Figure 2: HYLS-1 is required for IFT recruitment and ciliary entry.
From: The hydrolethalus syndrome protein HYLS-1 regulates formation of the ciliary gate
(a) Localization of IFT components in WT and hyls-1 mutant phasmids. Schematic of phasmid sensory organ. Each phasmid contains two cilia whose distal segments bundle together. Image panels show phasmid cilia in WT and hyls-1 mutants expressing GFP-tagged IFT components (OSM-6, the orthologue of IFT-B component IFT52; CHE-11, the orthologue of IFT-A component IFT140; BBS-7, the orthologue of BBSome component BBS7) alongside quantification of their fluorescence intensities inside the cilium proper. IFT signal is significantly reduced in hyls-1 mutants compared with that in WT. In contrast to the strong accumulation in WT, hyls-1 mutants further show no enrichment of IFT components at the ciliary base. Asterisks indicate cilia base. n represents number of cilia analysed. (b) Representative images of WT and hyls-1 cilia co-labelled with GFP-tagged BBS-7 and mCherry-tagged TZ marker MKS-5. BBS-7 strongly accumulates below the TZ in WT but not hyls-1 mutants. Corresponding fluorescence intensities are shown in right panels. Asterisks indicate cilia base. Red arrows indicate TZ, green arrows indicate TFs/BB. (c) BiFC reveals association between IFT-A and IFT-B components in both WT and hyls-1 mutant phasmids. However, complementation is restricted to the ciliary base in hyls-1 mutants as revealed by the quantification of BiFC signal within cilia (right panel). n represents number of cilia analysed. (d,e) Fluorescence recovery after photobleaching shows that ciliary entry of IFT is severely disrupted in hyls-1 mutants. (d) Representative images before/after photobleaching (boxed region) of GFP-tagged OSM-6 signal in WT and hyls-1 mutants. (e) Quantification and curve fit for experiment shown in d. (f) Representative images and corresponding kymographs illustrating GFP-tagged IFT particle movement. In hyls-1 mutants, IFT movement is either weak in cilia or non-detectable. (g) Only ∼20% hyls-1 cilia display detectable IFT movement. n represents number of cilia analysed. (h) Quantification reveals a significant reduction in anterograde IFT particle flux in hyls-1 mutants compared with that in WT. Each data point represents a single measurement. Scale bars, 5 μm. Error bars indicate s.d. Significant differences identified by Student’s t-test; *P<0.01 and ***P<0.001.
