Constructs, nematode culture and worm genetics
Worms were cultured using standard methods43
. The fluorescently labeled markers were introduced into single mutants by genetic crossing. The double mutants comprising dyf-6
, ift-74, tba-5 and tbb-4
were produced using genetic crosses and monitoring of the mutant background, Dyf phenotype or deletion sequence (by PCR). The double deletion mutant, tba-5(tm4200)
was facilitated using a dpy-6
marker linked to tbb-4
. Rescue of tba-5(qj14)
was performed by injection of a TBA-5 construct, which was made by cloning its cDNA and upstream 7.4 kb promoter region into pPD95.75. For observation of EBP-2 in dendrites and cilia, a construct of ebp-2::GFP driven by an osm-6
promoter was introduced into wild type worms.
Cloning of qj55, qj23, qj14 and dyf-12
Complementation tests between two dyf
mutants were done by crossing N2 male worms with one dyf
mutant to generate heterozygous males carrying the mutated gene (heterozygous males were used because most dyf
worms have low mating efficiency). These males were then mated with hermaphrodites of the second dyf mutant. The crossed progeny were analyzed by dye-filling assays to determine whether the two mutants are alleles or not. The SNP mappings were done based on documented single nucleotide polymorphisms between the N2 and the Hawaiian strains (CB4856)44
. In brief, a double mutant of qj23
with its linkage marker gene dpy-8
(the worms are dumpy) was made and allowed to mate with CB4856 to obtain the heterozygote worms. From their progeny, eleven Dpy non Dyf and five Dyf non Dpy recombinants were selected and analyzed by SNP markers. qj23
was narrowed down to a region containing nine genes. These genes were analyzed by sequencing to determine the mutation. The same SNP cloning strategy was applied to qj14
, and dpy-5
were used as their linkage markers.
Worms were washed off the culturing plates with M9 buffer and collected in a 15 ml tube by centrifugation at 3000 rpm for 3 min. The DiI (Molecular Probes, Invitrogen, Carlsbad, California, USA) solution was added to a final concentration of 10 μg ml−1. After incubation for 2–4 h, the stained worms were spun down and washed three times with M9 buffer. The worms were then transferred to 2% agarose pads with a drop of 10 mM NaN3 and observed under a compound microscope with a 60× objective. The staining ratio is the number of stained amphids or phasmids divided by the total number of amphids or phasmids.
Animals were prepared and sectioned for electron microscopy using standard methods45
. Imaging was performed with an FEI Tecnai G2 Spirit BioTwin transmission electron microscope equipped with a Gatan 4K × 4K digital camera.
IFT assay and cilium length measurement
IFT and cilium morphology were assayed as described previously19,21,46
. The worms were immobilized on a 2% agarose pad by anesthetizing them in 10 mM levamisole. The images were collected with an Olympus microscope equipped with a 100×, 1.35NA objective and an Ultraview spinning disc confocal head. The IFT was recorded at 300 ms/frame at 21°C for 3 min using a CCD camera (ORCA-ER; Hamamatsu, Bridgewater, New Jersey, USA). Acquired images were analyzed in MetaMorph (Molecular Devices, Sunnyvale, California, United States) to create kymographs and calculate the transport rate. For the transport assay of TBB-4::YFP, OSM-9::GFP and DYF-1::GFP, the recorded movies were processed using the basic filters (Sharpen High and Low pass) before creating kymographs. Cilia lengths were measured on projection images, created in MetaMorph from recorded z-stacks of the cilia. Shown are projection images edited in Adobe Photoshop 7.0 and assembled in Adobe Illustrator 10. During editing, the brightness and contrast of projection images were slightly adjusted in Photoshop.
The yeast strain used in this study was PJ69-4A (genotype
: MATa; trp1-901; leu2-3,112; ura3-52; his3-200; gal4D; gal80D; GAL2-2ADE;. LYS2::GAL1-HIS3; met2::GAL7-lacZ). The yeast two hybrid plasmids were pGAD-C1, pGBD-C1 containing GAL4 AD (activation domain) and GAL4 BD (DNA binding domain) respectively47
. The ift-81
gene was cloned from cDNA of C. elegans
, and the ift-74, osm-3, and dyf-6
genes were cloned from their EST clones. To eliminate self activation of the expression of His reporter, the genes cloned in pGAD-C1 were co-transformed with empty pGBD-C1 and genes cloned in pGBD-C1 were co-transformed with empty pGAD-C1. Combinations of pGAD-C1 and pGBD-C1 plasmids each carrying one gene to be tested were co-transformed into yeast strain PJ69-4A. Six transformant colonies from each selective plate were streaked onto Leu-, Trp- and His-lacking selective plate to detect the activation of His reporter. In each set of experiments both positive and negative controls were included.
The domain analyses of DYF-6 and IFT-81 were performed using SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1
) and Coils (e.g.48
). TBA-5 or TBB-4 and their orthologs were aligned with Clustal X249
. The heterodimeric structure of TBA-5 and TBB-4 was predicted with Modller9v6 using the porcine brain tubulin heterodimer structure 1JFF33
as a template. The predicted structure was visualized with PyMOL 0.99 (http://www.pymol.org/
FRAP experiments were performed on a laser-scanning Olympus confocal microscope (FV1000) with a 60× 1.40 NA objective at 23°C, and images were acquired using the Fluoview software (version 1.5; Olympus). A 405 nm laser at 40% power was used for photo-bleaching and images were acquired with a 514 nm laser every 3 or 5 s. The data were normalized to the fluorescence before the bleach. The recovery curve was fit with an exponential equation F(t)=F0
), where F(t) is the total fluorescence at time t after the bleach, and k is a constant describing the rate of recovery. F0
is the fluorescence immediately after the bleach and Finf is the maximum recovered fluorescence. The recovery half time was calculated by t1/2=ln2/k and the percentage of fluorescence recovery was given by (Finf−F0
, where Fpre is the fluorescence intensity before the bleach. It is difficult to determine the exact area of recovery directly so we used linescans along the cilia to determine the maximum recovered intensity i.e. Finf in the equation above.
Estimating the diffusion coefficient of GFP in the cilium
Worms expressing free GFP were photobleached and fluorescence profiles along the ciliary length were obtained before and after the bleach. The postbleach fluorescence profiles were subtracted from the prebleach profile. The difference profiles obtained were then fitted to a Gaussian curve. Diffusion coefficients were obtained from these plots, by fitting the normalized bleach depth over time as described in 51
. We estimate a value of ~ 1–5 μm2
Primers used for cloning the genes and identifying the mutants
For tba-5: pKP1056F, CCTCGGAGGAATTTCAAACG; pKP1056R, AGCTCCGTAAAGCAGCTTC; pKP1057F, ATCATTCTCCAGGCCACGTTAC; pKP1057R, CTGAACTAGTCGAACAAACCCC; pKP1082F, AATGAGATGCAAGACCGGGACC; pKP1082R, CTTTCCCACGACCTTTCTTGC; pKP1114F, AGATTGAGGCTGAAATATGGTG; pKP1114R, GTCGAGCAGCACCAGTTATTG; pKP1058F, CCAGTGTCCCGATAGAAAAC; pKP1058R, GAATCACCGCCAACATGAGA; pKP1059F, CATCTGGGACGTTCTTTCAC; pKP1059R, TTCAGGCTCCACTTTATGCC; pKP1117F, CGAATCCATATCGATGCGAC; pKP1117R, ACATCTCTGCGTGGCTCTTC; pKP1119F, TCAAATTTGGCACGTCATCAG; pKP1119R, CTCCATTTTGGAACTCCCAG; CE1-153F, CCGTGAAGCAAGTTCAAATGC; CE1-153R, CTTAACAAGAATTGGTGACCAAC; CE1-170F, CATGTCCGGCGAATGGATTC; CE1-170R, AGCCATGGAATCAGCTGTGG; F10D11.2F, CGCAGATTTGATGACTCCAC; F10D11.2R, TGGGAACTGGATAAACTGGC; uCE1-969F, ATACAGTCTAGTGGGGATTGC; uCE1-969R, CTCAGTGTTACTTGCAGCGG; F02E9F, AGAGAAGCTTATGCGGTTCG; F02E9R, AGTGCCGATTTACGATCTCG; F16D3.1-1, CTATGAGTACCTTCAAACCTG; F16D3.1-2, AAACTTGGCACTCCGTGTAC; F16D3.1-3, AAACTTGGCACTCCGTGTAC; F16D3.1-4, GTTGGACTTCTGACACCTAG; F16D3.1-5, CAGCAATGGTAGAGCCATAC; F16D3.1-6, TGTTCTAAGCCTATCTTGACC; F16D3.1-7, GCAATTTCGCTTGTTCTAACG; F16D3.1-8, TCCAAATGAACCCTTGTGCC; F16D3.1-9F(PstI), AAAActgcagGAGCATGAAGTAGTGTCCTTG; F16D3.1-10R(BamHI), CACGGGATCCCATTTTTCCATTTGGAGCCATGG; F16D3.1-11F(SalI), AAAAgtcgacggatccATGCGTGAAATAGTTTCGATTC; F16D3.1-12R(BamHI), CACGggatccTTTTCCATTTGGAGCCATGG; F16D3.1-12R(XmaI), CCTACCCGGGGATATTCTTCATCATTTGGATCGA; F16D3.1-13F(SphI), GAAAGACCTCGCATGCAAATTTA; F16D3.1-14R(BglII), CCTAagatctCTAATATTCTTCATCATTTGGATCGA; F16D3.1-14R(SphI), TAAATTTGCATGCGAGGTCTTTC; F16D3.1-15F, CGGAAATGtCTGTTGGGAACTG; F16D3.1-16R, CAGTTCCCAACAGACATTTCCG; F16D3.1-17F, TATCAACCACtGACTGTTGTT; F16D3.1-18R, AACAACAGTCAGTGGTTGATA. For tbb-4, pKP6127F, TTGGATCTCCGTAGACGTCAC; pKP6127R, GTCTTCATTGCATGATGTGGC; pKP6112F, GCGTGAGGGCAACTTTTTTG; pKP6112R, TAGGGATTCTCGCGTCATTG; pKP6135F, TCTTTGCTTGTGAGCCAATTGG; pKP6135R, CGGCACGTGTTTTCAAAATAAC; uCE6-1111F, TCTACATGACCTACATGTCTG; uCE6-1111R, TGGACATTTACACAGAACCTG; pas23221F, GTCGAGAAGTTATGTGTGCAG; pas23221R, AAGATGTCCATCTATGGACCG; uCE6-1120F, CAACCACATCGGATATGGTAG; uCE6-1120R, CGTTGGCTTTGACGTACGTTC; tbb4-1F, GCCATTTAAGGACACACCTCC; tbb4-2R, CACGCGTAAGGCGTTGAACC; tbb4-3F, CGGAAATTGAGCGACATCTCC; tbb4-4R, GCCATCATATTCTTGGCGTCG; tbb4-5F, TCCAGAGGAAGCCAGCAATAC; tbb4-6R, CTATGCATTTGTAGTAATGTATTACTG. For ift-81: pKP6103F, TGTCTAGTTCAAAAGCCCGG; pKP6103R, TTGTAGCAGATCCTACCCTACC; pKP6104F, TCCAATGTTACGCTACCAGC; pKP6104R, TGACAAGGCAACCACCATTG; pKP6120F, GATTCAGATCAAACAGAGGTGG; pKP6120R, TCGTGGCACCATAAAAGTG; pKP6151F, AGCAATTATAGTGTCATTGCCG; pKP6151R, TTAAAAGCTGGCTCTAGTGTTG; pKP6150F, AATCGTCCTAGTTATCCACGG; pKP6150R, TGGGGGTGAAAGAGATATGTC; uCE6-907F, GCAGACATGGGAAGAAGATG; uCE6-907R, GTGACGCATGAATGGCTGG; uCE6-929F, CGATGGAATTGAGTACTTCGATG; uCE6-929R, GTACATTTACTTACCTCCCACAC; pas16937F, AACGTGGTGAGAACGTGATG; pas16937R, GTACTGAACTCATCTCTGCC; Y34B4A-F,CTCAGATTCAGCTGTACCTC; Y34B4A-R, TCATTCCATTCTGCCGAAGG; pas16936F, ATCTAATTGTCTCGAGTGCG; pas16936R, GTCTCGCTCATTGAAATCTG; IFT-81-1F, ATAGCAAAGAGCCCAGCAAC; IFT-81-2R, CGCACATTGTAACTTTGTGCC; IFT-81-3F, TATCAGCAGGTCCACTTGGG; IFT-81-4R, CTAACACGATGAATTCAGATAGC; IFT-81-5F, AAGTAAGGGAGTTCTTTAGCG; IFT-81-6R, CTGTCGGCTGCACATTTATC; IFT-81-7F, AATGGCTTCAGACGTCAGAG; IFT-81-8R, ACGCAGATTGTGTCTCTTAGC; IFT-81-9F, AAGCAAAACCAGGTGATGAAC; IFT-81-10R, GTTAGCAGAGGTATCTGATAC; IFT-81-11F, TGCGTTCCCGATTTTGCAAG; IFT-81-12R, TGAAATGTCACTCTGCAACTG. For dyf-6: Dyf-6-1F, CTCAATGACCTAATATGCTC; Dyf-6-2R, AGAATGTCAGAAACGTCTGC; Dyf-6-3F, TTTGAATCCGTTTCTTCGGG; Dyf-6-4R, GTCActgcagCAGGTGACTCTATTCATTGAAGC; Dyf-6-5F, CTAGcccgggAAGTTCCAATCTGTCCATTGTTTC; Dyf-6-6R, CAGTCCCGGGCTCGCATGCGAGCTCCATTGGATTTTCCAATGCCTG; Dyf-6-7F, TTAAgagctcATGGCGGCAAACGGAGAGT; Dyf-6-8R(XmaI), CTAGCCCGGGAAAGTTCCAATCTGTCCATTGTTTC; Dyf-6-9F, CGTTGAATCCGACAGATACC.