CFAP 54 is required for proper ciliary motility and assembly of the central pair apparatus in mice

Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD. INTRODUCTION The motile cilium is an organelle vital to human health. Motile cilia extend from the basal bodies of epithelial cells in the respiratory system and the oviduct and ependymal cells in the brain (IbanezTallon et al., 2003; Satir and Christensen, 2007; Lee, 2011). Unlike the immotile primary cilia, motile cilia beat in a wave-like motion to clear mucus in the respiratory system, enable cerebrospinal fluid (CSF) flow in the ventricular system of the brain, and assist in egg and sperm transport through the oviduct (Ibanez-Tallon et al., 2003; Satir and Christensen, 2007; Berbari et al., 2009; Lee, 2011). The core, also known as the axoneme, of motile cilia consists of a 9+2 microtubule structure with nine doublet microtubules surrounding a central microtubule pair (Ibanez-Tallon et al., 2003; Satir and Christensen, 2007; Lee, 2011). The ciliary motor force is generated by dynein arms associated with the outer doublet microtubules. The central microtubule apparatus is believed to play a crucial role in regulation of the dynein motor force through its association with the radial spokes that connect the outer doublets to the central pair (Smith and Lefebvre, 1997; Mitchell, 2004). Motile 9+0 cilia lacking a central pair are found on the embryonic node and play a role in establishing left–right asymmetry (Ibanez-Tallon et al., 2003; Satir and Christensen, 2007; Lee, 2011). Sperm flagella have a 9+2 axonemal structure similar to motile cilia and are required for sperm motility (Ibanez-Tallon et al., 2003; Satir and Christensen, 2007; Lee, 2011). Proteomic analyses of eukaryotic motile cilia have demonstrated a remarkable complexity (Ostrowski et al., 2002; Monitoring Editor Stephen Doxsey University of Massachusetts Received: Feb 27, 2015 Revised: Jul 21, 2015 Accepted: Jul 22, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E15-02-0121) on July 29, 2015. Address correspondence to: Lance Lee (Lance.Lee@sanfordhealth.org). © 2015 McKenzie et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by -nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: 129, 129S6/SvEvTac; AK7, adenylate kinase 7; B6, C57BL/6J; CBF, ciliary beat frequency; CFAP221, ciliary and flagellar protein 221; CFAP54, ciliary and flagellar protein 54; CSF, cerebrospinal fluid; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; PCD, primary ciliary dyskinesia; PCDP1, primary ciliary dyskinesia protein 1; qRT-PCR, quantitative reverse transcription PCR; WT, wild type. Casey W. McKenziea, Branch Craigeb, Tiffany V. Kroegera, Rozzy Finna, Todd A. Wyattc, Joseph H. Sissonc, Jacqueline A. Pavlikc, Lara Strittmatterb, Gregory M. Hendricksb, George B. Witmanb, and Lance Leea,d aChildren’s Health Research Center, Sanford Research, Sioux Falls, SD 57104; bDepartment of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655; cDepartment of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198; dDepartment of Pediatrics, Sanford School of Medicine of the University of South Dakota, Sioux Falls, SD 57105 http://www.molbiolcell.org/content/suppl/2015/07/27/mbc.E15-02-0121v1.DC1. html Supplemental Material can be found at: