The hydrolethalus syndrome protein HYLS-1 regulates formation of the ciliary gate

Abstract

Transition fibres (TFs), together with the transition zone (TZ), are basal ciliary structures thought to be crucial for cilium biogenesis and function by acting as a ciliary gate to regulate selective protein entry and exit. Here we demonstrate that the centriolar and basal body protein HYLS-1, the C. elegans orthologue of hydrolethalus syndrome protein 1, is required for TF formation, TZ organization and ciliary gating. Loss of HYLS-1 compromises the docking and entry of intraflagellar transport (IFT) particles, ciliary gating for both membrane and soluble proteins, and axoneme assembly. Additional depletion of the TF component DYF-19 in hyls-1 mutants further exacerbates TZ anomalies and completely abrogates ciliogenesis. Our data support an important role for HYLS-1 and TFs in establishment of the ciliary gate and underline the importance of selective protein entry for cilia assembly.

Figure 1. HYLS-1 promotes TF assembly.

(a) Localization of DYF-19, HYLS-1, GASR-8 and K10G6.4 in tail phasmid cilia. Each phasmid organ contains two cilia bundled together. Images show the ciliary base of one set of two phasmid cilia expressing mCherry-tagged DYF-19 and GFP-tagged candidate proteins as indicated. HYLS-1 and GASR-8 localize proximal to DYF-19 in the region of the degenerated basal body, whereas K10G6.4 completely co-localizes with DYF-19 on TFs. (b) Cartoon illustrating the relative localization of DYF-19, HYLS-1, GASR-8 and K10G6.4 at the ciliary base. (c) Dye-filling of phasmid cilia was used to analyse cilia integrity. hyls-1 and dyf-19 mutants are dye-fill defective, whereas gasr-8 and k10g6.4 mutants possess apparently normal cilia. More than 200 worms analysed for each genetic background. (d–i) HYLS-1 is required for proper localization of DYF-19, GASR-8 and K10G6.4. Phasmid cilia expressing fluorescently tagged DYF-19 (d), GASR-8 (f) and K10G6.4 (h) in WT and hyls-1 mutants. NPHP-1 (d,f) and MKS-5 (h) were used to label the TZ. Quantification of relative fluorescence intensities of DYF-19 (e), GASR-8 (g) and K10G6.4 (i) in cilia of WT and hyls-1 mutants. n represents number of cilia analysed. (j) BB remnant and TFs are missing at the base of hyls-1 amphid cilia. Scalebars, 200 nm (j), 1 μm (other panels). Error bars indicate s.d. Student's t-test indicates significant differences; *P<0.01 and ***P<0.001.

Figure 2. HYLS-1 is required for IFT recruitment and ciliary entry.

(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.

Figure 3. Additional deletion of DYF-19 in hyls-1 mutants completely abrogates ciliogenesis.

(a) hyls-1 mutants exhibit variable defects in axoneme elongation. Phasmid cilia were labelled with the mCherry-tagged axoneme marker β-tubulin TBB-4. Asterisks indicate cilia base. (b) Quantification of cilia length in WT and hyls-1 mutants. Average of three independent experiments. n>100 cilia for each genetic background in each experiment. (c) Representative images of phasmid cilia co-labelled with mCherry-tagged TBB-4 and GFP-tagged DYF-19. The severity of cilia truncation in hyls-1 mutants is correlated with the level of residual DYF-19 at the cilia base. (d) Quantification of DYF-19 signal in WT and hyls-1 mutants. n represents the number of cilia analysed. (e) Additional deletion of DYF-19 in hyls-1 mutants completely abrogates ciliogenesis. Cilia are labelled with TBB-4. Asterisks indicate cilia base. (f) Quantification of phasmid neurons without visible axoneme based on OSM-6::GFP in WT, hyls-1, dyf-19 and hyls-1; dyf-19 double mutants, respectively. n represents the number of phasmid neurons analysed. (g) TEM analysis of the proximal axoneme (immediately distal to the TZ) of amphid neurons. Compared with WT and dyf-19 axonemes that consist of nine doublet microtubules, hyls-1 mutants frequently display missing B-tubules (red stars). dyf-19; hyls-1 double mutants show severely compromised and stunted axonemes that possess only one or two microtubules and immediately terminate only one TEM section (∼80 nm) beyond the TZ. Scale bars, 200 nm (g), 5 μm (a,c,e). Error bars indicate s.d. Student's t-test indicates significant differences; *P<0.01 and ***P<0.001.

Figure 4. HYLS-1 is required for TZ integrity and function.

(a) TEM analysis of the TZ. hyls-1 mutants display a range of anomalies in the TZ, including missing B-tubules (red stars), putative broken Y-links (blue arrows) and displaced singlet microtubules (black stars). Some Y-links still form and associate with the membrane (black arrows). hyls-1; dyf-19 double mutants display more severe defects with an increased number of incomplete doublet microtubules. (b) Quantification of incomplete doublet microtubules in the TZ in WT, hyls-1 and hyls-1; dyf-19 double mutants. See Supplementary Table 1 for the numbers of cilia analysed for each genetic background. Error bars indicate s.d. Student's t-test for significance; *P<0.01. (c) Fluorescence images of phasmid cilia co-expressing GFP-tagged BBS-7 and mCherry-tagged MKS-5 in WT and hyls-1 mutants. MKS-5 signal exclusively enriches around the TZ in WT cilia, but diffuses to a larger area including the cilia proper in hyls-1 mutants. (d) Dendrite extension is compromised in hyls-1; nphp-1 but not hyls-1; mks-6 double mutants. Representative images of dendrites visualized by expression of GFP-tagged IFT-B component OSM-6 illustrate genetic interaction between HYLS-1 and TZ proteins. See also Supplementary Fig. 4b. (e–g) Ciliary gating for both membrane-associated and soluble proteins is perturbed in hyls-1 mutants. Representative images of phasmid cilia co-expressing GFP-tagged proteins and mCherry-tagged MKS-5 as a TZ marker. (e) The non-ciliary membrane protein RPI-2 is restricted to below the TZ in WT but abnormally enters phasmid cilia in hyls-1 mutants. (f) OSM-9, a membrane receptor, enriches in the cilia of OLQ (outer labial quadrant) neurons in WT worms, but mislocalizes along dendrites in hyls-1 mutants. (g) The non-ciliary cytoplasmic protein PICC-1 is restricted below the TZ in WT but abnormally enters phasmid cilia in hyls-1 mutants. Scale bars, 200 nm (a), 5 μm (c–g).

Figure 5. Working model of HYLS-1 function in ciliogenesis.

Ciliogenesis involves a hierarchy of steps: TF maturation, basal body docking, TZ formation, IFT anchoring and entry and axoneme elongation. TFs and the TZ are proposed as important parts of the ciliary gate. HYLS-1 is essential for basal body stability and TF assembly. hyls-1 mutants also show defects in TZ integrity, compromised cilia gating and truncated axonemes. The formation of truncated cilia in hyls-1 mutants correlates with residual targeting of the TF component DYF-19 to the ciliary base. Depletion of DYF-19 in hyls-1 mutants abrogates ciliogenesis, potentially by abolishing IFT entry. TZ structural anomalies are also exacerbated in hyls-1; dyf-19 double mutants, suggesting that TFs contribute to ciliary entry of proteins required for TZ integrity.