Intraflagellar Transport and Ciliary Protein Localization Analyses To measure the integrity of IFT within the daf-25(m362) mutant, kymograph analyses were performed using GFP-tagged CHE-11 and CHE-2 IFT markers. Time-lapse movies were obtained for the different strains, including N2, and kymographs were generated from the resulting stacked tiff images using Metamorph software (Universal Imaging, West Chester, PA). Rates of fluorescent IFT particle motility along middle and distal segments were measured as described previously [35,50]. To assess how disrupting IFT affects the ciliary localization of DAF- 25::GFP, mEX179 was crossed into che-11 mutants and visualized by microscopy essentially as described [35]. Fluorescence intensity was measured by analyzing images in ImageJ by highlighting the entire head region for each animal, then measuring pixel density minus the pixel density for an equal sized adjacent region. The localization of several GFP–tagged proteins in daf-25(m362) animals, namely DAF-11, OSM-9, TAX-4, CHE-2, CHE-11, CHE-13, BBS-8, OSM-5 and XBX-1, were ascertained by crossing the reporter into the mutant, followed by visualization using standard microscopy. Co-localization was carried out by injecting the osm-5p::XBX-1::tdTomato into daf-25(m362);daf- 12(sa204) and crossing it into TJ9386 which carries the DAF- 11::GFP reporter [12]. Electron Microscopy Staged N2 and daf-25 L2 larvae were produced by harvesting eggs from gravid adults by alkaline hypochlorite treatment, followed by overnight hatching in M9 buffer, and subsequent incubation of hatched L1 larvae on seeded NGM plates for 26 hours at 16uC. Worms were then 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 dissecting microscope, transferred to 1.5 ml Ependorf tubes and fixed for one hour at room temperature. Samples were then centrifuged at 3,000 rpm for two minutes, the supernatant removed and the pellet washed for ten minutes in 0.1 M Sorensen phosphate buffer. The worms were then post-fixed in 1% osmium tetroxide in 0.1 M Sorensen phosphate buffer for one hour at room temperature. Following washing in Sorensen phosphate buffer, specimens were processed for electron microscopy by standard methods. Briefly, they were dehydrated in ascending grades of alcohol to 100%, infiltrated with Epon and placed in aluminum planchetes orientated in a longitudinal aspect and polymerized at 60uC for 24 hours. Using a Leica UC6 ultramicrotome individual worms were sectioned in cross section from anterior tip, at 1 mm until the area of interest was located as judged by examining the sections stained with toluidine blue by light microscopy. Thereafter, serial ultra-thin 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 a Tecnai Twin (FEI) electron microscope, sections were examined to locate, in the first instance, the most distal (anterior) region of the cilia, then to the more proximal regions of the ciliary apparatus. At each strategic point, distal segment, middle segment and transition zone/fiber regions were tilted using the Compustage of the Tecnai to ensure that the axonemal microtubules 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 mm, using a MegaView 3 digital recording system. DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling Co-Expression and Co-Immunoprecipitation of Ankmy2 and Guanylate Cyclase 1 (GC1) Mouse ankmy2 cDNA, amplified from retinal RNA, was engineered to contain a sequence encoding a 9 amino acid 1D4 C-terminal epitope as previously described [40]. Ankmy2-1D4 and either human GC1 or the retinal ABC transporter ABCA4 as a control were co-expressed in HEK 293 cell. HEK 293 cell extracts were solubilized in 18 mM CHAPS in TBS (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM MgCl2 and Complete inhibitor). The solution was stirred at 4uC for 20 minutes and subsequently centrifuged in an Optima TLA100.4 rotor (Beck- man) for 10 minutes at 40,000 rpm to remove any residual unsolubilized material. The solubilized extract was applied to an immunoaffinity resin consisting of the Rho 1D4 antibody conjugated to Sepharose 2B [31]. After incubation at 4uC for one hour, the resin was extensively washed with TBS to remove unbound protein, and the bound proteins were eluted with 0.2 mg/ml of the 1D4 competing peptide in TBS for analysis by Western blot labeled with Rho 1D4 antibody for the detection Ankmy2-1D4 and antibodies to GC1 or ABCA4. Supporting Information Figure S1 Lifespan phenotype of daf-25. Lifespan of daf- 25(m362) does not significantly differ from wild type N2. Mean lifespan was 12.3 for daf-25 (n = 96) compared to 13.2 for N2 (n = 88) while the maximum lifespan was 20 days for both (p = 0.08, t-test). Shown is one replicate of two. Survival was assayed at 25uC. Found at: doi:10.1371/journal.pgen.1001199.s001 (1.56 MB TIF) Figure S2 Dye filling of daf-25 mutants. Dye filling assay showing daf-25(m362) and daf-25(m98) compared to the wild type N2. Worms were incubated for 1 hour in 0.1% DiI in M9 buffer. No difference was detected between the two daf-25 alleles and the wild type N2. Found at: doi:10.1371/journal.pgen.1001199.s002 (1.56 MB TIF) Figure S3 Cilium ultrastructure is normal in daf-25 mutants. Shown are TEM serial cross sections of an amphid channel from N2 and daf-25(m362) L2-staged worms. In the six pairs of images, low magnification images (B, D, F, H, J, L) are presented on the left and one axoneme from the left image is shown in high magnification on the right (C, E, G, I, K, M). (A) Schematic of an amphid pore and channel from wild-type adult N2 worms. 10 ciliary axonemes (only three shown in longitudinal section) extend from the distal dendrite tips (den) into the lumen of the amphid pore, which is created by channel cilia invaginating surrounding support cells (sheath, socket). Channel have a ,1 mm long transition zone (tz) at the ciliary base, consisting of a constricted ring of 9 outer doublet microtubules (MTs), connected to the ciliary membrane via Y-link connections. This is followed by a ‘middle segment’ of ,4 mm, consisting of a ring of 9 outer doublet MTs, along with a varying number of inner singlet MTs. At the middle segment tip, the B-tubule of each doublet MT terminates, with the A-tubule extending to form the characteristic singlet MT structure of the ‘distal segment’. (B–E) Distal segment region of amphid cilia showing that N2 (B, C) and daf-25 (C–E) worms both possess 10 MT-singlet containing axonemes. (F–I) 4 mm (N2) or 5 mm (daf-25) proximal to B–E (through middle segments). Both N2 and daf-25 animals possess axonemes of similar number and MT ultrastructure (e.g., doublet MTs). Interestingly, 9 outer doublet MTs are not always observed in N2 and daf-25 worms (F, H), indicating that L2-staged worms lack a full complement of MTs (currently under investigation in Blacque lab). (J–M) 6 mm PLoS Genetics | www.plosgenetics.org 10 November 2010 | Volume 6 | Issue 11 | e1001199