DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling Figure 3. daf-25 is expressed in many ciliated cells and encodes a novel ciliary protein. A reporter construct joined the 2.0 kb promoter region 59 of the AUG for daf-25 to the daf-25 cDNA with the C-terminal GFP coding sequence. Expression is seen in many anterior chemosensory neurons in (A) including AFD, ASK, ASI, ASH, ASJ, ASG, ASE, ADF, AWA, AWB, AWC and IL2. There is a strong DAF-25::GFP signal localized in the sensory cilia (B). Expression of DAF-25::GFP is shown in the PQR neuron (C) and in the male tale neuron 7A (D). doi:10.1371/journal.pgen.1001199.g003 (mean DAF-25::GFP fluorescence in che-11 (8.7E12) compared to N2 (1.4E13), p,0.00001, n = 9 for both), suggesting that the protein is associated with IFT (Figure 4). To test for a possible role for DAF-25 in the core IFT complex, GFP translational fusion constructs of two IFT proteins, CHE-2 and CHE-11 [30], were crossed into the daf-25(m362) mutant background and analyzed by time-lapse microscopy. The velocities of IFT transport of CHE-2 and CHE-11, as determined by kymograph analysis, were unchanged in daf-25 compared to that of wild type animals (Figure 4). Specifically, transport velocities in the middle segment were ,0.7 mm/s, and in the distal segments ,1.2 mm/s, exactly as reported for all studied IFT proteins [36]. Collectively, our data show that DAF-25 is not essential for IFT, and is therefore unlikely to be a core component of IFT transport particles—consistent with the findings that the ciliary ultrastructure of the daf-25 mutant is intact (Figure S3). However, its accumulation within cilia in the retrograde IFT mutant does suggest that it is associated with (i.e., transported by) the IFT machinery. DAF-25 Is Required for DAF-11 Localization to Cilia The phenotype of daf-25 is most similar to that of daf-11, and our epistasis results placed daf-25 at the same position in the genetic pathway previously reported for daf-11 [37]. To test for possible functional interactions, a strain harboring DAF-11::GFP (gift from Dr. James Thomas), which is known to localize to cilia [12], was crossed with two daf-25 mutants (m98 and m362). In wild-type animals, the DAF-11::GFP protein localized to the sensory cilia of the olfactory neuron pairs ASI, ASJ, ASK, AWB and AWC (Figure 5A), all of which express DAF-25::GFP (Figure 3). In both daf-25 mutants, the DAF-11::GFP protein was observed only in a region near the base of cilia, rather than along their length (Figure 5B). To assess more precisely where the DAF-11::GFP protein is mislocalized, we introduced into the same strain a ciliary (IFT) marker, namely tdTomato-tagged XBX-1 (a gift from Dr. B. Yoder), which localizes at basal bodies and along the ciliary axoneme [38]. Visualization of the two fluorescently- tagged proteins in the daf-25 mutant revealed that DAF-11::GFP accumulates at the very distal end of dendrites, with little or no localization to the basal body-ciliary structures (Figure 5E). This indicates that DAF-25 is required for the proper localization of DAF-11 to the cilia, providing a likely explanation for the similarities between the daf-11 and daf-25 mutant phenotypes. To test if the DAF-25-DAF-11 functional interaction is specific, GFP- tagged ciliary channel proteins (OSM-9/TRPV4 and TAX-4/ CNGA1) and IFT-associated proteins (CHE-2/IFT80, CHE-11/ IFT140, CHE-13/IFT57, BBS-8/TTC8, OSM-5/IFT88 and PLoS Genetics | www.plosgenetics.org 5 November 2010 | Volume 6 | Issue 11 | e1001199