C. elegans strains
All nematode strains were maintained and cultured at 20°C using standard techniques (Brenner, 1974
). Nontransgenic strains are as follows: Bristol, N2
; 2322, arl-13(tm2322)
; PR802, osm-3(p802)
; VC1228, klp-11(tm324)
; PR813, osm-5(p813)
; CB1033, che-2(e1033)
; CB3330, che-11(e1810)
; MX52, bbs-8(nx77)
; and RB1146, dyf-5(ok1170)
, and pkd-2(sy606);him-5(e1490)
Transgenic strains expressing ARL-13(WT)::GFP and ARL-13::GFP variants are as follows: OEB223, N2;oqEx58[arl-13::gfp+pRF4]; OEB161, arl-13(tm2322);oqEx58[arl-13::gfp+pRF4]; OEB97, N2;oqEx52[arl-13(delPal)::gfp+pRF4]; OEB96, N2;oqEx51[arl-13(rPal)::gfp+pRF4]; and OEB99, N2;oqEx55[arl-13(del203–370)::gfp+pRF4].
Transgenic strains expressing ciliated cell–specific transcriptional GFP markers are as follows: N2;gmIs13[srb-6p::gfp+pRF4]; CX3553, kyIs104[str-1p::gfp]; CX3695, lin-15;kyIs136[str-2p::gfp+lin-15(+)]; OH3192, kyIs164[gcy-5p::gfp] and pkIs519[gpa-6p::gfp]; OEB141, arl-13(tm2322);gmIs13[srb-6p::gfp+pRF4]; OEB49, arl-13(tm2322);klp-11(tm324);gmIs13[srb-6p::gfp+pRF4]; OEB224, arl-13(tm2322);osm-3(p802);gmIs13[srb-6p::gfp+pRF4]; OEB174, arl-13(tm2322);che-11(e1810);gmIs13[srb-6p::gfp+pRF4]; OEB51, arl-13(tm2322);bbs-8(nx77);gmIs13[srb-6p::gfp+pRF4]; OEB89, arl-13(tm2322);nph-4(tm925);gmIs13[srb-6p::gfp+pRF4]; OEB338, nph-4(tm925);gmIs13[srb-6p::gfp+pRF4]; OEB324, arl-13(tm2322);dyf-5(ok1170);gmIs13[srb-6p::gfp+pRF4]; OEB339, dyf-5(ok1170);gmIs13[srb-6p::gfp+pRF4]; OEB53, arl-13(tm2322);kyIs164[gcy-5p::gfp]; OEB180, arl-13(tm2322);kyIs104[str-1p::gfp]; OEB61, arl-13(tm2322);kyIs136[str-2p::gfp+lin-15(+)]; and OEB210, arl-13;pkIs519[gpa-6p::gfp].
Transgenic strains expressing GFP-tagged ciliary membrane protein markers are as follows: CX3260, kyIs53[odr-10::gfp]; CX3716, kyIs141[osm-9::gfp+lin-15(+)] and kyIs[tax-2::gfp+lin-15(+)]; PT572, myIs1[PKD-2::GFP+unc-122p::GFP] (gift from M. Barr, Rutgers University, Piscataway, NJ); OEB152, arl-13(tm2322);kyIs53[odr-10::gfp]; OEB175, arl-13;kyIs141[osm-9::gfp+lin-15(+)]; OEB177, arl-13(tm2322);kyIs111[tax-2::gfp]; and OEB289, arl-13;myIs1[PKD-2::GFP;unc-122p::GFP].
Transgenic strains expressing IFT::GFP markers are as follows: MX255, N2;ejEx[osm-3::gfp+pRF4] (gift from J. Scholey, University of California, Davis, Davis, CA), N2;ejEx[kap-1::gfp+pRF4] (gift from J. Scholey), xbx-1(ok279);Ex[xbx-1::gfp+pRF4], N2;yhEx[che-13::gfp+pRF4], and N2;Ex[che-12p::che-13::mcherry+unc-122p::gfp] (gift from S. Shaham, The Rockefeller University, New York, NY); PT47, N2;myEx[che-2::gfp+pRF4], N2;ejEx[dyf-1::gfp+pRF4] (gift from G. Jansen, Erasmus Medical Center, Rotterdam, Netherlands), N2;ofEx46[dyf-2::gfp+pRF4], and N2;myEx10[che-11::gfp+pRF4]; MX231, N2;nxEx[ifta-1::gfp+dpy-5(+)] (gift from M. Leroux, Simon Fraser University, Burnaby, British Columbia, Canada); MX76, dpy-5;nxEx25[bbs-7::gfp+dpy-5(+)] (gift from M. Leroux); MX68, dpy-5;nxEx[bbs-8::gfp+dpy-5(+)] (gift from M. Leroux); OEB134, arl-13(tm2322);ejEx[osm-3::gfp+pRF4]; OEB284, arl-13(tm2322);klp-11(tm324);ejEx[osm-3::gfp+pRF4]; OEB248, klp-11(tm324);ejEx[osm-3::gfp+pRF4]; OEB132, arl-13;ejEx[kap-1::gfp+pRF4]; OEB110, arl-13(tm2322);dyf-5(ok1170);ejEx[kap-1::gfp+pRF4]; OEB226, arl-13(tm2322);bbs-8(nx77);ejEx[kap-1::gfp+pRF4]; OEB133, arl-13(tm2322);nxEx[xbx-1::gfp+pRF4]; OEB163, arl-13(tm2322);mnIs17[osm-6::gfp+unc-36(+)]; OEB159, arl-13(tm2322);yhEx[che-13::gfp+pRF4]; OEB157, arl-13(tm2322);myEx[che-2::gfp+pRF4]; OEB160, arl-13(tm2322);ejEx[dyf-1::gfp+pRF4]; OEB130, arl-13(tm2322);myEx10[che-11::gfp+pRF4]; OEB117, arl-13(tm2322);osm-3(p802);myEx10[che-11::gfp+pRF4]; OEB137, arl-13(tm2322);nxEx[ifta-1::gfp+dpy-5(+)]; OEB135, arl-13(tm2322);nxEx25[bbs-7::gfp+dpy-5(+)]; OEB136, arl-13(tm2322);nxEx[bbs-8::gfp+dpy-5(+)]; OEB156, arl-13(tm2322);ofEx46[dyf-2::gfp+pRF4]; OEB95, arl-13(tm2322);klp-11(tm324);ofEx46[dyf-2::gfp+pRF4]; OEB227, arl-13(tm2322);dyf-5(ok1170);ofEx46[dyf-2::gfp+pRF4]; and OEB115, arl-13(tm2322);bbs-8(nx77);ofEx46[dyf-2::gfp+pRF4] and bbs-8(nx77);ofEx46[dyf-2::gfp+pRF4].
Standard genetic crossing techniques were used to make double mutants and to introduce transgenes into various genetic backgrounds. PCR using primers that flank deletions were used to follow the arl-13(tm2322), klp-11(tm324), bbs-8(nx77), and nph-4(tm925) deletion mutations. All other mutations were followed using the dye-filling assay.
Generation of arl-13::gfp constructs
All translational arl-13::gfp
constructs (WT and variant) were generated using fusion PCR as previously described (Hobert, 2002
). For the arl-13(WT)
reporter, a genomic DNA fragment containing 300 bp of 5′ untranslated region (promoter) plus the entire exonic and intronic sequence of arl-13
was fused in frame with gfp
. Similarly, for the C-terminal truncation variant (del203–370), gfp
was fused in frame with genomic DNA fragments containing 300 bp of arl-13
5′ untranslated region plus exonic/intronic arl-13
sequence (nucleotides 1–2701 of arl-13
genomic sequence). For the Pal motif variants (delPal, deletion of C12–C15; and rPal, replacement of C12–C15 with ASAS), two fusion reactions were performed. In the first reaction, PCR fragments containing the arl-13
promotor (214 bp) plus 33 bp of exon 1 were fused to nucleotides 34–3300 of arl-13
, where nucleotides 34–45 (TGCTGTTGTTGC) were altered by primer design to (TCCGCTTCTGCC; rPal) or deleted altogether (delPal). Resulting PCR products were subsequently fused in frame with gfp
. All constructs were coinjected at 1–10 ng/µl with 50 ng/µl pRF4 into N2 worms to generate roller transgenic animals harboring arl-13::gfp
(WT and variant) extrachromosomal arrays.
Worms were placed in 200 µl of DiI (or DiO) solution (Invitrogen; diluted 1:200 with M9 buffer). After 1-h incubation, worms were recovered on seeded nematode growth medium plates for a further hour and then mounted on slides. Epifluorescence wide-field imaging under red (DiI) or green filters (DiO) was used to image DiI/DiO uptake into ciliated amphid/phasmid cells.
Chemosensory behavioral assays
Chemotaxis assay toward isoamyl alcohol was performed on 85-mm round plates. 1 µl of 1 M NaN3 (anesthetic) was applied to two points at opposite ends of the plate (5 mm from the edge). Then, 1 µl of attractant (diluted 1:100 in 95% ethanol) was spotted at one of these points, and 1 µl of ethanol was spotted at the other as a control. Young adult worms (~100), washed three times with M9 buffer and once with deionized water, were then applied to plate center (~3.5 cm from attractant). After excess liquid removal, worms were allowed to partition across the plate for 30 and 60 min. The chemotaxis index was calculated at each time point as (a − b)/n, where a equals the number of worms within 1.5 cm of the attractant, b equals the number of worms within 1.5 cm of the ethanol counterattractant, and n equals the total number of worms in the assay.
Worms were washed directly into a primary fixative of 2.5% glutaraldehyde in 0.1 M Sorensen phosphate buffer. To facilitate rapid ingress of fixative, worms were cut in half using a razor blade under a dissection microscope, transferred to Eppendorf tubes, and fixed for 1 h at room temperature. Samples were then centrifuged at 3,000 rpm for 2 min, and supernatant was removed and washed for 10 min in 0.1 M Sorensen phosphate buffer. The worms were then postfixed in 1% osmium tetroxide in 0.1 M Sorensen phosphate buffer for 1 h at room temperature. After washing in buffer, specimens were processed for electron microscopy by standard methods; in brief, they were dehydrated in ascending grades of alcohol to 100% infiltrated with epon and placed in aluminum planchettes orientated in a longitudinal aspect and polymerized at 60°C for 24 h. Using an ultramicrotome (UC6; Leica), individual worms were sectioned in cross section, from anterior tip, at 1 µm until the area of interest was located, as judged by examining the sections stained with toluidine blue by light microscopy. Thereafter, serial ultrathin sections of 80 nm were taken for electron microscopical examination. These were picked up onto 100-mesh copper grids and stained with uranyl acetate and lead citrate. Using an electron microscope (Tecnai Twin; FEI), sections were examined to locate, in the first instance, the most distal region of the ciliary region and subsequently from that point to the more proximal regions of the ciliary apparatus. At each strategic point, DSs, MSs, and transition zone/fiber regions were tilted using the Compustage of the Tecnai to ensure that the axonemal MTs were imaged in an exact geometrical normalcy to the imaging system. All images were recorded at an accelerating voltage (120 kV) and objective aperture of 10 µm using a MegaView 3 digital recording system (Olympus).
C. elegans fluorescence microscopy and IFT motility assays
Worms were anesthetized in 10 mM levamisole and mounted on 2% agarose pads for analysis on a microscope (DM5000B; Leica), fitted with epifluorescence, and a 1.4 NA 100× Plan-Apochromat lens. Images were captured using an electron-multiplying charge-coupled device camera (DV885; Andor Technology) and iQ2.0 software (Andor Technology). iQ2.0 was also used to measure signal intensities of PKD-2::GFP in the cell body and cilia of N2 and arl-13(tm2322)
mutants. The region of interest was always of identical size, and background signals were subtracted to normalize values. For IFT motility assays, time-lapse videos of IFT along amphid and phasmid cilia were taken no more than 30–45 min after slide preparation, and in each case, the same exposure times (300 ms), gain, and frame rate (3 frames/s) were used. Resulting stacked multi tiff images were processed into kymographs using the multiple kymograph plug-in of ImageJ 1.38× software (National Institutes of Health; http://www.embl-heidelberg.de/eamnet/html/body_kymograph.html
). Particle velocities were determined from the kymographs using the ImageJ “read velocities from tsp” macro. The frequency of IFT particles was also determined from the kymographs by counting the number of lines detected.
Mammalian cell culture and immunofluorescence microscopy
MDCKII cells were cultured on a coverglass (15-mm diameter) in DME supplemented with 10% (vol/vol) fetal bovine serum for 7 d after full confluence, with the medium changed every 3 d. RPE1 cells were transiently transfected with Arl13b-GFP expression vectors using Fugene6 (Roche) and cultured for 48 h in a starvation medium consisting of DME/F12[1:1] (Invitrogen) and 0.1% (wt/vol) BSA. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde in PBS for 10 min, followed by incubation with ice-cold methanol for 5 min. After permeabilization with 0.2% Triton X-100 in PBS for 10 min, cells were treated with 5% BSA in TBS (TBS/5% BSA) and further incubated with primary antibodies in TBS/5% BSA at 37°C for 2 h. Cells were then washed three times with PBS and incubated with an Alexa Fluor 488/568–conjugated secondary antibody (Invitrogen) and DAPI for 30 min in TBS/5% BSA. After washing three times with PBS, the coverglass was mounted onto a glass slide in Permafluor mounting medium (Immunon) and viewed under a confocal microscope (LSM 510; Carl Zeiss, Inc.). The rabbit anti-Arl13b polyclonal antibody was described previously (Hori et al., 2008
). Anti–acetylated tubulin, anti–γ-tubulin, and anti-Flag M2 antibodies were purchased from Sigma-Aldrich.
Subcellular fractionation and immunoblotting
293T cells cultured on a 10-cm dish (~70% confluent) were transfected with 8 µg of expression vectors encoding WT or mutant versions of Arl13b (Flag tagged) using Lipofectamine 2000 (Invitrogen). 2 d after transfection, cells were washed twice with ice-cold wash buffer (10 mM triethanolamine–acetic acid, pH 7.6, and 250 mM sucrose) and suspended to 1 ml of ice-cold homogenization buffer (10 mM triethanolamine–acetic acid, pH 7.6, 250 mM sucrose, 5 mM MgSO4
, 1 mM DTT, and 2 µg/ml aprotinin). The cell suspension was homogenized by 10 strokes in a chilled cell homogenizer with a tungsten-carbide ball (clearance of 10 µm; Isobiotec) and then subjected to standard subcellular fractionation by centrifugation (Graham and Rickman, 1997
), followed by immunoblotting with anti-Flag M2, anti–µ-calpain (#MA3-940; Thermo Fisher Scientific), and anti-Na+
ATPase α-1 (Millipore) antibodies.
Metabolic labeling with [3H]palmitic acid and fluorography
Palmitoylated proteins were detected as described previously (Fukata, 2006
; Tsutsumi, 2009
). 293T cells cultured on a 6-well dish (~70% confluent) were transfected with 3 µg of expression vectors encoding WT or mutant versions of human GFP-tagged Arl13b using Lipofectamine 2000. 2 d after transfection, the cells were preincubated for 30 min in serum-free DME supplemented with 0.1% fatty acid–free BSA (Sigma-Aldrich). The cells were then metabolically labeled for 4 h with serum-free DME containing 0.25 mCi/ml [3
H]palmitate (PerkinElmer). After washing twice with ice-cold PBS, the cells were extracted with 100 µl of ice-cold extraction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, 5 mM MgCl2
, 1 mM DTT, 2 µg/ml aprotinin, and 2 µg/ml leupeptin). After centrifugation at 20,000 g
for 20 min, the supernatants were subjected to SDS-PAGE. After fixing the gels for 30 min in a fixing solution (methanol/acetic acid/water, 3:1:6), the gels were treated with Amplify fluorographic reagent (GE Healthcare) for 30 min, dried under vacuum, and exposed to the x-ray film (RX-U; Fujifilm) without intensifying screen at −80°C for 72 h. The supernatants were also subjected to Western blot analysis with anti-GFP antibody.
Online supplemental material
Fig. S1 shows that C. elegans
ARL-13 is a sequence homologue of human JS-associated Arl13b. Fig. S2 shows that overexpression of arl-13(delPal)
in N2 worms does not disrupt PKD-2 ciliary abundance. Fig. S3 shows anterograde rate profiles of various IFT proteins along the MSs of arl-13(tm2322)
mutants. Videos 1–10 show time-lapse recordings of KAP-1::GFP in amphid/phasmid channel cilia of arl-13
(Video 1), dyf-5
(Video 3), and arl-13;dyf-5
(Video 6) and DYF-2::GFP in amphid/phasmid channel cilia of arl-13
(Video 2), dyf-5
(Video 4), bbs-8
(Video 5), arl-13;dyf-5
(Videos 7 and 8), and arl-13;bbs-8
(Videos 9 and 10). Table S1 shows that overexpression of high levels but not low levels of a che-13::mCherry
transgene enhances the Dyf defect of arl-13
mutants. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200908133/DC1