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The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery

Nature Genetics volume 48, pages 648–656 (2016)Cite this article

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A Corrigendum to this article was published on 27 July 2016

This article has been updated

Abstract

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 1: The CPLANE interactome.
Figure 2: Jbts17 localizes to the base of cilia and is required for ciliogenesis and cilia-mediated patterning.
Figure 3: Jbts17 is necessary for recruitment of peripheral IFT-A proteins to basal bodies.
Figure 4: Jbts17 is required for bidirectional axonemal transport of IFT-B particles but not the IFT-A core.
Figure 5: CPLANE-mutant mice display diagnostic features of human OFD6.
Figure 6: CPLANE gene mutations in human ciliopathies.
Figure 7: Models for CPLANE function and structure.

Change history

  • 30 May 2016

    In the version of this article initially published, the name of author Daniela A. Braun was misspelled. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank the patients and their families, the IntegraGen society for exome analysis and the NHLBI GO Exome Sequencing Project, which produced and provided exome variant calls for comparison: Lung GO Sequencing Project (HL-102923), WHI Sequencing Project (HL-102924), Broad GO Sequencing Project (HL-102925), Seattle GO Sequencing Project (HL-102926) and Heart GO Sequencing Project (HL-103010). We thank the Biological Resources Center–Ferdinand Cabanne (Dijon, France) for fibroblast centralization and storage. Sequencing was provided by the University of Washington Center for Mendelian Genomics (UW CMG) and was funded by the NHGRI and NHLBI (grant 1U54 HG006493) to D. Nickerson, J. Shendure and M. Bamshad. This work was supported by grants from the following: Uehara Memorial Foundation Fellowship to M.T.; an NIDCR NRSA to J.M.T.; the French Rare Diseases Foundation, the French Ministry of Health (PHRC national 2010-A01014-35 to C.T.-R.) and the Regional Council of Burgundy to C.T.-R.; NIDDK (DK1068306) to F.H., who is a Howard Hughes Medical Institute investigator, a Doris Duke Distinguished Clinical Scientist and the Warren E. Grupe Professor; NIAMS (AR061485) to J.C.; BBSRC (BB/K010492/1) and MRC (MR/L017237/1) to K.J.L.; NIH, NSF, CPRIT and the Welch Foundation (F-1515) to E.M.M.; R01 AR066124, March of Dimes and the Joseph Drown Foundation, NIH/NCATS UCLA CTSI grant UL1TR000124 to D.K.; R01 AR062651 to D.H.C.; NIGMS (GM114276), Baxter Laboratory, the Stanford Department of Research and NIGMS (GM114276) to P.K.J.; and NIGMS (GM104853) and NHLBI (HL117164) to J.B.W., who was a Howard Hughes Medical Institute Early Career Scientist.

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Author notes

  1. Tae Joo Park

    Present address: Present address: UNIST, Ulsan, Korea.,

Authors and Affiliations

  1. Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA

    Michinori Toriyama, Chanjae Lee, Jacqueline M Tabler, Kevin Drew, Sukyoung Kim, Tae Joo Park, Edward M Marcotte & John B Wallingford

  2. Department of Orthopaedic Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

    S Paige Taylor & Ivan Duran

  3. Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

    S Paige Taylor & Ivan Duran

  4. Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

    S Paige Taylor & Ivan Duran

  5. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California, USA

    Daniel H Cohn & Deborah Krakow

  6. EA4271GAD Genetics of Developmental Anomalies, FHU-TRANSLAD, Medecine Faculty, Burgundy University, Dijon, France

    Ange-Line Bruel, Yannis Duffourd, Laurence Faivre, Jean-Baptiste Rivière & Christel Thauvin-Robinet

  7. Department of Microbiology and Immunology, Baxter Laboratory, Stanford University School of Medicine, Stanford, California, USA

    Marcus R Kelly & Peter K Jackson

  8. Howard Hughes Medical Institute, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Daniela A Braun & Friedhelm Hildebrandt

  9. Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Daniela A Braun & Friedhelm Hildebrandt

  10. Clinical Genetics Centre, University Hospital Center, Liège, Belgium

    Ghislaine Pierquin

  11. Pediatric Unit, Hospital Center, Luxemburg, Luxembourg

    Armand Biver

  12. Cardiological Pediatric Unit, Hospital Center, Luxemburg, Luxembourg

    Kerstin Wagner

  13. Clinic of Pediatric Respiratory Diseases, Universitair Ziekenhuis Brussel, Brussels, Belgium

    Anne Malfroot

  14. Infectious Diseases, Travel Clinic, Universitair Ziekenhuis Brussel, Brussels, Belgium

    Anne Malfroot

  15. Cystic Fibrosis Clinic, Universitair Ziekenhuis Brussel, Brussels, Belgium

    Anne Malfroot

  16. Department of Pediatrics Advanced, Pediatric Centre Pigmer, Chandigarh, India

    Inusha Panigrahi

  17. Division of Pediatrics, Department of Medical Translational Sciences, Federico II University of Naples, Naples, Italy

    Brunella Franco

  18. Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

    Brunella Franco

  19. Department of Craniofacial and Stem Cell Biology, Dental Institute, King's College London, London, UK

    Hadeel Adel Al-lami, Yvonne Yeung & Karen J Liu

  20. Department of Pathology, Stony Brook University, Stony Brook, New York, USA

    Yeon Ja Choi & Jiang Chen

  21. Department of Dermatology, Stony Brook University, Stony Brook, New York, USA

    Yeon Ja Choi & Jiang Chen

  22. Clinical Genetics Centre, FHU-TRANSLAD, Children Hospital, CHU Dijon, Dijon, France

    Laurence Faivre

  23. Eastern Referral Centre for Developmental Anomalies and Malformative Syndromes, FHU-TRANSLAD, Children Hospital, CHU Dijon, Dijon, France

    Laurence Faivre

  24. Laboratory of Molecular Genetics, FHU-TRANSLAD, PTB, CHU Dijon, Dijon, France

    Jean-Baptiste Rivière & Christel Thauvin-Robinet

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  1. Michinori Toriyama
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Consortia

University of Washington Center for Mendelian Genomics

Contributions

M.T. contributed to the design, execution and interpretation of the overall research plan, with special emphasis on all Xenopus embryo experiments and in vitro binding assays. M.T. also contributed to writing the manuscript. C.L. designed, performed and interpreted live imaging of IFT particles in axonemes and contributed to other imaging experiments in Xenopus. K.D. and E.M.M. provided protein structural models. J.M.T., J.C. and K.J.L. contributed to the design, execution and interpretation of mouse genetic data. M.R.K. contributed to the execution and analysis of the proteomic data. S.K. contributed targeted coimmunoprecipitation data that confirmed CPLANE interactions. T.J.P. contributed to Xenopus studies. S.P.T., I.D., D.H.C., A.-L.B., D.A.B., G.P., A.B., K.W., A.M., I.P., B.F., H.A.A., Y.Y., Y.J.C., the University of Washington Center for Mendelian Genomics, Y.D., L.F. and J.-B.R. contributed to the collection of human patient and sequencing data. F.H., C.T.-R. and D.K. contributed to the design, execution and interpretation of human genetic data. P.K.J. contributed to coordinating the overall research effort with a focus on the design and interpretation of the proteomic screen and contributed to writing the manuscript. J.B.W. coordinated the overall research effort, oversaw experimental design and interpretation, and wrote the manuscript.

Corresponding author

Correspondence to John B Wallingford.

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Competing interests

The authors declare no competing financial interests.

Additional information

A full list of members is available at http://uwcmg.org/#/.

Integrated supplementary information

Supplementary Figure 1 CPLANE pulldowns.

(a) Tandem affinity purification of Inturned interacting proteins. Lysates from mouse IMCD3 collecting duct cells stably expressing LAP-Intu were subjected to tandem affinity purification and silver stain to visualize major interacting species. Molecular mass markers are indicated on the left. (b) Tandem affinity purification of interacting proteins for Inturned, Fuz and Wdpcp. “sindicates the tagged proteins. Molecular mass markers are indicated on the left. The calculated molecular masses for these proteins are as follows: mIntu, 104.8 kDa; mFuz, 45.5 kDa; mWdpcp, 81.7 kDa. (c–e) Tables showing an extracted subset of data from Supplementary Data 1 and highlighting key findings reported here. Numbers indicate peptide spectral matches for preys retrieved after pulldowns with the indicated baits. The “Other Ciliopathy” column reflects combined data from pulldowns of Ahi1, Cep290, Invs, Iqcb1 and Nphp4.

Supplementary Figure 2 CPLANE interactors.

(a) Venn diagram showing overlap between the interactomes of Intu, Fuz and Wdpcp. The intersection of the three (“combined CPLANE interactome”) contains ~250 proteins. (b) Additional components of the extended CPLANE protein network built using data from tandem affinity purification of Intu, Fuz, Wdpcp, IFT-A and the published NPHP network and thresholded for most likely network members. (c) The CPLANE proteins associate with IFT-A proteins and poorly characterized cilia-related proteins (arrows indicates bait → prey in Fuz, Wdpcp and Intu pulldowns). (d) The core CPLANE complex was identified by reciprocal pulldowns. (e) Coimmunoprecipitation with in vitro–translated proteins confirms CPLANE interactions with Jbts17 (a fragment of Jbts17 was used, as the very large size of this protein made in vitro translation intractable).

Supplementary Figure 3 Basal body localization of CPLANE proteins.

(a) Dorsal views of stage 19 Xenopus embryos show neural tube closure defects after Jbts17 knockdown that are rescued by expression of wild-type Jbts17 but not the Joubert syndrome–associated truncation (R1569*). The graph shows average distance between neural folds. (b) In situ hybridization to stage 30 Xenopus embryos shows loss of expression of vax1, a gene downstream of sonic hedgehog, after Jbts17 knockdown; numbers in each panel indicate the number of embryos showing the phenotype. (c–f) The fluorescence micrographs show the localization of CPLANE proteins at basal bodies in multiciliated cells with Inturned (c), Fuzzy (d), Wdpcp (e) or Rsg1 (f) knockdown. Green and red signals represent GFP-tagged CPLANE proteins and centrin4-RFP, respectively. The graph to the right in each figure shows the fluorescence intensity of GFP signals normalized against Centrin4-RFP (ns, not significant; **P < 0.01, ***P < 0.001). (g) The fluorescence micrographs show the localization of GFP-tagged Cep164, Ofd1, Hook2 and Mks1 in control multiciliated cells and after Jbts17 knockdown. The graph to the right shows the normalized fluorescence intensity for the indicated proteins, as described for c–f. (h) CRISPR-mediated disruption of Jbts17 phenocopies MO knockdown. The gels to the left demonstrate targeting of Jbts17 (see the Online Methods for details); the images to the right show disruption of ciliogenesis in MCCs.

Supplementary Figure 4 Effect of a Joubert syndrome–associated CPLANE allele.

(a) The Jbts17 truncated mutant and fragment constructs used in this study. (b) Fluorescence images of multiciliated cells expressing GFP-tagged wild-type Jbts17, R1569* and R2406* mutant Jbts17, and amino acids 1770–2318 of Jbts17. (c,d) Fluorescence images of GFP-tagged Inturned (c) and Ift43 (d) in control multiciliated cells and cells from Jbts17-knockdown embryos with no exogenous expression or expressing wild-type or truncated mutant (R1569*) Jbts17. The graphs show the normalized fluorescence intensity of GFP-Inturned (c) and GFP-IFT43 (d) at basal bodies, as described in Supplementary Figure 2. Scale bars, 10 μm.

Supplementary Figure 5 CPLANE and IFT.

(a,b) The fluorescence micrographs show the localization of the indicated GFP-tagged IFT-A (a) and IFT-B (b) proteins at basal bodies in multiciliated cells. The bottom panels in each figure show high-magnification images of basal bodies. (c–e) Live images of multiciliated cells expressing GFP-tagged IFTs (green) and membrane-RFP (magenta) in control and Jbts17 morphants. (f,g) Kymographs representing the movements of IFT particles in control, Jbts17 and WDPCP morphants. (f) GFP-IFT20. (g) GFP-IFT122. (h) Plot showing the mean intensity of IFT protein fusions in axonemes. Scale bars, 10 μm.

Supplementary Figure 6 CPLANE mutations in human patients.

(a) The D54 residue of WDPCP that is mutated in OFD is invariant from human to fish. (b) Y-shaped metacarpals in a human patient with OFD harboring a mutation in INTU; this is patient 2 from Panigrahi et al. (2013). (c) An INTU mutation segregates with the OFD phenotype. (d) An INTU missense mutation segregates with NPHP in another family. (e) The A452 residue of INTU that is mutated in NPHP is not well conserved and may be hypomorphic, consistent with the more restricted phenotype in this patient. (f) The E500 residue of INTU that is mutated in SRP is invariant from human to fish. (g,h) Radiographs showing the phenotype of a patient with SRP transheterozygous for mutations in INTU and WDR35 (Ift121), as indicated by the pedigree in i. (j) The W311 residue of WDR35 that is mutated in SRP is invariant from human to fish.

Supplementary Figure 7 Models of CPLANE protein structures.

(a) Fuz. (b) Wdpcp. (c) Intu. (d) Rsg1. (e) Jbts17. As outlined in the Discussion, modeling predicts similarities between CPLANE proteins and vesicle trafficking machinery.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–3. (PDF 3107 kb)

Supplementary Data 1

CPLANE pulldown interactions. (XLS 1176 kb)

Supplementary Data 2

Cytoscape network of CPLANE pulldown interactions. (ZIP 153 kb)

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Toriyama, M., Lee, C., Taylor, S. et al. The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery. Nat Genet 48, 648–656 (2016). https://doi.org/10.1038/ng.3558

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