Establishment of the early cilia preassembly protein complex during motile ciliogenesis

Abstract

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

Role of the Novel Hsp90 Co-Chaperones in Dynein Arms’ Preassembly

“…On the other hand, preassembly genes such as DNAAF1, DNAAF3, DNAAF4, LRRC6, DNAAF6/PIH1D3/TWISTER/TWI, and DNAAF7/ZMYND10, were expressed at a later stage of cell differentiation, similar to dynein DNAI1 [65]. Based on these data, it can be assumed that R2SP is involved in the early phase of the formation of dynein arms when SPAG1 binds to DNAAF2.…”

“…SPAG1, like other proteins with TPR domains, (Table 1) mediates the delivery of clients from Hsp70 to Hsp90, thanks to simultaneous anchoring of the chaperones via EEVD residues within Hsp70 and Hsp90 C-terminal end to the designed TPR domains of SPAG1 [64]. SPAG1, besides RuvBL1, RuvBL2, and PIH1D2, interacts (possibly indirectly) with DNAAF5/HEATR2 (HEAT Repeat-Containing) and DNAAF2 (Table 1), and co-localizes with them in DynAPs [27,65]. Recent studies on ciliogenesis in human primary airway epithelial cells have shown that DNAAF5, SPAG1, and DNAAF2 undergo transcription during the early stages of airway epithelial cell differentiation.…”

“…Recent studies on ciliogenesis in human primary airway epithelial cells have shown that DNAAF5, SPAG1, and DNAAF2 undergo transcription during the early stages of airway epithelial cell differentiation. The analyses of the epithelial respiratory cells obtained from PCD patients during biopsies have shown that mutations in DNAAF5 do not disrupt direct interaction with SPAG1, but cause the accumulation of large DNAAF5-SPAG1-DNAAF2 aggregates and degradation of all three proteins [65].…”

Primary ciliary dyskinesia (PCD): A genetic disorder of motile cilia

“…Other PCD genes encoding cytoplasmic proteins that are associated with absence of ODA and IDA when mutated include: DNAAF1 (LRRC50, ODA7, MIM 613190) [90], LRRC6 (MIM 614930) [64,83], DNAAF5 (HEATR2; MIM 614864) [62], DNAAF3 (PF22; MIM 614566) [100], DNAAF4 (DYX1C1; MIM 608706) [145], SPAG1 (MIM 603395) [81], CCDC103 (MIM 614677) [121], ZMYND10 (MIM 607070) [102,155], CFAP298 (C21orf59; Kurly; MIM 615494)) [4], CFAP300 (C11orf70; MIM 618058)) [59] and PIH1D3; MIM 300933) [112,119] (Table 1). The exact function of these proteins is not known, but evidence points to close interactions among several proteins, as well as protein complexes and heat-shock proteins suggesting that they may function as chaperones during dynein arm protein assembly [65,92], e.g., LRRC6 for instance interacts with ZMYND10 [155]; DNAAF5 interacts with SPAG1 and DNAAF2 during the earliest stages of cilia preassembly [65]; DNAAF2 interacts with DYX1C1 and PIH1D3 at later stages of cilia preassembly, possibly acting as linker proteins [65,119,145]. These proteins also interact with protein chaperones HSP70 and HSP90, facilitating the organization of dynein arm assembly [25,115,120,145].…”

Mutation ofCFAP57causes primary ciliary dyskinesia by disrupting the asymmetric targeting of a subset of ciliary inner dynein arms

“…The most common genetic lesions that cause PCD are those that affect components of the ODA, including DNAH5, DNAI1, and DNAH11 (2)(3)(4). Another group of PCD associated genes encode ODA-docking complex components (CCDC114 and CCDC151) (5)(6)(7), or the proteins that play a role in the cytoplasmic assembly of dynein (SPAG1, DNAAF1-3, HEATR2, and DYX1C1) (8)(9)(10)(11)(12)(13)(14). In addition, mutations in two genes, CCNO (15) and MCIDAS (16), cause a PCD-like phenotype by greatly reducing the number of motile cilia.…”