functions.
It has recently been proposed that C. elegans IFT machinery has a modular structure consisting of IFT complex A, IFT complex B and BBS proteins that may function interdependently, as well as motor and cargo modules. An IFT-associated molecule can be assigned into distinct modules based on its transport and mutant profiles (Ou et al. 2007). We showed that dyf-11 mutants have short and occasionally malformed cilia and that fluorescent protein-tagged DYF-11 are transported at velocities comparable to that of OSM-3 kinesin in the bbs mutants. These results are consistent with the idea that DYF-11 is associated with IFT complex B. The results of cell- and stage-specific rescue experiments also fit this idea because a previously known IFT-B protein CHE-2/IFT80 shows similar properties (Fujiwara et al. 1999). DYF-11 may be an integral component of the IFT-B particle. Alternatively, DYF-11 may be a cargo protein carried by the OSM-3/IFT-B complex that is critically required for ciliogenesis. Binding assay or purification of IFT particles is required to further attribute DYF-11 to any of the modular components.
Tubulins are the fundamental structural component of cilia. tbb-4, which encodes one of the six β-tubulin isoforms in C. elegans, is selectively expressed in CSNs. Furthermore, the promoter region of tbb-4 contains a symmetric X-box. These observations raised a possibility that TBB-4 is a component of ciliary microtubules (Portman & Emmons 2004). Because DYF-11 is an orthologue of β-tubulin-binding protein, we hypothesized that it may be involved in ciliary localization of TBB-4. To examine this possibility, we observed the intracellular localization of TBB-4 :: GFP in wild-type and dyf-11 mutant backgrounds. The fusion proteins localized to the cell bodies, axons, dendrites and cilia of CSNs in wild-type animals. The intracellular localization pattern of TBB-4 :: GFP in dyf-11 mutants was mostly similar to that of wild-type animals; in the cilia of dyf-11(pe554) mutants, GFP-tagged TBB-4 still entered the residual cilia (our unpublished results). Together with the observation that N-terminal tubulin-binding domain of DYF-11 is not required for ciliogenesis, it seems unlikely that DYF-11 is involved in the transport of TBB-4. Our results showed that the C-terminal coiled-coil domain of DYF-11 is critical for its ciliogenic function. Many of IFT-associated proteins are known to contain protein–protein interaction domains such as WD40 repeats, TPR motifs or coiled-coil domains. These domains are considered to be involved in formation of the IFT particles and interaction with cargo proteins. The coiled-coil domain of DYF-11 is likely to serve similar functions for its interaction with other IFT particle components or cargo proteins.
IL-13 signaling is critical in type 2 helper T cell-induced inflammation and therefore considered to be an important target of therapies for allergic disorders (Wynn 2003). Recent studies have shown that the mammalian orthologue of DYF-11, Traf3ip1/MIP-T3 binds to the 1 subunit of IL-13 receptor (IL-13R1) and modulates the activation of STAT6. STAT6 is also known to localize to cilia and bind to polycystin-1 (PC1, the autosomal-dominant PKD-associated ciliary protein) in renal cells (Niu et al. 2003; Low et al. 2006). Here, we showed that Traf3ip1 localizes to cilia in polarized MDCK cells. Considering that DYF-11 is involved in IFT, DYF-11/Traf3ip1 may function as an adaptor that carries some classes of receptors to cilia and further might be involved in signal transduction by modulating signal transducers; in mammalian cells, we expect that IL-13R1 is targeted to cilia by IFT in association with Traf3ip1, although it needs to be verified experimentally.
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Experimental procedures |
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Growth and culture of C. elegans was carried out by standard methods (Brenner 1974), except that the Escherichia coli strain NA22 was used as food source. Standard mating procedures were used to transfer reporter constructs from the original transgenic worms to various mutant backgrounds. Although dyf-11 mutants show reduced male mating, which is 10%–30% efficient as wild type (Starich et al. 1995), cross-progenies were successfully obtained throughout this study. Genotypes of the resulting strains were determined by reporter expression and fluorescent dye-filling (Dyf) phenotype. The Bristol N2 was used as wild type. Following alleles were used in this study: linkage group (LG) II: daf-19(m86); LG III: osm-12(n1606); LG IV: daf-10(p821); LG V: bbs-8(nx77), odr-3(n2150); LG X: che-2(e1033), dyf-11(mn392), dyf-11(pe554). Transgenes mnIs17[osm-6 :: gfp], ntIs1[gcy-5 :: gfp], and Ex[sra-6 :: venus] were used for visualizing sensory cilia. All strains used and construction details are available on request.
Assays
Staining of living nematode with lipophilic fluorescent dye was carried out essentially as described (Hedgecock et al. 1985), except that 4-(p-Dihexadecylaminostyryl)-N-methylquinolinium iodide (DiQ, Sigma, St Louis, MO) was used at the concentration of 10 µg/mL in M9 buffer. Chemotaxis assays were performed as described (Bargmann et al. 1993; Saeki et al. 2001) with modifications (Matsuki et al. 2006). Osmotic avoidance assays were carried out as described (Culotti & Russell 1978) with minor modifications (Fujiwara et al. 1999). Behavioral assays were performed at least 6 times in each experiment. Heat-shock experiments were performed as described (Fujiwara et al. 1999).
Isolation of C02H7.1/dyf-11(pe554) deletion allele
The deletion allele pe554 was obtained from a UV/TMP-induced deletion library as described previously (Gengyo-Ando & Mitani 2000). The original mutated strain was outcrossed 4 times with N2, resulting in the homozygous strain JN554: dyf-11(pe554) X. This strain was then used in further characterization and genetic crosses. Fluorescent dye-filling assay revealed that Dyf phenotype is linked to linkage group X and that dyf-11(pe554) is fully recessive. Nested primer pairs used for the PCR screening of the dyf-11 deletion mutations were 5'-TAAAAGTGCGATGAGCGTTG-3' and 5'-GGGCATTTCCAAGTTTTTCA-3' for the first round, and 5'-TTTCACGACCACCATTCAAA-3' and 5'-TGATCCTGTCCTCCTCCATC-3' for the second round reaction, respectively.
Complementation test
Complementation between C02H7.1(pe554) and dyf-11(mn392) was performed as follows. Males of C02H7.1(pe554) were cultured with hermaphrodites of dyf-11(mn392) overnight. The mated mn392 hermaphrodites were separated and individually cultured for 3 days to obtain their progeny. Hermaphrodites were picked from the progeny that contained significant ratio of males and tested for dye-filling. Heterozygosity of the tested animals was confirmed by single-worm PCR. We examined 38 pe554/mn392 heterozygotes that resulted from three independent crosses and found that all of them exhibited defective dye-filling.
Cloning and molecular characterization of the C02H7.1/dyf-11 gene
A 5-kb genome DNA fragment, which contains the entire C02H7.1 coding region, was amplified from dyf-11(mn392) worms using the primers 5'-AGCGTGGCTGCTCATTA-3' and 5'-AGTGTTGGACCTAAACAAA-3', and cloned into pBluescript II vector. The nucleotide sequence of all exons was determined and compared with that of wild-type worms to determine the mutation point. The result was confirmed by sequencing two independently amplified PCR fragments. The same genome region obtained from both wild-type and dyf-11(mn392) animals was used to rescue dyf-11(mn392) (see text). A cDNA clone for dyf-11, yk91d12, was provided by the C. elegans cDNA project led by Yuji Kohara. Nucleotide sequence of this clone verified the predicted exon–intron structure (The WormBase: <http://www.wormbase.org/>, Chen et al. 2005), whereas it just lacked initiation codon. We obtained the missing 5' part of cDNA by RT-PCR with a trans-splicing leader sequence SL1 as the forward primer and 5'-GTTCCCAACATTTGCAGCA-3' as the reverse primer. The full-length cDNA thus obtained was cloned into a Gateway destination vector (Invitrogen, Carlsbad, CA) and used to generate rescue constructs in combination with various promoters.
Plasmid construction and generation of transgenic worms
The Gateway cloning technology (Invitrogen) was used to generate plasmids. Two different lengths (4.1 and 2.0-kb) of promoter region of dyf-11 were subcloned into pENTR1A entry vector. Promoters were then transferred to a destination vector, pDEST-venus, which carries the coding sequence for a variant of GFP, VENUS. Thus transcriptional reporter fusions, pGdyf11p(–4.1) :: venus and pGdyf11p(–2.0) :: venus were generated. Similarly, authentic promoter-driven and heat shock-inducible dyf-11(+) expression vectors used in Figs 2G, and 5B and C were generated by combining the dyf-11 full-length cDNA with the dyf-11 (4.1-kb) promoter or the heat-inducible hsp-16.2 promoter, respectively. A destination vector for expressing VENUS :: DYF-11 fusion protein was generated based on the full-length cDNA construct. The coding sequence for VENUS without a termination codon (gift of T. Ishihara) was inserted in frame into the translation start site of dyf-11. The resulting plasmid was then recombined with the entry clones that harbor the 4.1-kb dyf-11 promoter, generating pGdyf11p(-4.1) :: venus :: dyf11. Tissue specific VENUS :: DYF-11 expressing constructs used in Figs 2D, E and 5A were generated in a similar fashion. DYF-11 deletion constructs used in the functional domain analyses were generated by PCR to remove particular dyf-11 region from the pGdyf11p(-4.1) :: venus :: dyf11.
Reporter constructs were introduced into worms by germ-line transformation with standard method (Mello et al. 1991). For expression analyses, wild-type animals were transformed with 25 ng/µL of test DNA with 50 ng/µL pRF4, a transformation marker carrying the dominant rol-6(su1006) gene (Mello et al. 1991). In rescue experiments, test DNA was injected at a concentration of 5 ng/µL. The myo-3 :: venus transcriptional fusion, which enables the transformants recognizable under dissecting fluorescence microscope, was used as a transformation marker in behavioral assays.
For localization studies of VENUS :: DYF-11, wild-type animals were transformed with pGdyf11p(-4.1) :: venus :: dyf11 at 5 ng/µL along with 50 ng/µL pRF4. To obtain the transgenic mutant strains used in motility assays, adult Rol males carrying the extrachromosomal array were mated to dyf-11(pe554), osm-12(n1606) or bbs-8(nx77) hermaphrodites. F1 hermaphrodites were screened for Rol phenotype and allowed to self-fertilize. Homozygous transgenic strains were screened by the presence of Rol phenotype and defects in dye-filling.
Cell culture and immunofluorescence
MDCK cells were grown in MEM Eagle's medium supplemented with 10% fetal bovine serum and 0.1 mM MEM non-essential amino acids (Gibco, Carlsbad, CA). Cells were allowed to polarize on Transwell (Corning, NY) for 7 days with daily media change. Affinity-purified anti-MIP-T3/Traf3ip1 antibodies were obtained from Santa Cruz Biotechnology. Immunofluorescence staining was performed as described (Hirabayashi et al. 2003). Anti-MIP-T3 antibodies diluted 1 : 100 were preincubated with 50 mg/mL of either control (3xFLAG, Sigma) or competing (MIP-T3, Santa Cruz, Santa Cruz, CA) peptide prior to application to fixed cells along with anti-acetylated tubulin (Sigma). Images were obtained with a 40x objective on a Zeiss Axioplan2 microscope equipped with CSU21 confocal scanning unit (Yokogawa, Musashino, Tokyo, Japan). Three-dimensional reconstruction of the serial sections was processed by Metamorph software (Molecular Devices, Downingtown, PA). Western blotting was performed using the anti-MIP-T3 antibodies diluted 1 : 100 as primary antibodies.
Imaging and IFT motility assay
Cellular expression patterns of the reporter constructs were determined as described (Kunitomo et al. 2005). For IFT motility assays, worms were anesthetized and mounted as described (Orozco et al. 1999; Signor et al. 1999; Zhou et al. 2001). Time-lapse images of the sensory cilia expressing VENUS :: DYF-11 was obtained using a 100x objective on a Zeiss Axioplan2 fluorescence microscope equipped with ORCA-ER CCD camera (Hamamatsu, Shizuoka, Japan). Images were acquired at two frames per second for 15 s and processed by Metamorph software. Velocities of particle movement were determined as described (Haycraft et al. 2003).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: iino{at}biochem.s.u-tokyo.ac.jp
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Received: 3 August 2007
Accepted: 26 September 2007
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