The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

Abstract

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

Strongly truncated Dnaaf4 plays a conserved role in Drosophila ciliary dynein assembly as part of an R2TP-like co-chaperone complex with Dnaaf6

“…However, of the other associated proteins identified, none appeared to have TPR domains or other features that would help clarify Dnaaf4 function. Filtering the data for proteins associated with motile cilia (zur Lage et al, 2021), we found two proteins of interest to be associated but at a P value that is below the threshold for significance (Fig. 4B).…”

“…Pontin of R2TP is significantly associated (arrow). (B) The same dataset filtered to extract proteins associated with motile cilia (zur Lage et al, 2021). Pontin is the only associated protein to reach statistical significance.…”

“…To assess the auditory function of chordotonal neurons, a larval hearing assay was performed. Third-instar larvae normally respond to a 1000-Hz sine wave tone by momentarily contracting, a behaviour that requires functional dynein motors for mechanotransduction within chordotonal neuron cilia (zur Lage et al, 2021). Larvae homozygous for Dnaaf4 or Dnaaf6 mutations did not respond to a tone stimulus, consistent with functionally impaired chordotonal neurons in vibration sensing (Fig.…”

“…Drosophila testis fixing and staining was carried out according to Sitaram et al (Sitaram et al, 2014). The following primary antibodies were used: goat anti-GFP antibody (1:500, ab6673), rabbit anti-GFP antibody (1:500, Life Technologies, A11122), mouse anti-Futsch antibody (1:200, Developmental Studies Hybridoma Bank, 22C10), mouse anti-pan polyglycylated tubulin (1:100, Merck, MABS276), rabbit anti-Sas-4 (1:350, gift from Jordan Raff) and rabbit anti-Dnah5 antibody (1:2000, (zur Lage et al, 2021)). The following secondary antibodies were used: goat anti-Rabbit antibody (1:500, Alexa Fluor 488, Life Technologies, A11008) and goat anti-Mouse antibody (1:500, Alexa Fluor 568, Life Technologies, A11019), donkey anti-goat antibody (1:500, Alexa Fluor 488, Life Technologies, A11055), donkey anti-mouse antibody (1:500, Alexa Fluor 568, Life Technologies, A10037), and donkey anti-rabbit antibody (1:500, Alexa Fluor 647, Life Technologies, A31573).…”

“…In the case of Drosophila , ciliary motility is confined to the sensory cilium of mechanosensory neurons (chordotonal neurons) and the sperm flagellum. Flies with dysfunctional dyneins are therefore deaf, uncoordinated and have immotile sperm, which makes the fly a convenient model for analysis of motile ciliogenesis (Diggle et al, 2014; Moore et al, 2013; zur Lage et al, 2018; zur Lage et al, 2021).…”