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 homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. 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.

The dynamics of protein localisation to restricted zones within Drosophila mechanosensory cilia

“…We first analysed flies with a CRISPR-engineered null mutation of Dnaaf3 (CG17669), the orthologue of the human dynein assembly factor and PCD gene, DNAAF3. Human DNAAF3 is required for all dynein assembly, and Drosophila Dnaaf3 CR mutant flies show absence of dynein arms from the chordotonal neuron cilia (zur Lage et al, 2021). However, we found that Iav localisation was unaltered in Dnaaf3 CR mutant antennae (Fig.…”

“…They were mounted in a 2.5% n-Propyl gallate/glycerol mix. The following primary antibodies were used: Goat anti-GFP (1:500: Abcam, ab6673), Rabbit anti-mCherry (1:500; Abcam, ab167453), Mouse anti-NompC (1:250; Liang et al 2011), Rabbit anti-Sas4 (1: 350; gift from Jordan Raff), Rabbit anti-Dnah5 (1:2000; (zur Lage et al, 2021). The secondary antibodies (Life Technologies) were all used at a 1:500 concentration and included: Donkey anti goat (Alexa Fluor® 488, A11055), Donkey anti mouse (Alexa Fluor® 568, A10037), Donkey anti rabbit (Alexa Fluor® 647, A31573).…”

“…Although the role of DNAAFs in cytoplasmic pre-assembly within chordotonal neurons has been studied (Diggle et al, 2014;Moore et al, 2013;zur Lage et al, 2018zur Lage et al, , 2021, the later stages of dynein motor complex assembly have not been addressed previously. To follow IDA complex localisation, we used an mVenus fusion reporter for Dnali1, hereafter referred to as IDA-Dnali1 for clarity (Moore et al, 2013).…”

“…(B) Knockdown of Ccdc151 and Dhc93AB (scaGal4, UAS-RNAi) result in impaired performance in climbing assay compared to control (scaGal4, KK control line). For comparison, knockdown of Dnaaf3 is also included, in which dynein arms are completely absent from the cilia (zur Lage et al, 2021). Significance was determined by one-way Anova followed by Dunnett’s test for multiple comparisons: CG14127: p = 0.0246; Dhc93AB: P < 0.0001; Dnaaf3: P<0.0001. n = 10 batches of 15 flies.…”

“…1C). These specialised 9+0 cilia are notable for two reasons: (1) They have the features of motile cilia, including axonemal dynein motor complexes (zur Lage et al, 2019) the function of which is critical to their mechanotransduction mechanism (Karak et al, 2015; zur Lage et al, 2021); (2) They have structurally distinct zones with specialised functions: a sensory ‘distal zone’ closely connected with an external ciliary cap and containing the mechanosensory TRP channel, NompC, and a motile ‘proximal zone’ containing heteromeric TRPV channels (Inactive/Nanchung or lav/Nan) as well as the axonemal dynein motors (Fig. 1D).…”