Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration inChlamydomonas reinhardtii

Motile cilia are complex organelles comprised of >800 structural proteins. Variants in 58 of these genes cause primary ciliary dyskinesia (PCD) in humans. We used a second-site non-complementation approach in diploid Chlamydomonas reinhardtii strains to investigate whether reduced dosage of different combinations of motile cilia proteins affects cilia growth / regeneration. Temperature-sensitive mutants in intraflagellar transport genes fail to complement in heterozygous diploids at the restrictive temperature, which may be due to poisonous interactions or a reduction in protein levels. Diploid strains heterozygous for 211 double mutant combinations in 21 genes are phenotypically recessive when unperturbed and do not show SSNC. Consequently, we developed a sensitized screen. When protein synthesis is inhibited, cells utilize the existing cytoplasmic pool of proteins, which is insufficient to build full-length cilia. Six double heterozygous strains in dynein arm preassembly factors (DNAAFs) pf13, pf23, wdr92, and oda8 regrow cilia that are shorter than wild-type diploids, which suggests that double heterozygosity limits the pool of assembled dynein arms further. We isolated a new null allele ( pf23-4) that shows a more severe loss of ODAs and IDAs in cilia than the previously studied pf23-1 hypomorphic allele. We also identified a new role for PF23 in cytoplasmic modification of IC138, protein of the I1/ f inner dynein arm. In our SSNC assay, the pf23-4 allele also exhibits a more severe phenotype than pf23-1 . Whole cell extract immunoblots show a reduction of PF23 protein to one-half of wild-type levels in both single and double heterozygous genotypes. The pf13 mutant shows SSNC with mutants in two outer dynein arm structural proteins, ODA6 , and ODA9. We suggest that reduction of multiple dynein preassembly factors limits the pool of assembled dynein arms needed for cilia assembly and regeneration. Our data support PF23 as a scaffolding hub protein involved in dynein arm assembly. Author Summary Motile cilia are essential for movement of cells and fluids. In humans, motile cilia defects cause primary ciliary dyskinesia (PCD), a rare disease characterized by recurring respiratory infections, left-right asymmetry defects, ear infections, and infertility. Many genes are involved in motile cilia function and assembly. We use Chlamydomonas reinhardtii to study the effects of changes in protein levels in different combinations of proteins needed for motile cilia. Our results show that the proteins that help fold and assemble the dynein motors in the cytoplasm are sensitive to changes in the dosage of these preassembly factors. Reductions in proteins involved in multiple steps of the pathway prevents efficient dynein preassembly and cilia regeneration under stressful cellular conditions.