SummaryCilia/flagella are highly conserved organelles that play diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia/flagella often result in primary ciliary dyskinesia (PCD). However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a novel gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in PCD patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.

Primary Ciliary Dyskinesia (PCD) most often arises from loss of the dynein motors that power ciliary beating. Here we show that PF22/DNAAF3, a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes prior to their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in patients from families with situs inversus and defects in assembly of inner and outer dynein arms. Zebrafish dnaaf3 knockdown likewise disrupts dynein arm assembly and ciliary motility, causing PCD phenotypes including hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a null mutant fails to assemble outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests PF22/DNAAF3 acts at a similar stage to other preassembly proteins, PF13/KTU and ODA7/LRRC50, in the dynein preassembly pathway. These results support the existence of a conserved multi-step pathway for cytoplasmic formation of assembly-competent ciliary dynein complexes.

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCbeta. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr approximately 2 x 10(6) dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.