Fig 2: Ubiquitous FKBP8 actively participates in axonemal dynein heavy chain stability via its interaction with ZMYND10.(A) Summary of mutations generated in ZMYND10-tGFP by site-directed mutagenesis to disrupt the binding interface for FKBP8, including the W423A mutation predicted to functionally disrupt one of two Zn2+-fingers in the MYND domain and the T379C PCD patient mutation (Zariwala et al., 2013), lying just before the MYND domain. (B) Extracts from transiently transfected HEK293 cells affinity purified against turboGFP shows C-terminal mutations interrupt endogenous FKBP8 and HSP90 binding to ZMYND10. (C) Healthy human donor tracheal epithelial cultures (MucilAir) before ciliation (D17, post-ALI) or fully ciliated (D60, post-ALI), were cultured for 24 hr in control (vehicle only) or DM-CHX (concentrations indicated in µM) before harvesting protein extracts. Immature cultures (D17) were more sensitive to effects of specific PPIase inhibitor DM-CHX, destabilizing dynein components, whilst mature cultures were minimally affected. (D) Quantification of band intensities for DNAH5 (blue) or DNAI1 (yellow) from (C) were normalized to loading control, and plotted as a fold change after 24 hr. (E) Z projections of whole-mount immunofluorescence of mouse tracheal epithelial cultures (MTECs) stained with DNAH5 (magenta), DNAI2 (green) and DAPI (blue) after treatment of three days post-airlift (ALI) with either 100 µm DM-CHX or vehicle control for 14 days in culture to differentiate, followed by switching cultures from control to 100 µm DM-CHX (mature: cilial beat visualized) or from DM-CHX into vehicle control (no cilia beat) and culturing an additional 7 days. Treatment of mature ciliated cultures had little effect on DNAH5 and DNAI2 levels, whilst treatment during differentiation disrupted expression of dynein subunits, as evidenced by lack of axonemal staining in most cells (right panels) but release allows recovery of dynein pre-assembly, mostly cytoplasmic after 7 days.

Fig 3: ZMYND10 specifies dynein heavy chains as clients in a chaperone relay during dynein pre-assembly.(A) Extracts from transiently transfected RPE-1 cells with ZMYND10-turboGFP variants and human LRRC6-myc were affinity purified against turboGFP (upper panels). Inclusion of LRRC6 destabilizes binding of wild type ZMYND10 to endogenous FKBP8. C-terminal ZMYND10 mutations do not affect its interaction with LRRC6. Expression of interactors is confirmed in input cell lysates (lower panels). (B) During dynein arm assembly in the cytoplasm, ZMYND10 interaction with co-chaperone PPIase FKBP8 and HSP90 is required for the stabilization and folding of dynein heavy chains (DNAH). Competitive binding of LRRC6 to ZMYND10 may advance the HSP90-FKBP8 chaperone cycle taking the client DNAHs to the next stage of assembly. We propose either R2TP or a specialized R2TP-like complex may function in parallel to assemble, scaffold or tether the DNAI1-DNAI2 heterodimeric complex via the PIH-domain proteins, such as DNAAF6, DNAAF2 and PIH1D2. The TPR-domain containing SPAG1 or DNAAF4 could recruit HSP90 via its MEEVD domain and together with RUVBL1/RUVBL2 these assembly factors could form a stable platform to promote HC-IC subunit interactions. This R2TP or R2TP-like complex likely operates distinctly to ZMYND10 as DNAI1/2 heterodimers are detected in Zmynd10 mutants, however, these are likely degraded if fully functional mature complexes (with heavy chains) cannot be assembled. This chaperone-relay system comprises several discrete chaperone complexes overseeing the folding and stability of discrete dynein subunits. Folding intermediates are handed off to successive complexes to promote stable interactions between subunits all the while preventing spurious interactions. Stable dynein complexes, once formed are targeted to cilia via transport adaptors and intraflagellar transport (IFT).