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Genes to Cells (2007) 12, 593-602. doi:10.1111/j.1365-2443.2007.01076.x
© 2007 Blackwell Publishing or its licensors
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IFT-81 and IFT-74 are required for intraflagellar transport in C. elegans

 Tetsuo Kobayashi1,2, Keiko Gengyo-Ando1,2, Takeshi Ishihara3, Isao Katsura4,5 and Shohei Mitani1,2,*

1 Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
2 CREST, JST, Japan
3 Deptatment of Biology, Faculty of Science, Kyushu University Graduate School, Fukuoka, 812-8581, Japan
4 Structural Biology Center, National Institute of Genetics, Mishima, 411-8540, Japan
5 Department of Genetics, The Graduate University for Advanced Studies


   Abstract
Top
 Abstract
Introduction
 Results
 Discussions
 Experimental procedures
 References
 
Intraflagellar transport (IFT) is essential machinery for biogenesis and maintenance of cilia in many eukaryotic and prokaryotic cells. A large number of polypeptides are known to be involved in IFT, but the physiological role of each component is not fully elucidated. Here, we identified a C. elegans orthologue of a Chlamydomonas reinhardtii IFT component, IFT-81, and found that its loss-of-function mutants show an unusual behavioral property and small body size. IFT-81 is expressed in sensory neurons, and localized at the base of cilia. The similar phenotypes with ift-81 mutants were also observed in several IFT mutants, suggesting these defects are caused by inability of IFT. We also demonstrated that IFT-81 interacts and co-localizes with IFT-74, which is another putative component of IFT. The ift-74 loss-of-function mutants showed phenocopies with ift-81 mutants, suggesting IFT-81 and IFT-74 play comparable functions. Moreover, ift-81 and ift-74 mutants similarly exhibited weak anomalies in cilia formation and obvious disruptions of transport in mature cilia. Thus, we conclude that IFT-81 and IFT-74 coordinately act in IFT in C. elegans sensory cilia.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussions
 Experimental procedures
 References
 
Many eukaryotic cells have cilia, which play diverse roles in many physiological processes, such as cell and fluid movement, sensory perception and development (Inglis et al. 2006; Marshall & Nonaka 2006). Recently, aberrant cilia functions have been revealed to underlie the large number of human disorders (Pazour & Rosenbaum 2002; Ibanez-Tallon et al. 2003; Afzelius 2004; Beales 2005; Pan et al. 2005). The phenotypes of these diseases include cystic kidneys, retinal degeneration and retinitis pigmentosa, situs invs., anosmia, respiratory problem, infertility, hydrocephalus, obesity, diabetes, hypertension, heart defects, sensory deficits, skeletal, neurological and development anomalies (Pazour & Rosenbaum 2002; Ibanez-Tallon et al. 2003; Afzelius 2004; Beales 2005; Pan et al. 2005).

Cilium biogenesis and maintenance depend on a microtuble associated transport machinery: intraflagellar transport (IFT) (Kozminski et al. 1993; Rosenbaum & Witman 2002; Scholey 2003). The IFT machinery conveys cargo, such as receptors and channels, to the ciliary tip by kinesin-II motors and recycles components to the basal body by a dynein motor (Rosenbaum & Witman 2002; Cole 2003; Scholey 2003; Inglis et al. 2006). The motors cooperate with a multiprotein complex, IFT particle, which consists of two subcomplexes, A and B (Piperno & Mead 1997; Cole et al. 1998). Although the IFT particle has been reported to contain 16 proteins by biochemical analysis in the unicellular green alga Chlamydomonas reinhardtii (Piperno & Mead 1997; Cole et al. 1998), the physiological role of each component yet remains obscure. Here, we used a model organism, C. elegans. In C. elegans, 60 of the 302 neurons of the hermaphrodite are ciliated sensory neurons, and several works have demonstrated that the mutants of IFT-related genes show defects in sensory functions (Cole et al. 1998; Signor et al. 1999; Qin et al. 2001; Haycraft et al. 2003; Schafer et al. 2003; Blacque et al. 2004; Scholey et al. 2004; Snow et al. 2004; Ou et al. 2005; Bell et al. 2006; Evans et al. 2006). In the present work, we identified nematode IFT-81 and IFT-74, whose C. reinhardtii orthologues were recently shown to form core complex in subcomplex B (Lucker et al. 2005), and attempted to elucidate their physiological roles in C. elegans.


   Results
Top
 Abstract
 Introduction
 Results
Discussions
 Experimental procedures
 References
 
Identification of the ift-81 gene on the C. elegans genome and its mutant phenotypes

To investigate the function of C. elegans orthologue of IFT-81, we performed a database search. A putative F32A6.2 polypeptide has homology with the N-terminal region of C. reinhardtii IFT-81. In addition, the protein sequence of a neighbor gene F32A6.1 is similar to the C-terminal region of C. reinhardtii IFT-81. We assumed a possibility that F32A6.1 and F32A6.2 form one gene. Since a DNA fragment could be amplified by PCR using F32A6.2 5' primer and F32A6.1 3' primer from N2 cDNA (data not shown), we determined the DNA sequence of the coding region of amplified cDNA. A comparison of the determined cDNA and genomic sequences revealed a new gene that consists of 17 exons encoding a polypeptide of 638 amino acids (Supplementary Fig. S1A). This protein shares 28% and 23% identity with Homo sapiens and C. reinhardtii IFT-81, respectively.

To analyze the physiological roles of this putative gene, we isolated deletion alleles, tm2355 and tm2356 disrupting F32A6.2, and tm2389 disrupting F32A6.1 (Supplementary Fig. S1A). Although these mutant worms appear visibly normal, we found that they show decreased moving range. When single N2 worm was placed on the seeded NGM plate and incubated overnight, the tracking pattern usually spreads over the whole surface of bacteria (Fig. 1A). Under the same condition, the tracking patterns of tm2355, tm2356 and tm2389 worms were restricted to very narrow area on food (Fig. 1C and data not shown). To assess this behavioral feature, we quantitatively analyzed the track of worms. After the photography of the tracking pattern of single worm on food, the picture was binarized to quantify the area that the worm passed (Fig. 1B,D). Then, the ratio of the marked region per seeded region was calculated. As shown in Fig. 1E, the estimated values obtained for tm2355, tm2356 and tm2389 mutants were clearly lower than that of N2. This result suggests that the region from F32A6.2 to F32A6.1 encodes one gene, which is probably an orthologue of C. reinhardtii IFT-81, and this protein is required for controlling moving range. We assigned a CGC name ift-81 to this gene. To investigate whether the unusual tracking mode of these mutants are caused by locomotion defect, we compared tracking pattern of ift-81(tm2355) mutant and an Unc mutant. unc-86(eDf25) worms showed slightly lower frequency of body bend but exhibited more normal tracking pattern than ift-81(tm2355) worms (Supplementary Fig. S2). This result suggests that the confined tracking pattern of ift-81 mutant is not explained by an inability of movement.


Figure 1
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  		Figure 1  ift-81 and ift-74 mutant worms show abnormalities in tracking pattern and body size. (A–D) Tracking patterns of N2 (A, B) and ift-81(tm2355) worms (C, D). Left panels (A, C) exhibit tracking patterns of each strain on food, and right panels (B, D) show processed image of left panels. Scale bar: 2 mm. (E) Graph for quantified tracking data of N2, ift-81(tm2355), ift-81(tm2356), ift-81(tm2389), ift-74(tm2394), ift-74(tm2397) and ift-74; ift-81(tm2394; tm2355). Error bars indicate SEM. **P < 0.01; *P < 0.05, comparing each mutant strain with N2. (F) Growth curves of N2, ift-81(tm2355), ift-81(tm2356), ift-81(tm2389), ift-74(tm2394), ift-74(tm2397) and ift-74; ift-81(tm2394; tm2355). Error bars indicate SEM. P < 0.01, comparing each mutant strain with N2 at 96, 144, 192 h.

 
We observed that ift-81 mutants have smaller bodies than N2. As shown in Fig. 1F, the body length of ift-81 mutant worms (tm2355, tm2356, tm2389) is shorter than N2 worms from the L4 stage to late adulthood. This result suggests that IFT-81 is needed for normal growth. We confirmed that extrachromosomal array of ift-81::venus translational fusion construct almost rescues these abnormalities in behavior and body size (Fig. 2 and Supplementary Fig. S1C).


Figure 2
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  		Figure 2  Tracking and body size defects of ift-81 and ift-74 mutants are rescued by expressing ift-81 and ift-74 genes, respectively. (A) Graph for quantified tracking data of each strain. Error bars indicate SEM. *P < 0.01. (B) Graph for body length of each strain at 144 h post-hatch. Error bars indicate SEM. *P < 0.01.

 
Expression of IFT-81/Venus in sensory cilia

Since C. reinhardtii IFT-81 is a component of IFT-particle subcomplex B, we conceived that C. elegans IFT-81 is also involved in IFT. We then analyzed the expression pattern of ift-81::venus translational fusion construct (Supplementary Fig. S1C). As shown in Fig. 3A–D, the punctuate fluorescent signals were observed in the cell bodies of the head and tail neurons. These signals were detected in all of the dye-filling assay positive cells (data not shown), suggesting that IFT-81 is expressed in ADL, ASH, ASI, ASJ, ASK, AWB, PHA, PHB ciliary sensory neurons. To more clarify IFT-81 expressing cells, we next did co-expression analysis of IFT-81/DsRed and OSM-6/venus. OSM-6 is a member of IFT subcomplex B, and known to be expressed in all of the ciliated neurons except the BAG and FLP neurons (Collet et al. 1998). As shown in Fig. 3E–H, the fluorescent signals of DsRed and venus were observed in the cell body of almost the same cells. In these cells, subset of OSM-6/venus overlapped with IFT-81/DsRed signals (Fig. 3E,G). These results suggest that IFT-81 is expressed in almost all of the ciliated sensory neurons and may form complex with OSM-6 in these cells. Moreover, we observed co-distribution of IFT-81/DsRed and OSM-6/venus at the transition zone at the base of cilia in amphid (Fig. 3I–L) and phasmid neurons (Fig. 3M–P) like many IFT-related proteins. These results suggest that IFT-81 is involved in ciliary functions, which is probably IFT.


Figure 3
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  		Figure 3  IFT-81 is expressed in sensory neurons. (A–D) Expression patterns of IFT-81/venus in head region (A, B) and tail region (C, D). Fluorescent images (A, C) and DIC images (B, D) are shown. (E–H) Expression patterns of IFT-81/DsRed and OSM-6/venus in head region (E, F) and tail region (G, H). Merged fluorescent images (E, G) and DIC images (F, H) are shown. (I–P) Expression patterns of IFT-81/DsRed and OSM-6/venus in amphid cilia (I–L) and phasmid cilia (M–P). DsRed (I, M), venus (J, N), merged (K, O), and DIC (L, P) images are shown. Arrowheads indicate transition zone. Scale bar: 20 µm (A–H), 5 µm (I–P). (Q, R) Schematics of the head (Q) and tail (R) show the position of cilial structures at the dendric endings of amphid and phasmid neurons. Arrowheads indicate transition zone.

 
Sensory perception mutants show phenotypes similar to ift-81 mutants

The above properties of ift-81 mutants resemble the phenotypes of some chemosensory mutants. These mutants were reported to show an altered locomotory behavior and small body size, suggesting defects in sensory perception cause abnormal behavior and growth (Fujiwara et al. 2002). In addition, some of these gene-products, such as CHE-2 and OSM-6, are putative orthologues of C. reinhardtii IFT components involved in subcomplex B (Rosenbaum & Witman 2002; Scholey et al. 2004).

In C. elegans, several proteins, such as OSM-1, OSM-5, OSM-6, CHE-2 and CHE-13, have been considered as putative members of subcomplex B (Rosenbaum & Witman 2002; Scholey et al. 2004). We investigated whether the mutants of these genes also show unusual tracking pattern and small body in a similar fashion with ift-81 mutants. As shown in Fig. 4, these mutants exhibited the unusual behavior and small body size like the ift-81 mutant. These results suggest that the subcomplex B mutants generally exhibit abnormalities in tracking pattern and growth, and support the idea that IFT-81 plays similar roles with the components of subcomplex B in C. elegans.


Figure 4
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  		Figure 4  IFT subcomplex B mutants in C. elegans exhibit tracking and body size defects. (A) Graph for quantified tracking data of each strain. Error bars indicate SEM. *P < 0.01. (B) Graph for body length of each strain at 144 h post-hatch. Small body size of osm-6(p811) was previously shown by Fujiwara et al. (2002). Error bars indicate SEM. *P < 0.01.

 
IFT-74 as another component which interacts with IFT-81

It was recently reported that Chlamydomonas IFT-81 directly interacts with Chlamydomonas IFT-74/72, which are components of subcomplex B (Lucker et al. 2005). IFT-74 and IFT-72 are derived from the same gene in C. reinhardtii, and database search elucidated that a putative nematode orthologue is encoded by C18H9.8 on C. elegans genome. We assigned C18H9.8 a CGC name ift-74, and first investigated whether nematode IFT-81 associates with IFT-74 by yeast two-hybrid assay. As shown in Fig. 5, yeast colonies containing ift-81 and ift-74 genes specifically showed blue color in ß-gal assay. This result suggests that IFT-81 binds to IFT-74 in C. elegans. Next, we analyzed expression pattern of IFT-74 using ift-74::venus translational fusion construct (Supplementary Fig. S1D). Fluorescent signals of IFT-81/DsRed and IFT-74/venus overlapped in head and tail region (Fig. 5B–I). Moreover, the co-localization was also detected in the transition zone of cilia (Fig. 5J–Q). These results suggest that IFT-81 and IFT-74 form a complex in nematode ciliary neurons. We then isolated deletion mutants of ift-74, tm2394 and tm2397 (Supplementary Fig. S1B), and investigated the phenotypes. These ift-74 mutants worms (tm2394, tm2397) showed tracking defect and small body size like ift-81 mutant worms (tm2355, tm2356, tm2389) (Fig. 1E,F). We confirmed that extrachromosomal array of ift-74::venus translational fusion construct almost rescues these abnormalities in tm2394 (Fig. 2 and Supplementary Fig. S1D). An ift-74; ift-81(tm2394; tm2355) double mutant also exhibited the defects analogous to those of each single mutant in both assays (Fig. 1E,F). All these results suggest that IFT-74 is required for the normal behavior and growth like IFT-81, and IFT-81 and IFT-74 cooperatively act as components of IFT in nematode sensory cilia.


Figure 5
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  		Figure 5  IFT-74 binds and co-localizes with IFT-81. (A) Yeast strains carrying indicated plasmids were streaked on a filter paper and subjected to the ß-gal assay. (B–I) Expression patterns of IFT-81/DsRed and IFT-74/venus in the head region (B–E) and the tail region (F–I). Venus (B, F), DsRed (C, G), merged (D, H), and DIC (E, I) images are shown. (J–Q) Expression patterns of IFT-81/DsRed and IFT-74/venus in amphid cilia (J–M) and phasmid cilia (N–Q). Venus (J, N), DsRed (K, O), merged (L, P), and DIC (M, Q) images are shown. Arrowheads indicate transition zone. Scale bar: 20 µm (B–I), 5 µm (J–M).

 
ift-81 and ift-74 mutants show apparent chemotaxis, but only mild ciliogenesis-defects

C. elegans IFT subcomplex B mutants have morphologically aberrant cilia, and hence these worms show defects in chemotaxis (Scholey et al. 2004). We then performed the chemotaxis assay to clarify whether IFT-81 and IFT-74 are necessary for chemotaxis. As shown in Fig. 6A, ift-81 and ift-74 mutant worms showed defects in chemotaxis for Diacetyl and Isoamyl alcohol, suggesting IFT-81 and IFT-74 are required for the normal function of sensory cilia. Next, we did dye-filling assay to elucidate whether the ciliary dysfunction in ift-81 and ift-74 mutant worms are caused by the morphological abnormalities. In the dye-filling assay, wild-type animals can absorb fluorescent dye in the medium, whereas cilia assembly mutants are unable to uptake the dye. We used synchronously cultured young adult worms, and analyzed the uptake of fluorescent dye. As shown in Fig. 7A, ift-81 and ift-74 mutants (tm2355, tm2356, tm2389, tm2394, tm2394; tm2355, except for tm2397 which harbors an in-frame deletion) showed partial but evident deficiencies in absorbance of fluorescent dye compared with N2. On the other hand, all strains of other IFT complex B mutants could not absorb the dye under the same condition. These results suggest that IFT-81 and IFT-74 are surely necessary for the cilia morphogenesis, but the importance of these proteins might be somewhat lower than the other components of IFT complex B. These notions are supported by the result that cilium structure of ift-81(tm2355) and ift-74(tm2394) worms is apparently indistinguishable from that of N2 (Fig. 7B–D).


Figure 6
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  		Figure 6  IFT-81 and IFT-74 are required for correct chemotaxis and IFT. (A) ift-81 and ift-74 mutants show defects in chemotaxis. Black, gray and white columns are N2, ift-81(tm2355) and ift-74(tm2394), respectively. Error bars indicate SEM. *P < 0.01. (B–D) Ciliary distribution of OSM-9/venus in N2 (B), ift-81(tm2355) (C) and ift-74(tm2394) (D). An arrow indicates normal distribution of OSM-9/venus in N2 (B). Arrowheads indicate unusual pattern of OSM-9/venus in ift-81(tm2355) and ift-74(tm2394) (C, D). The relative ratios of ift-81 and ift-74 mutants against N2 which showed correct OSM-9/venus distribution worms were only 45% and 41%, respectively. Scale bar: 5 µm. (E) A schematic of the head shows the position of cilial structures at the dendric endings of amphid neurons. An arrow indicates the cilia.

 

Figure 7
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  		Figure 7  ift-81 and ift-74 mutants show marginal defects in cilia formation. (A) ift-81 and ift-74 mutants show mild abnormalities in dye-filling assay. White box: worms showing normal uptake. Gray box: worms showing partial or weak uptake. Black box: worms showing no uptake. For each strain, more than 50 worms were observed. (B–D) Fluorescent patterns of ift-81p::venus in N2 (B), ift-81(tm2355) (C) and ift-74(tm2394) (D). Phasmid cilia are indicated. Scale bar: 5 µm. (E) A schematic of the tail shows the position of cilium structure at the dendric ending of a phasmid neuron. The phasmid cilium is indicated.

 
ift-81 and ift-74 mutants show defects in IFT

Recently, it was reported that GFP-tagged transient receptor potential vanilloid (TRPV) channel OSM-9 localizes along the cilia in an IFT dependent manner, and the distribution is ruined in IFT mutants possessing morphologically normal cilia (Qin et al. 2005). To investigate whether the IFT-81 and IFT-74 are necessary for the IFT in developed cilia as well as ciliogenesis, we attempted to monitor OSM-9/venus in the mutant worms. We constructed a strain that have an extrachromosomal array of osm-9::venus translational fusion construct, and observed the distribution of OSM-9/venus in ift-81 and ift-74 mutants. The dye-filling positive worms were used to evaluate the capability of IFT in morphologically normal cilia. In wild-type worms, the fluorescent signals of OSM-9/venus were detected along the cilia (Fig. 6B). On the other hand, in ift-81 and ift-74 mutants, OSM-9/venus was not precisely localized at cilia even in dye-filling positive worms (Fig. 6C,D). These results suggest that IFT-81 and IFT-74 are responsible for the IFT in mature cilia.


   Discussions
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 Abstract
 Introduction
 Results
 Discussions
Experimental procedures
 References
 
So far, significant information about IFT has been mainly obtained by the works of C. reinhardtii and C. elegans. Although insertional mutagenesis studies using C. reinhardtii have indicated the physiological necessities of several IFT factors for flagellar assembly (Cole 2003), the role of IFT-81 and IFT-74 remains unclear. In the present study, we isolated C. elegans ift-81 and ift-74 mutants, and demonstrated that these worms show the phenotypes caused by the ciliary dysfunction, suggesting IFT-81 and IFT-74 are essential for the normal feature of cilia. Our findings about ift-81 mutant are consistent with the report that the zebrafish ift-81 mutant has cystic kidney, which is also considered to be caused by the defects of cilia formation and/or function (Sun et al. 2004).

We exhibited that nematode IFT-81 and IFT-74 associate with each other like C. reinhardtii and H. sapiens (Lucker et al. 2005). We also found that IFT-81 and IFT-74 co-localize in sensory cilia, and these mutants showed similar characters. Furthermore, we showed that IFT-81 co-localizes with OSM-6, and ift-81 mutant shows similar abnormalities in tracking and body size with those of IFT subcomplex B mutants. These data strongly suggest that IFT-81 and IFT-74 cooperatively function as a complex, and are involved in IFT subcomplex B in C. elegans. This notion is consistent with the knowledge that IFT-81 and IFT-74 form core complex in C. reinhardtii IFT subcomplex B (Lucker et al. 2005). On the other hand, all ift-81 and ift-74 mutants, even an ift-74; ift-81 double mutant, did not show the severe defect of cilia formation unlike other IFT subcomplex B mutants (Fig. 7). This result was somewhat unexpected considering the above report in C. reinhardtii (Lucker et al. 2005). Although it cannot be denied that all C. elegans ift-81 and ift-74 mutants are not null mutants and hence the defect is weak, it is conceivable that the position of IFT-81/IFT-74 in IFT subcomplex B in multicellular C. elegans may be different from that in unicellular organism C. reinhardtii.

Many comprehensive studies using bioinformatics, genomics and proteomics have identified numerous putative ciliary proteins (Ostrowski et al. 2002; Avidor-Reiss et al. 2004; Li et al. 2004; Blacque et al. 2005; Efimenko et al. 2005; Pazour et al. 2005; Smith et al. 2005; Stolc et al. 2005). A recent work showed that a cGMP-dependent protein kinase (PKG) depending on IFT machinery participate in the flagellar-adhesion pathway in the alga Chlamydomonas, suggesting IFT has a direct role in cilium-based signaling (Scholey & Anderson 2006; Wang et al. 2006; Yoder 2006). These studies allow us to support that there are yet undiscovered factors and/or novel signal cascades to be essential for healthy ciliary functions. In the viewpoint of human diseases, to unravel the ciliary feature in multicellular organism is significant, and we are currently working on this important challenge in combination with C. elegans genomics.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussions
 Experimental procedures
References
 
Strains

Strains were basically maintained under standard conditions. Several deletion alleles used in this work were isolated as previously described (Gengyo-Ando & Mitani 2000). The strains include: N2 Bristol wild-type, ift-81(tm2355), ift-81(tm2356), ift-81(tm2389), ift-74(tm2394), ift-74(tm2397), tm2394; tm2355, unc-86(eDf25), osm-1(p808), osm-1(p816), osm-5(p803), osm-5(ok451), osm-6(p811), che-2(e1033), che-13(e1805), che-13(n1520), tm2355; tmEx1455[pFX_venusT-ift-81(FL) (ift-81::venus), pFX_YFPT-unc-122], tm2355; tmEx1457[pFX_venusT, pFX_YFPT-unc-122], tm2394; tmEx1458[pFX_venusT-ift-74(FL) (ift-74::venus), pFX_YFPT-unc-122] and tm2394; tmEx1460[pFX_venusT, pFX_YFPT-unc-122], tmEx1446[pFX_venusT-ift-81(FL) (ift-81::venus), pRF4], tmEx1452[pFX_DsRedxT-ift-81(FL) (ift-81::DsRed), pFX_venusT-osm-6(FL) (osm-6::venus), pRF4], tmEx1415[pFX_DsRedxT-ift-81(FL), pFX_venusT-ift-74(FL) (ift-74::venus), pRF4], tmEx1444[pFX_venusT-osm-9(FL) (osm-9::venus), pRF4], tm2355; tmEx1444, tm2394; tmEx1444, tmEx1407[pFX_venusT-ift-81(aa1) (ift-81p::venus), pRF4], tm2355; tmEx1407 and tm2394; tmEx1407.

Plasmids

ift-81(FL), ift-74(FL), osm-6(FL) and osm-9(FL) were amplified from N2 genomic DNA, and include promoter region and whole genomic region without stop codon. ift-81(FL), ift-74(FL), osm-6(FL) and osm-9(FL) contain ~3.0, ~1.1, ~0.9 and ~5.0 kbp promoter region, respectively. ift-81(aa1) contains promoter region of ift-81(FL). These DNA fragments were inserted into pFX_venusT and pFX_DsRedxT vectors (Gengyo-Ando et al. 2006). ift-81(cds) and ift-74(cds) were amplified from N2 cDNA, and inserted into pGBKT7 and pGADT7, respectively. ift-81(cds) contains whole coding sequence of ift-81 (amino acids 1–638), and ift-74(cds) contains deleted coding sequence of ift-74 (amino acids 51–624).

Tracking assay

Synchronously cultured single L4-worm was transferred to 3.5 cm seeded NGM plate, and allowed to crawl freely at 20 °C. We used NGM plates that have almost similar bacterial area. After 18 h, the picture of tracking pattern was taken under a dissecting microscope with C5060WZ (Olympus). The image was binaryzed and the area that a worm passed on food was measured using the IPLAB software (Scanalytics). Statistical analysis was performed by using Student's t-test.

Body size measurement

The worms of each strain were fed on at 20 °C to induce the laying of embryos. After the parent adults were removed, remaining eggs were incubated for 2 h at 20 °C. The newly hatched L1-worms were harvested, and cultured at 20 °C. At indicated time points, body length of worms was measured with the scale-bar which was attached to a dissecting microscope. Although the measured values were varied in each experiment, the ratio of mutant body size to N2 was almost the same. Statistical analysis was performed by using Student's t-test.

Microscopy

Fluorescence images were obtained using a BX51 microscope (Olympus) equipped with a DP30BW camera (Olympus) or a Axio Imager.M1 (ZEISS) equipped with a AxioCam HRm camera (ZEISS).

Yeast two-hybrid assay

Yeast two-hybrid assay was performed using the Matchmaker two-hybrid system 3 (Clontech). Yeast cells were grown in standard culture media and transformed with DNA by the lithium acetate method. ß-gal assay was performed as described in the manufacturer's protocol. For each experiment, four independently derived colonies were tested.

Chemotaxis assay

Chemotaxis assays were basically performed as previous works (Bargmann & Horvitz 1991; Bargmann et al. 1993). The concentrations of each compound are described below; Diacetyl 1% (v/v) and Isoamyl alcohol 1% (v/v). The index was calculated as (number of worms at attractant—number of worms at reference)/(number of total worms). Statistical analysis was performed by using Student's t-test.

Dye-filling assay

The young adult worms were incubated in the DiI solution (10 µg/mL diluted with M9 buffer) for 2 h, and then allowed to crawl freely on NGM plate for 1 h (Hedgecock et al. 1985).


   Acknowledgements
 
We thank A. Miyawaki for the venus cDNA; the Caenorhabditis Genetics Center for strains; T. Awaji and A. Sugimoto for discussions. This work was supported by Grants from Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Science and Technology Agency to S.M. and K. G-A.


   Footnotes
 
Communicated by: Masayuki Yamamoto

* Correspondence: E-mail: mitani1{at}research.twmu.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussions
 Experimental procedures
 References
 
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Received: 12 October 2006
Accepted: 1 February 2007




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