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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References
Coren, M.E., Meeks, M., Morrison, I., Buchdahl, R.M. & Bush, A. Primary ciliary dyskinesia: age at diagnosis and symptom history. Acta Paediatr. 91, 667–669 (2002).
Bush, A., Hogg, C., Mitchison, H.M., Nisbet, M. & Wilson, R. Update in primary ciliary dyskinesia. Clin. Pulm. Med. 16, 219–225 (2009).
Bush, A. Congenital heart disease in primary ciliary dyskinesia. Pediatr. Cardiol. 19, 191 (1998).
Kennedy, M.P. et al. Congenital heart disease and other heterotaxic defects in a large cohort of patients with primary ciliary dyskinesia. Circulation 115, 2814–2821 (2007).
Tan, S.Y. et al. Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia. J. Clin. Invest. 117, 3742–3752 (2007).
Barbato, A. et al. Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. Eur. Respir. J. 34, 1264–1276 (2009).
Ibañez-Tallon, I. et al. Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation. Hum. Mol. Genet. 13, 2133–2141 (2004).
Kosaki, K. et al. Absent inner dynein arms in a fetus with familial hydrocephalus-situs abnormality. Am. J. Med. Genet. A. 129A, 308–311 (2004).
Yang, P. et al. Radial spoke proteins of Chlamydomonas flagella. J. Cell Sci. 119, 1165–1174 (2006).
Castleman, V.H. et al. Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular-pair abnormalities. Am. J. Hum. Genet. 84, 197–209 (2009).
Heuser, T., Raytchev, M., Krell, J., Porter, M.E. & Nicastro, D. The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella. J. Cell Biol. 187, 921–933 (2009).
Papon, J.F. et al. A 20-year experience of electron microscopy in the diagnosis of primary ciliary dyskinesia. Eur. Respir. J. 35, 1057–1063 (2010).
El Zein, L., Omran, H. & Bouvagnet, P. Lateralization defects and ciliary dyskinesia: lessons from algae. Trends Genet. 19, 162–167 (2003).
Olbrich, H. et al. Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right and asymmetry. Nat. Genet. 30, 143–144 (2002).
Bartoloni, L. et al. Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia. Proc. Natl. Acad. Sci. USA 99, 10282–10286 (2002).
Pennarun, G. et al. Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia. Am. J. Hum. Genet. 65, 1508–1519 (1999).
Loges, N.T. et al. DNAI2 mutations cause primary ciliary dyskinesia with defects in the outer dynein arm. Am. J. Hum. Genet. 83, 547–558 (2008).
Mazor, M. et al. Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1. Am. J. Hum. Genet. 88, 599–607 (2011).
Duriez, B. et al. A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia. Proc. Natl. Acad. Sci. USA 104, 3336–3341 (2007).
Omran, H. et al. Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins. Nature 456, 611–616 (2008).
Duquesnoy, P. et al. Loss-of-function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia. Am. J. Hum. Genet. 85, 890–896 (2009).
Loges, N.T. et al. Deletions and point mutations of LRRC50 cause primary ciliary dyskinesia due to dynein arm defects. Am. J. Hum. Genet. 85, 883–889 (2009).
Kamiya, R. Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii. J. Cell Biol. 107, 2253–2258 (1988).
Huang, B., Piperno, G. & Luck, D.J.L. Paralyzed flagella mutants of Chlamydomonas reinhardtii. J. Biol. Chem. 254, 3091–3099 (1979).
Piperno, G., Mead, K. & Shestak, W. The inner dynein arms I2 interact with a “dynein regulatory complex” in Chlamydomonas flagella. J. Cell Biol. 118, 1455–1463 (1992).
Piperno, G. & Ramanis, Z. The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms. J. Cell Biol. 112, 701–709 (1991).
Mitchell, D.R. & Kang, Y. Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene. J. Cell Biol. 113, 835–842 (1991).
Kamiya, R. Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants. Int. Rev. Cytol. 219, 115–155 (2002).
Freshour, J., Yokoyama, R. & Mitchell, D.R. Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins. J. Biol. Chem. 282, 5404–5412 (2007).
Hom, E.F. et al. A unified taxonomy for ciliary dyneins. Cytoskeleton (Hoboken) 68, 555–565 (2011).
Fowkes, M.E. & Mitchell, D.R. The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits. Mol. Biol. Cell 9, 2337–2347 (1998).
Ahmed, N.T., Gao, C., Lucker, B.F., Cole, D.G. & Mitchell, D.R. ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. J. Cell Biol. 183, 313–322 (2008).
Yamamoto, R., Hirono, M. & Kamiya, R. Discrete PIH proteins function in the cytoplasmic preassembly of different subsets of axonemal dyneins. J. Cell Biol. 190, 65–71 (2010).
Ahmed, N.T. & Mitchell, D.R. ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly. Mol. Biol. Cell 16, 5004–5012 (2005).
Geremek, M. et al. Gene expression studies in cells from primary ciliary dyskinesia patients identify 208 potential ciliary genes. Hum. Genet. 129, 283–293 (2011).
Ross, A.J., Dailey, L.A., Brighton, L.E. & Devlin, R.B. Transcriptional profiling of mucociliary differentiation in human airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 37, 169–185 (2007).
Meeks, M. et al. A locus for primary ciliary dyskinesia maps to chromosome 19q. J. Med. Genet. 37, 241–244 (2000).
Adzhubei, I.A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).
Kumar, P., Henikoff, S. & Ng, P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081 (2009).
Fliegauf, M. et al. Mislocalization of DNAH5 and DNAH9 in respiratory cells from patients with primary ciliary dyskinesia. Am. J. Respir. Crit. Care Med. 171, 1343–1349 (2005).
Kramer-Zucker, A.G. et al. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis. Development 132, 1907–1921 (2005).
van Rooijen, E. et al. LRRC50, a conserved ciliary protein implicated in polycystic kidney disease. J. Am. Soc. Nephrol. 19, 1128–1138 (2008).
Gao, C., Wang, G., Amack, J.D. & Mitchell, D.R. Oda16/Wdr69 is essential for axonemal dynein assembly and ciliary motility during zebrafish embryogenesis. Dev. Dyn. 239, 2190–2197 (2010).
Lunt, S.C., Haynes, T. & Perkins, B.D. Zebrafish ift57, ift88, and ift172 intraflagellar transport mutants disrupt cilia but do not affect hedgehog signaling. Dev. Dyn. 238, 1744–1759 (2009).
Colantonio, J.R. et al. The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear. Nature 457, 205–209 (2009).
Rosenbaum, J.L., Moulder, J.E. & Ringo, D.L. Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins. J. Cell Biol. 41, 600–619 (1969).
Zhao, R. et al. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120, 715–727 (2005).
Liu, L., Srikakulam, R. & Winkelmann, D.A. Unc45 activates Hsp90-dependent folding of the myosin motor domain. J. Biol. Chem. 283, 13185–13193 (2008).
Wilkerson, C.G., King, S.M., Koutoulis, A., Pazour, G.J. & Witman, G.B. The 78,000 Mr intermediate chain of Chlamydomonas outer arm dynein is a WD-repeat protein required for arm assembly. J. Cell Biol. 129, 169–178 (1995).
Mitchell, D.R. & Brown, K.S. Sequence analysis of the Chlamydomonas α and β dynein heavy chain genes. J. Cell Sci. 107, 635–644 (1994).
Takada, S. & Kamiya, R. Functional reconstitution of Chlamydomonas outer dynein arms from α-β and γ subunits: requirement of a third factor. J. Cell Biol. 126, 737–745 (1994).
Fowkes, M.E. The Role of a 70 kDa Intermediate Chain in Flagellar Outer Row Dynein Assembly. Thesis, State University of New York Health Science Center (1999).
Boulon, S. et al. HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol. Cell 39, 912–924 (2010).
Horejsí, Z. et al. CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability. Mol. Cell 39, 839–850 (2010).
Sullivan-Brown, J. et al. Zebrafish mutations affecting cilia motility share similar cystic phenotypes and suggest a mechanism of cyst formation that differs from pkd2 morphants. Dev. Biol. 314, 261–275 (2008).
Rymarquis, L.A., Handley, J.M., Thomas, M. & Stern, D.B. Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii. Plant Physiol. 137, 557–566 (2005).
Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Edgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
Gouy, M., Guindon, S. & Gascuel, O. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27, 221–224 (2010).
Mitchell, D.R. & Rosenbaum, J.L. Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms. Cell Motil. Cytoskeleton 6, 510–520 (1986).
King, S.M., Otter, T. & Witman, G.B. Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting. Proc. Natl. Acad. Sci. USA 82, 4717–4721 (1985).
Yang, P. & Sale, W.S. The Mr 140,000 intermediate chain of Chlamydomonas flagellar inner arm dynein is a WD-repeat protein implicated in dynein arm anchoring. Mol. Biol. Cell 9, 3335–3349 (1998).
Yagi, T., Uematsu, K., Liu, Z. & Kamiya, R. Identification of dyneins that localize exclusively to the proximal portion of Chlamydomonas flagella. J. Cell Sci. 122, 1306–1314 (2009).
Rashid, S. et al. The murine Dnali1 gene encodes a flagellar protein that interacts with the cytoplasmic dynein heavy chain 1. Mol. Reprod. Dev. 73, 784–794 (2006).
Ferrante, M.I. et al. Convergent extension movements and ciliary function are mediated by ofd1, a zebrafish orthologue of the human oral-facial-digital type 1 syndrome gene. Hum. Mol. Genet. 18, 289–303 (2009).
Barth, K.A. & Wilson, S.W. Expression of zebrafish nkx2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain. Development 121, 1755–1768 (1995).
Yelon, D., Horne, S.A. & Stainier, D.Y. Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev. Biol. 214, 23–37 (1999).
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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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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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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DOI: https://doi.org/10.1038/ng.1106
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