HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

Abstract

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

ZMYND10 functions in a chaperone relay during axonemal dynein assembly

“…We previously reported distinct morphological and dynein staining characteristics observed among human multiciliated nasal brush biopsied cells depending on their maturity 11 . In cells isolated from nasal turbinates of control mice, ‘immature’ multiciliated cells were rounder with higher cytoplasmic dynein immunostaining and shorter cilia in keeping with cytoplasmic pre-assembly of the motility machinery.…”

“…Firstly, we did not detect interactions with IFT-B proteins involved in ODA transport ( Figure S4 ). Next, we investigated whether ZMYND10 associates with chaperones or other dynein assembly factors ( Figure 6A ), namely DNAAF2 which was shown to interact with DNAAF4, which also co-precipitates several chaperone proteins including the CCT3 subunit of the TriC chaperonin complex 8 as well as the non-canonical co-chaperone DNAAF5, whose HEAT repeats have been proposed to function as scaffolds in multisubunit assembly 11,19 . We failed to detect interactions with HSP70, DNAAF2, CCT3 or DNAAF5 by endogenous ZMYND10 affinity purification from mouse testes or oviduct extracts ( Figure 6A,C ).…”

“…Alexa-488 phalloidin (Thermo Fischer) or rat anti-GRP94 (Thermo Fischer) counterstaining was done post-PLA protocol, prior to mounting in Duolink ® In Situ Mounting Medium with DAPI (Sigma Aldrich). Trachea, testes and oviducts were dissected and immersion fixed in 4% paraformaldehyde (from 16% solution, Thermo Fischer) overnight and cryosectioned for immunofluorescence 11 . Nasal turbinates were similarly fixed and processed for paraffin sectioning stained with H&E to reveal mucus plugs.…”

“…Interactions between LRRC6, DNAAF1/LRRC50 and C21ORF59/Kurly were recently reported which, coupled with the phenotypic analysis of Lrrc6 mutant mice, suggests that these assembly factors may primarily function in apical targeting/trafficking of dynein complexes 9,10 . The functions of DNAAF5/HEATR2 which has no reported links to chaperones, remain elusive 11 .…”

C11orf70 mutations causing primary ciliary dyskinesia disrupt a conserved step in the intraflagellar transport-dependent assembly of multiple axonemal dyneins

“…[10][11][12][13][14][15][16] Perhaps more surprising than the finding that mutations in cilia structural components are a frequent cause of PCD has been a growing number of PCD loci that do not encode parts of the cilium per se, but rather encode proteins that act in the apparently complicated process of the assembly and transport of ciliary dynein motors. 17 Many such assembly factors work strictly in the cytoplasm in a chaperoning step and cause PCD when mutated, such as DNAAF1, 18-20 DNAAF2, 21 DNAAF3,22 DYX1C1 (DNAAF4), 23; 24 HEATR2 (DNAAF5), 25; 26 LRRC6 27-29 and PIH1D3. 30; 31 Other factors, identified in both the human population through analysis of PCD patients and in genetic screens for cilia motility defects in model organisms, resemble chaperones in that they affect the assembly of multiple ciliary dynein isoforms, but also resemble transport factors based on their presence in a detergent-soluble ciliary compartment.…”