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The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans

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Abstract

Primary ciliary dyskinesia (PCD) is characterized by dysfunction of respiratory cilia and sperm flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the nexin-dynein regulatory complex (N-DRC), an axonemal structure critical for the regulation of dynein motors, and show that mutations in the gene encoding DRC1, CCDC164, are involved in PCD pathogenesis. Loss-of-function mutations disrupting DRC1 result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas reinhardtii and humans. Our results highlight a role for N-DRC integrity in regulating ciliary beating and provide the first direct evidence that mutations in DRC genes cause human disease.

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Figure 1: Location of protein alterations in Chlamydomonas and humans.
Figure 2: The Chlamydomonas pf3 mutant is defective in DRC1, resulting in disruption of N-DRC integrity.
Figure 3: Mutations in the CCDC164 gene in humans result in defects in the N-DRC links.
Figure 4: CCDC164 mutations result in defective N-DRC assembly in respiratory cilia.
Figure 5: Comparison of axonemal bending patterns in Chlamydomonas and human respiratory cilia.

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Change history

  • 08 February 2013

    In the version of this article initially published online, authors Unne Stenram and Birgitta Carlén were listed with the incorrect departmental affiliation. Their correct affiliation is the Department of Pathology, Lund University and Skane University Hospital, Lund, Sweden, not the Department of Otorhinolaryngology–Head and Neck Surgery, Lund University and Skane University Hospital, Lund, Sweden, as originally stated. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank G. Piperno for providing the peptide sequences used to identify the Chlamydomonas DRC1 gene. M.E.P. also thanks C. Perrone, T. vy Le, A. Bostrom, J. Mueller and J.A. Knott for assistance with mapping the DRC1 locus, P. Kathir, C. Silflow and S. Dutcher for advice on RFLP mapping and T. Markowski and B. Witthun for assistance with mass spectrometry and spectral counting. We thank the German patient support group Kartagener Syndrom und Primaere Ciliaere Dyskinesie e.V. We also thank A. Heer and C. Westermann for excellent technical assistance. We thank M. Laudon and the Chlamydomonas Genetics Center for strains. For antibodies, we thank R. Linck (University of Minnesota) for antibody to Rib72, M. Sanders (University of Minnesota) for MC1 antibody to centrin, R. Kamiya (University of Tokyo) for antibodies to tektin, p44 and p38, T. Yagi (Kyoto University) for antibodies to DHC5, DHC9 and DHC11, P. Yang (Marquette University) for antibody to RSP16, E. Smith (Dartmouth College) for antibody to CaM-IP3 and G. Piperno (Mount Sinai School of Medicine) for antibody to p28). This work was supported by US National Institutes of Health (NIH) grants to M.E.P. (GM-55667) and W.S.S. (GM-051173), a National Research Service Award (NRSA) postdoctoral fellowship to M.W. (GM-075446), funding to W.S.S. from the Children's Healthcare of Atlanta and Emory University School of Medicine Pediatric Research Center and funding to H. Omran (Deutsche Forschungsgemeinschaft DFG Om 6/4 and Om 6/5, GRK1104, SFB592, IZKF Muenster and the Cell Dynamics and Disease (CEDAD) graduate school as well as SYSCILIA from the European Community). The Center for Mass Spectrometry and Proteomics at the University of Minnesota is supported by multiple grants, including National Science Foundation (NSF) Major Research Instrumentation grants 9871237 and NSF-DBI-0215759. Technical and software support was also provided by the Minnesota Supercomputing Institute.

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Authors and Affiliations

Authors

Contributions

D.T. and M.W. cloned the Chlamydomonas DRC1 gene and generated the antibody against the DRC1 protein. D.T. identified the mutation in the pf3 strain. R.B. performed biochemical studies on Chlamydomonas axonemes. M.E.P. evaluated the spectral counting and pf3 mapping data. H. Olbrich evaluated linkage analysis and performed sequencing of human subjects with PCD. C.W. evaluated clinical data from individuals with PCD and performed high-speed video microscopy analysis and nasal NO measurements on OP-26II1. N.T.L. performed high-resolution immunofluorescence microscopy of PCD samples. P.P. generated in situ hybridization of Ccdc164 at the mouse embryonic node and performed immunofluorescence. H. Olbrich, N.T.L., P.P., D.T., R.B. and M.W. prepared the figures. S.L., U.S. and B.C. provided clinical data, TEM and DNA from OP-39 and OP-56. S.L. performed surgery on OP-59II1. E.H. provided clinical data and DNA from OP-26. G.K. performed TEM. P.N. and G.N. performed linkage and haplotype analyses. H. Omran evaluated all TEM analyses. H. Omran, W.S.S. and M.E.P. coordinated the study. M.W., M.E.P. and H. Omran wrote the manuscript.

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Correspondence to Mary E Porter or Heymut Omran.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–5 and Supplementary Note (PDF 1440 kb)

Supplementary Video 1

Respiratory cilia of individual OP-26II1 in real time (AVI 15106 kb)

Supplementary Video 2

Respiratory cilia of individual OP-26II1 in slow motion (1/8 speed) (WMV 1770 kb)

Supplementary Video 3

Respiratory cilia of a healthy control in real time (WMV 1120 kb)

Supplementary Video 4

Respiratory cilia of a healthy control in slow motion (1/8 speed) (WMV 4355 kb)

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Wirschell, M., Olbrich, H., Werner, C. et al. The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans. Nat Genet 45, 262–268 (2013). https://doi.org/10.1038/ng.2533

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