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Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

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19358 -19366). Peptide sequence data (16/16 residues correct) implicate the M r ‫؍‬ 14,000 polypeptide as Tctex-1, a protein encoded within the mouse t-complex. Tctex-1 cosediments with microtubules and is eluted with ATP or salt but not with GTP as expected for a dynein subunit. The ATP-eluted protein precisely cosediments with known cytoplasmic dynein proteins in sucrose density gradients. Tctex-1 also is immunoprecipitated from brain and other tissue homogenates by a monoclonal antibody raised against the 74-kDa cytoplasmic dynein intermediate chain. Quantitative densitometry indicates that Tctex-1 is a stoichiometric component of the dynein complex. As Tctex-1 is a candidate for involvement in the transmission ratio distortion (meiotic drive) of mouse t-haplotypes, these results suggest that cytoplasmic dynein dysfunction may play an important role in non-mendelian chromosome segregation.Dyneins are microtubule-based molecular motors. Axonemal dyneins are responsible for generation and propagation of ciliary/flagellar bends (see Ref. 1), whereas cytoplasmic dynein is involved in a wide range of intracellular motile events including retrograde vesicle transport in axons, membrane trafficking, nuclear migration, and both the positioning and anaphase movement of the mitotic spindle (2-9). Although involved in diverse motile systems, recent molecular analyses have revealed that the flagellar and cytoplasmic dynein isozymes are related both in terms of their component polypeptides and in their overall structural organization (for review, see Refs. 10 and 11).Cytoplasmic dynein is a multimeric complex containing two dynein heavy chains (DHCs) 1 (ϳ530 kDa) that exhibit both ATPase and motor activities; these components form the globular heads and stems seen in the soluble particle (reviewed in Ref. 10). A recent report suggests that there are at least three cytoplasmic DHC isoforms in mammals that are associated with discrete intracellular structures (12). Thus, different cytoplasmic DHC-containing isozymes may have distinct functions, as do their flagellar counterparts (reviewed in Refs. 11 and 13).Associated with these large motor subunits are 2-3 copies of a 74-kDa intermediate chain (IC74) (14) that contains five repeats of the WD motif (15) and is therefore likely a member of the ␤-propeller family of proteins (16). IC74 apparently is involved in attaching the cytoplasmic dynein motor to its cargo by mediating the interaction between dynein and dynactin (17, 18). Multiple alternatively spliced and phosphorylated variants of this component have been identified (14, 19 -21), and these may be involved in targeting the motor to various intracellular cargoes. The cytoplasmic ICs are related both structurally and functionally to their counterpart proteins found within flagellar dyneins (reviewed in Ref. 11). For example, the M r ϭ 78,000 IC (IC78) of Chlamydomonas outer arm dynein is also a WDrepeat protein that mediates the ATP-insensitive (cargo) attachment of that enzyme to the flagellar doublet microtubules (22...

Abstract. A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-Mr intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with e~-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995. J. Cell Biol. 129:169-178 provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.