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Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia

Nature Genetics volume 44, pages 381–389 (2012)Cite this article

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Abstract

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

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Figure 1: The Chlamydomonas PF22 locus encodes a conserved cytoplasmic protein that is essential for axonemal dynein assembly.
Figure 2: Identification of DNAAF3 mutations in individuals with PCD with dynein assembly defects.
Figure 3: An absence of outer dynein arms in respiratory epithelial cell cilia from individuals with PCD carrying DNAAF3 mutations.
Figure 4: An absence of the inner row dynein subunit DNALI1 in respiratory epithelial cell cilia from individuals with PCD carrying DNAAF3 mutations.
Figure 5: Morpholino knockdown of dnaaf3 in zebrafish embryos results in axis curvature defects, kidney cysts, hydrocephalus, perturbed otolith development and laterality defects.
Figure 6: Altered ODA subunit abundance in the Chlamydomonas pf22 mutant cytoplasm.
Figure 7: The Chlamydomonas pf22 mutant does not correctly assemble outer dynein arms in the cytoplasm.
Figure 8: Hypothesized pathway of the cytoplasmic assembly of axonemal outer row dynein.

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NCBI Reference Sequence

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Acknowledgements

We thank the patients and their families for their participation and the physicians involved, particularly H. Simpson, J. Clarke and D. Spencer. We are grateful to M. Turmaine (University College London) for zebrafish electron microscopy. W. Sale (Emory University) and S. King (University of Connecticut) provided antibodies to Chlamydomonas dynein subunits. We thank R.M. Gardiner, S. Spiden, M. Meeks, D. Antony and D. Osborn for advice and assistance. We also thank A. Heer, D. Nergenau, C. Reinhard, C. Kopp, K. Sutter, C. Tessmer, T. de Ledezma and S. Franz for excellent technical assistance. The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under contract number DE-AC02-05CH11231. D.R.M. was supported by US National Institutes of Health grant R01-GM044228. H.M.M. received support from the PCD Family Support Group (UK) and funding from the Fondation Milena Carvajal Pro-Kartagener, the Medical Research Council UK, the Wellcome Trust and Action Medical Research. H. Omran was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (Om 6/4, GRK1104, BIOSS and SFB592).

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Authors and Affiliations

  1. Molecular Medicine Unit, University College London Institute of Child Health, London, UK

    Hannah M Mitchison, Miriam Schmidts, Athina Dritsoula & Philip L Beales

  2. Department of Pediatrics and Adolescent Medicine, University Hospital Muenster, Muenster, Germany

    Niki T Loges, Heike Olbrich & Heymut Omran

  3. Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York, USA

    Judy Freshour & David R Mitchell

  4. Department of Infection, Immunity and Inflammation, University of Leicester, Leicester Royal Infirmary, Leicester, UK

    Rob A Hirst & Christopher O'Callaghan

  5. Pulmonary Unit, Schneider Children's Medical Center of Israel, Petah-Tikva, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel

    Hannah Blau & Huda Mussaffi

  6. Department of Pediatrics, Pulmonary Section, King Fahd Armed Forces Hospital, Jeddah, Saudi Arabia

    Maha Al Dabbagh

  7. Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

    Toshiki Yagi

  8. General and Adolescent Paediatric Unit, University College London Institute of Child Health, London, UK

    Eddie M K Chung

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Contributions

H.M.M., A.D., H.B., N.T.L., M.A.D., H. Olbrich, H.M., E.M.K.C. and H. Omran performed the studies on human samples. D.R.M. designed the Chlamydomonas studies, and D.R.M. and J.F. performed the experiments. T.Y. contributed essential reagents and data analysis. H.M.M. designed the zebrafish studies, and H.M.M., M.S., A.D., R.A.H., C.O. and P.L.B. performed the experiments. D.R.M. and H.M.M. wrote the manuscript.

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Correspondence to Hannah M Mitchison or David R Mitchell.

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Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Figures 1–6 and Supplementary Videos 1–7. (PDF 842 kb)

Supplementary Video 1

Wild type Chlamydomonas cells swimming under darkfield illumination. Most cells swim progressively. Occasional stationary cells have adhered to the glass surface through flagellar contact. Images were captured and displayed at 30 fps. (MOV 3300 kb)

Supplementary Video 2

Mutant Chlamydomonas strain pf22 cells fail to swim. Most cells are non-adherent but remain stationary due to lack of flagellar motility. Images were captured and displayed at 30 fps. (MOV 3124 kb)

Supplementary Video 3

Wild type swimming of the Chlamydomonas pf22 strain expressing a Myc-tagged PF22 protein. Most cells have full length flagella and swim progressively with a swimming pattern and velocity similar to wild type. Occasional stationary cells are adhering to the glass surface by their flagella. Images were captured and displayed at 30 fps. (MOV 2959 kb)

Supplementary Video 4

Olfactory placode cilia in dnaaf3MOex8 zebrafish embryo. (MOV 2189 kb)

Supplementary Video 5

Olfactory placode cilia in wildtype zebrafish embryo. (MOV 2402 kb)

Supplementary Video 6

Spinal cord canal cilia in dnaaf3MOex8 zebrafish embryo. (MOV 2404 kb)

Supplementary Video 7

Spinal cord canal cilia in wildtype zebrafish embryo. (MOV 2787 kb)

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Mitchison, H., Schmidts, M., Loges, N. et al. Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia. Nat Genet 44, 381–389 (2012). https://doi.org/10.1038/ng.1106

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