DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling Figure 4. DAF-25 depends on IFT for proper localization within cilia but is not essential for the IFT process. The DAF-25::GFP translational fusion was crossed into che-11(e1810) and assayed for protein accumulation. As seen in (A), DAF-25::GFP localized normally to the cilia in the N2 background, but in the che-11 background DAF-25::GFP accumulates in the cilia, indicating that when IFT is disrupted DAF-25 localization is also disrupted. We conclude that DAF-25 requires the IFT complex for proper transport and/or localization within cilia. Translational fusion reporters for CHE-2::GFP and CHE-11::GFP were crossed into daf-25(m362). As reported previously [34] both reporters localize to basal bodies and ciliary axonemes (B), and have normal velocities in both N2 and daf-25 mutants, as measured in the kymographs (C). Slopes in kymographs correlate with IFT complex speeds and were created as described previously [49]. In daf-25(m362) mutants there is no change in localization (B) or velocity (C) for either of the two reporters, indicating that DAF-25 is not required for normal rates of IFT transport, and is probably not a core IFT protein. cil = cilia, den = dendrite, TZ/BB = transition zone/basal body, asterisk indicates DAF-25::GFP accumulation, DS = distal segment, and MS = middle segment. doi:10.1371/journal.pgen.1001199.g004 XBX-1/D2LIC) were also crossed into the daf-25(m362) mutant background. All eight reporters showed normal localization to the olfactory cilia in the wild-type N2 and daf-25(m362) strains, indicating the possible specificity of DAF-25 for guanylyl cyclases (OSM-9::GFP localization in the daf-25 mutant shown in Figure 5C and 5D; the remaining constructs are presented in Figure S6). The mislocalization of DAF-11::GFP in daf-25(m362) was not suppressed by daf-12(sa204) (Figure S7). This indicates that it is the abrogation of DAF-25 rather than entry into dauer that controls the ciliary localization of DAF-11. PLoS Genetics | www.plosgenetics.org 6 November 2010 | Volume 6 | Issue 11 | e1001199