Transition fibre protein FBF1 is required for the ciliary entry of assembled intraflagellar transport complexes

Abstract

Sensory organelle cilia have critical roles in mammalian embryonic development and tissue homeostasis. Intraflagellar transport (IFT) machinery is required for the assembly and maintenance of cilia. Yet, how this large complex passes through the size-dependent barrier at the ciliary base remains enigmatic. Here we report that FBF1, a highly conserved transition fibre protein, is required for the ciliary import of assembled IFT particles at the ciliary base. We cloned dyf-19, the Caenorhabditis elegans homologue of human FBF1, in a whole-genome screen for ciliogenesis mutants. DYF-19 localizes specifically to transition fibres and interacts directly with the IFT-B component DYF-11/IFT54. Although not a structural component of transition fibres, DYF-19 is required for the transit of assembled IFT particles through the ciliary base. Furthermore, we found that human FBF1 shares conserved localization and function with its worm counterpart. We conclude that FBF1 is a key functional transition fibre component that actively facilitates the ciliary entry of assembled IFT machinery.

Figure 1. DYF-19 is a functional component of TFs

a, Schematic of Y43F8C.4 alleles. Jhu455 and jhu546 were retrieved from a genome-wide mutagenesis screen for ciliogenesis mutants. Jhu455 and jhu546 exhibit identical mutant phenotypes in all experiments we conducted. Hereafter, the jhu455 allele with an R294>Stop mutation is the reference allele used in this report. b, In C. elegans, the head amphids and tail phasmids are ciliated neurons and primary sensory organs. The amphid cilia consist of a bundle of 10 cilia, and the phasmid has 2 cilia. c, Fluorescence micrographs of cilia labeled with the GFP-tagged IFT-B component OSM-6 in Wt, dyf-19(jhu455) and dyf-19 rescue strain. dyf-19 show truncated cilia and abnormal accumulation of OSM-6::GFP at the tips of residual cilia. Arrows and arrowheads indicate the base and tip of cilia, respectively. Stars note the accumulation of OSM-6::GFP. d, The dye-filling defect in dyf-19(jhu455) is fully rescued by introducing a wild-type copy of the Y43F8C.4 gene. Data are represented as mean of 5 independent experiments (n=300) and error bars indicate s.d. Significant differences were identified by the Student’s t-test. *P<0.001. e, mCherry-tagged DYF-19 specifically localizes at the ciliary base. f, Spatial relationships between DYF-19 and TZ markers (MKS-5 and NPHP-1) and between DYF-19 and a PCMC marker (RPI-2). g, Cartoon illustrating the TF localization of DYF-19 at the ciliary base. h, Transmission electron microscopy reveals that dyf-19 mutants possess normal TFs. Scale bars: 200 nm in h and 5 μm in other micrographs.

Figure 2. The ciliary entry of IFT components is compromised in dyf-19 mutants

a, Fluorescence micrographs of cilia labeled with various IFT markers. In dyf-19, IFT-B components (OSM-5, CHE-2, IFT-20) and IFT-B–associated kinesin motor OSM-3/KIF17 show consistent ciliary tip accumulation similar to OSM-6 (Fig. 1c). In contrast, IFT-A components CHE-11, IFT-A–associated kinesin-II subunit KAP-1, dynein light chain XBX-1, and BBSome proteins are all excluded from the cilia. Arrows and arrowheads indicate the base and tip of cilia, respectively. Stars note accumulation. Co-labeling with TZ markers revealed that CHE-11 (b), XBX-1 (c), and BBS-7 (d) were restricted below the TZ in dyf-19 mutants. Scale bars, 5 μm.

Figure 3. DYF-19 regulates the ciliary entry of assembled IFT particles

a, At the base of Wt or dyf-19 cilia, the IFT-A component CHE-11 and the IFT-B component OSM-6 colocalize, presumably below the TZ. b, In BiFC analyses, fluorescence complementation of CHE-11::VN-IFT-20::VC, DYF-2::VN-VC::BBS-9, and DYF-2::VN-VC::BBS-9 occurs along the entire cilium in Wt, but only at the ciliary base in dyf-19 mutants. Reconstituted CHE-11::VN-IFT-20::VC (c) and DYF-2::VN-VC::BBS-9 (d) localize just below the TZ marker in dyf-19 mutants. VN: N-terminus of Venus YFP; VC: C-terminus of Venus YFP. Bars, 5 μm.

Figure 4. DYF-19 interacts directly with IFT component DYF-11

a, DYF-11 shows a unique mislocalization pattern in DYF-19-deficient cilia. b, The DYF-11 N-terminus interacts directly with DYF-19 in a GST pull-down assay. c, At the ciliary base, stable fluorescence complementation in BiFC assays occurs between DYF-19 and DYF-11 but no or very weak between DYF-19 and other IFT components below the TZ. d, Dyf-11 mutants show similar phenotypes to dyf-19 mutants for the ciliary entry of IFT components. e, Schematic of the role of TF component DYF-19 in regulating the ciliary entry of assembled IFT particles through a direct interaction with the IFT component DYF-11. VN: N-terminus of Venus YFP; VC: C-terminus of Venus YFP. Bars, 5 μm.

Figure 5. The localization and function of DYF-19 are highly conserved

Either endogenous (a) or overexpressed (b) FBF1, the mammalian homolog of worm DYF-19, localizes specifically on one centriole with a ring-like pattern in IMCD3 cells. c, FBF1 localizes at the ciliary base, above the basal body. d, In IMCD3 cells, FBF1 localizes above rootlet and subdistal appendage protein ODF2 and completely colocalizes with distal appendage protein CEP164. e, Immuno-EM demonstrates that FBF1 localizes specifically to distal appendages of mother centrioles. f–h, Knock-down of FBF1 leads to severely truncated cilia in most RNAi-treated hTERT-RPE cells. Data are represented as mean of 3 independent experiments (n=200) and error bars indicate s.d. Significant differences were identified by the Student’s t-test. *P<0.001. i and j, The IFT-B component IFT88, but not the IFT-A component IFT140, enters the truncated cilia of FBF1-knockdown hTERT-RPE cells. Arrows indicate the tips of truncated cilia. k, Endogenous IFT54 immunoprecipitates with FBF1 in hTERT-RPE cells. l, HEK293 cells were transiently transfected with FLAG-HA-tagged FBF1, and 48 hours later, cells were subjected to immunoprecipitation using normal mouse IgG (mIgG) or anti-IFT54 antibody. 50 μg protein were loaded into each lane. Bars: c, 1μm; e, 200 nm; others, 20 μm.