Drosophila Ttc12 genes are putative co-chaperones for dynein assembly and docking in motile cilia

Motile cilia have many developmental and physiological functions. The hallmark of motile cilia are their axonemal dynein motor proteins that generate movement. These large protein complexes require cytoplasmic pre-assembly prior to transport and docking in the cilium, together with an array of associated support complexes, including nexin-DRC and radial spokes. Motile cilia reside in a number of epithelial surfaces, ranging from respiratory airways to fallopian tubes and are similar in structure to sperm flagella. Defective motility in humans results in complex clinical manifestations including chronic bronchitis, situs inversus and infertility, which are classical symptoms of the motile ciliopathy primary ciliary dyskinesia (PCD). In human PCD patients and in model organisms, a number of ‘assembly factors’ have been identified that appear to act with molecular chaperones to guide dynein motor assembly, but the assembly of other complexes is poorly characterised. In a Drosophila transcriptomic screen for new candidate ciliogenic chaperones, CG6980 (Ttc12A) and CG34297 (Ttc12B), were found to be significantly enriched in motile-cilium-bearing cells. Both genes are co-orthologues of human TTC12, which encodes a tetratricopeptide repeat-containing protein predicted to bind the chaperone HSP90, but of unknown function. In this thesis, I demonstrate that Ttc12A/B are expressed exclusively in developing chordotonal mechanosensory neurons, and that TTC12A expression also occurs in spermatocytes, thereby correlating with the only two cell types in Drosophila that generate a motile cilium/flagellum. CRISPR-generated deletion mutants of each gene exhibit loss of chordotonal neuron mechanosensory response to sound or stretch, which normally requires ciliary motility. Mutation of Ttc12A additionally renders sperm immotile, with the mutant sperm showing a loss of dynein arms, as well as a disruption of the axoneme organisation. This is consistent with defects in trafficking and docking of dynein complexes. Reporter line data on chordotonal neuron cilia suggests that TTC12A and TTC12B proteins are required for correct protein trafficking and organisation on the axoneme. Proteomic analysis of Ttc12A null mutant spermatocytes corroborate these findings, as well as indicating a reduction in CCDC39/40 molecular ruler protein homologues, essential for accurate docking of inner dynein arms, nexin-DRC and radial spokes. Furthermore, a striking decline in expression of several dynein heavy chains and associated components were also noted. In conclusion, I hypothesise that Ttc12A/B encode co-chaperones involved in the assembly and trafficking of ciliary motility complexes