June 1, 2016;
The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery.
Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.
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Figure 2. (a) RT–PCR demonstrates disrupted jbts17 splicing after MO injection (Jbts17-KD). odc1 is the loading control. (b) In situ hybridization of the Sonic Hedgehog (SHH) direct target nkx2.2 in control embryos and ones with Jbts17 knockdown (stage 22). Scale bars, 2 mm. Fractions represent the fraction of embryos displaying the phenotype. (c) Expression of pitx2, which labels the left lateral plate, at stage 26. Arrows indicate signal in left lateral plate mesoderm (LPM); the graph represents pitx2 expression patterns in embryos. Scale bars, 1 mm. (d) Immunostaining for acetylated α-tubulin, which labels cilia, shows ciliogenesis defects after Jbts17 knockdown in the ventral neural tube (stage 22). Scale bars, 10 μm. (e) Immunostaining for acetylated α-tubulin shows that Jbts17 knockdown reduces cilia length; cilia numbers are unchanged. Scale bars, 10 μm; membrane-RFP labels membranes. The graphs in d and e each show pooled data from two independent experiments for cilia length (shown as means ± s.e.m.; ***P < 0.001). (f) MCCs from control embryos, embryos with Jbts17 knockdown showing disrupted cilia, and Jbts17-knockdown embryos rescued with untargeted jbts17 mRNA labeled by GFP-Cfap20 (green); membrane-RFP labels membranes. Scale bars, 10 μm. (g) GFP-tagged Jbts17 localizes near basal bodies (visualized with coexpressed Cetn4-RFP) in an MCC. Scale bar, 10 μm. (h) Super-resolution image of GFP-Jbts17 and mCherry-Cep164 at a single basal body; both form rings of ~260 nm in diameter around the basal body, visualized by Cetn4-BFP. Diameters are shown as means ± s.d. in each panel. The graph shows fluorescence intensities (in arbitrary units) for GFP-Jbts17, mCherry-Cep164 and Cetn4-BFP. Scale bar, 100 nm. (i) GFP-tagged CPLANE proteins (green) and basal bodies visualized by Cetn4-RFP (magenta) in control and Jbts17-knockdown MCCs. Scale bars, 1 μm. In box plots of CPLANE fluorescence intensities at basal bodies, boxes extend from the 25th to the 75th percentile, with a line at the median; whiskers indicate maximum and minimum values. **P = 0.0041, ***P < 0.001; NS, not significant. Con, control. (j) Table summarizing the localization of CPLANE proteins at basal bodies for each knockdown (Supplementary Fig. 2c–f).
Figure 3: Jbts17 is necessary for recruitment of peripheral IFT-A proteins to basal bodies.(a) Ift43 localization at basal bodies in Xenopus MCCs, as marked by Cetn4-RFP, is lost in MCCs after Jbts17 knockdown. Scale bars, 10 μm. (b) Peripheral IFT-A components are not recruited to Cetn4-RFP-labeled basal bodies after Jbts17 knockdown. IFT-A components are fused to GFP. Scale bars, 1 μm. (c) Quantification of IFT protein localization to basal bodies from two independent experiments. Box plots show fluorescence intensities of GFP fusions to indicated IFT proteins normalized against the intensity of Cetn4-RFP (Online Methods). Peripheral IFT-A proteins are specifically lost after Jbts17 knockdown. **P = 0.0013, ***P < 0.001; NS, not significant.
Figure 6: CPLANE gene mutations in human ciliopathies. (a) Pedigree showing WDPCP mutations in a patient with OFD. (b) The patient displays tongue hamartomas and dental anomalies. (c) When expressed in Xenopus embryos, the allele of human WDPCP encoding Asp54Ala produces less protein than the wild-type allele; the allele encoding Leu176Phefs*23 produces no protein. (d) Pedigree showing INTU mutations in an individual with SRPS. (e) X-ray of the affected individual. (f) Wild-type Xenopus Intu localizes to basal bodies, but the Xenopus cognate of human INTU Glu355* (Gln361*) fails to localize to basal bodies. Scale bars, 10 μm. (g,h) Expression of Xenopus Intu rescues Ift43 localization to basal bodies after Intu knockdown, but the Xenopus cognate of human INTU Glu550Ala (Xenopus Intu Glu506Ala; see Supplementary Fig. 6f) does not. Scale bars, 1 μm. Data shown in h are pooled from three independent experiments. *P < 0.05, ***P < 0.001; NS, not significant.