Abstract
Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia1,2. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth3,4,5, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation6,7. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.
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Acknowledgements
We thank L. Pelletier (Lunenfeld Institute, University of Toronto), M. Bornens (Institut Curie, Paris), C. Janke (Institut Curie, Orsay), T. Stearns (Stanford University) and A. Merdes (CNRS, Université de Toulouse) for antibodies against SASS6, ninein, glutamylated tubulin, CEP164 and PCM-1, respectively, A. Liu for the C2cd3-mutant MEFs and C2cd3 cDNA, J. Reiter for Ofd1 cDNA, S. Munro for pRFP-PACT, V. Meyer (Genoscope, Centre National de Génotypage), T. Hardy (University of Leicester) and N. Elkhartoufi (Necker, Paris) for technical support, F. Collman (Stanford University) for help with serial alignment, members of the Nachury laboratory for helpful discussions, and the patients and their families for their participation. This work was supported by grants from NIGMS (GM089933 to M.V.N.), the GIS–Institut des Maladies Rares (HTS), the French Ministry of Health (PHRC national 2010-A01014-35), the Regional Council of Burgundy (to C.T.-R.), the Wellcome Trust and Kidney Research UK (to A.M.F.), and the Fondazione Telethon (TGM11CB3) and the European Community's Seventh Framework Programme (FP7/2007-2013; 241955) (to B.F.). We also thank the National Heart, Lung, and Blood Institute (NHLBI) Grand Opportunity (GO) Exome Sequencing Project (see URLs) and its ongoing studies that produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the Women's Health Initiative (WHI) Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010).
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Contributions
M.V.N. conceived and supervised the functional characterization of C2CD3. C.T.-R. designed and conducted the gene identification strategy with assistance from T.A.-B., B.F., L.F., L.J., E.L., J.-B.R., C.A., N.G., B.A., A. Mégarbané, J.T., J.S.-O. and A.-L.B. J.-B.R. and S.S. performed mapping analysis, genetic screening and mutation analysis. A. Mégarbané, B.F., L.P., P.L. and C.T.-R. identified and recruited subjects. S.G.-G., A. Munnich, F.H. and M.V. gave technical support and conceptual advice. A.M.F. and C.A.M.L. provided OFD1 reagents. M.V.N., J.S.L. and T.S. designed, executed and analyzed the immunohistochemistry, overexpression and protein interaction assays. F.Y. designed and executed the centriole length measurement by light microscopy. V.H.-P. and J.M.G.-V. designed and executed electron microscopy imaging and analysis. C.T.-R. and M.V.N. wrote the manuscript. All authors reviewed the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Homozygosity mapping and characterization of C2CD3 mutations.
(a) A 4-Mb region containing 75 genes was identified at chromosome 11q between SNPs rs6592508 and rs7103374 by homozygosity mapping with the Affymetrix 500k SNPs chip. Analysis was performed with HomozigosityMapper (see URLs). (b) Chromatograms of genomic DNA sequencing showing: (left) the homozygous c.184C>T mutation in exon 2 of the C2CD3 gene (NM_015531.4) in patient 1 and at heterozygous status in his parents; (center) the missense variation c.3085T>G in exon 17 found heterozygous in patient 2 and his father; (right) a substitution c.3911–2A>T in the splice acceptor sequence of exon 22 heterozygous in patient 2 and his mother. (c,d) Chromatograms of C2CD3 cDNA sequencing at the junction between exons 21 and 22 in a control individual (c) and in patient 2 (d). (e) Nucleotide sequences of C2CD3 cDNA at the junction between exons 21 and 22 from the reference sequence (NM_015531.4), a control individual and patient 2.
Supplementary Figure 2 C2CD3 interacts with the BBSome and localizes to centriolar satellites.
(a) C2CD3 coimmunoprecipitates with most BBSome subunits. Plasmids encoding GFP-C2CD3 and a Myc-tagged BBSome subunit were cotransfected in HEK cells, and complexes were precipitated with anti-Myc antibody. Inputs and eluates were resolved by SDS-PAGE and immunoblotted for Myc or GFP. For the Myc immunoblot, 2.5 equivalents of eluate and 1 equivalent of lysate were run on the gel. Both Myc immunoblot panels are cropped from the same film exposure. For the GFP immunoblot, 25 equivalents of eluate and 1 equivalent of lysate were run on the gel. Both GFP immunoblot panels are cropped from the same film exposure. (b) C2CD3 localizes to centriolar satellites. IMCD3-[GFP-C2CD3] cells were stained for PCM-1 (red) and DNA (blue). Most foci of C2CD3 signal overlap with PCM-1 except for two spots in the center of the cell (pointed by arrowheads within the magnified region in the inset). Scale bars, 5 μm; the inset is 2 μm by 2 μm. (c) The distribution and overall number of centriolar satellites are not affected by the loss of C2CD3. Heterozygous MEFs or mutant MEFs were stained for PCM-1 (green) and DNA (blue). Scale bar, 5 μm.
Supplementary Figure 3 C2CD3 localization to centrioles does not depend on microtubules.
(a,b) IMCD3-[GFP-C2CD3] cells were incubated in the presence of nocodazole (b) or the equivalent amount of DMSO (a) for 4 h before fixation and processing for immunofluorescence. Cells were stained for tyrosinated tubulin (white), branched-glutamylated tubulin (GT335; pink) and PCM-1 (red). A magnified view of the centrioles is shown in the insets. Scale bars: 5 μm (main panels), 0.5 μm (insets).
Supplementary Figure 4 C2CD3 localizes to centrioles and procentrioles.
(a,b) IMCD3-[GFP-C2CD3] cells stained for (a) centrin (red) and glutamylated tubulin (GT335; pink). Centrin marks the centrioles and procentrioles where C2CD3 is also found. Meanwhile, glutamylated tubulin is only found at the mature centrioles. Importantly, C2CD3 appears to mark a distal structure of the centriole coinciding with centrin, while glutamylated tubulin marks the entire centriole and generates a diffraction-limited spot at the center of the centriole. (b) ODF2 (red; marks the subdistal appendages) and glutamylated tubulin (GT335; blue). All cells were treated with nocodazole before processing for immunofluorescence. All scale bars are 1 μm. (c) GST capture assays, supplement to Figure 2j. The same samples as in Figure 2j were resolved by SDS-PAGE and stained with Coomassie to assess the amounts of the GST-OFD1 fusion pulled down by glutathione-sepharose beads. Asterisks denote full-length GST-OFD1 truncations.
Supplementary Figure 5 Characterization of C2cd3-mutant MEFs.
(a) MEF extracts were immunoblotted for centrin and tubulin (DM1A mAb). (b) MEFs derived from heterozygous (C2cd3Gt/+) and homozygous C2cd3-mutant mice (C2cd3Hty/Hty and C2cd3Gt/Gt) were stained for ninein (green) and centrin (red).
Supplementary Figure 6 Extended gallery of TEM images of centrioles in C2cd3-mutant and control cells.
(a) Longitudinal TEM sections of C2cd3Gt/+ and C2cd3Hty/Hty centrioles. Centrioles from the mutant MEFs appear stunted and lack all appendages (arrows point to subdistal appendages; arrowheads point to distal appendages). Scale bar, 100 nm. (b) Serial transverse TEM sections of C2cd3Gt/+ and C2cd3Hty/Hty centrioles. C2cd3Gt/+ centrioles are contained within 5 to 6 sections, whereas C2cd3Hty/Hty centrioles span fewer than 3 sections. Scale bar, 100 nm.
Supplementary Figure 7 Characterization of the C2CD3-induced elongated structures.
(a) U2OS cells transfected with GFP-C2CD3 were treated with vehicle (top) or 5 μM nocodazole (bottom) for 1 h followed by fixation and staining for tubulin (DM1A; white), acetylated tubulin (red) and DNA. The diffuse tubulin staining in nocodazole-treated cells corresponds to the free tubulin dimers in the cytoplasm. Scale bars, 5 μm. (b) Serial sections of the hyperelongated centriole shown in Figure 5f. (c) RPE cells transfected with GFP-C2CD3 were stained for tubulin (red). GFP-C2CD3 colocalizes extensively with bundles of microtubules. Scale bar, 5 μm.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Tables 1 and 2. (PDF 62875 kb)
Hyperelongated centriole from Figure 5f.
The five serial sections of the hyperelongated centriole shown in Figure 5f and Supplementary Figure 7b are displayed as an animation. A separate structure that does not appear to be connected to the centriole can be seen in the last two sections. (MOV 858 kb)
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Thauvin-Robinet, C., Lee, J., Lopez, E. et al. The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation. Nat Genet 46, 905–911 (2014). https://doi.org/10.1038/ng.3031
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DOI: https://doi.org/10.1038/ng.3031
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