PMC:6288205 / 29232-33671 JSONTXT

{"project":"2_test","denotations":[{"id":"30471718-15750039-2047756","span":{"begin":263,"end":264},"obj":"15750039"},{"id":"30471718-26909801-2047757","span":{"begin":266,"end":267},"obj":"26909801"},{"id":"30471718-23872636-2047758","span":{"begin":583,"end":585},"obj":"23872636"},{"id":"30471718-20409784-2047759","span":{"begin":587,"end":589},"obj":"20409784"},{"id":"30471718-22387996-2047760","span":{"begin":1965,"end":1967},"obj":"22387996"}],"text":"In control human respiratory cilia, DNAH9 localizes exclusively to the distal part of the respiratory ciliary axoneme while the β-DHC paralog DNAH11 localizes only to the proximal half. In contrast, DNAH5 shows a panaxonemal distribution along the ciliary axoneme5, 6 (Figures 3B–3E). To assess the effect of DNAH9 variants on the axonemal localization of DNAH9 as well as localization of other ODA proteins, we performed high-resolution IF analysis of respiratory cilia obtained from individuals OP-2905 II1, OP-1226 II1, and MS-SI46 II1 and control subjects as previously described44, 45 using antibodies directed against the axonemal ODA heavy chains DNAH5, DNAH9, and DNAH11, the ODA intermediate chains DNAI1 and DNAI2, the ODA docking complex components TTC25 and CCDC114, as well as the N-DRC component GAS8. The anti-DNAH9 antibody (HPA052641, Atlas antibodies) recognizes an epitope that consists of amino acid residues 675–754. Consistent with loss-of-function mutations, DNAH9 was completely absent from the ciliary axoneme in OP-2905 II1, OP-1226 II1, and MS-SI46 II1 (Figure 4A). This indicates that despite moderately reduced scores predicted by Alamut for the wild-type splice site for the +4 splice site variant in OP-2905 II1, no wild-type transcript is present in multiciliated respiratory epithelial cells and probably the predicted alternative splice site causes a frameshift and/or the mRNA is subjected to nonsense-mediated decay. Additionally, DNAH5 was absent or severely reduced from the distal ciliary axoneme in all three individuals (Figures 4B and S6), and DNAI1 and DNAI2 were absent or severely reduced from the distal ciliary axonemes in individuals OP-2905 II1 and MS-SI46 II1 (Figures S7–S9). Cellular staining for ODA components such as DNAH5, DNAI1, and DNAI2 was also absent and results from protein degradation because ODA complexes cannot be correctly assembled in the cytoplasm and half-life of those components is shortened.46 Thus, all three analyzed DNAH9 mutant individuals depicted identical defects on the molecular (absence of DNAH9, DNAH5, DNAI1, and DNAI2) and sub-cellular (distal ciliary axoneme) levels. DNAH9 appears to be essential for assembly of ODAs type 2, because DNAH5 and DNAI2 are absent from the distal ciliary axonemes in DNAH9 mutant cilia. Vice versa, we next analyzed the localization of DNAH9 in cells from PCD-affected individuals with mutations in DNAH5, DNAI1, DNAI2, and DNAH11. DNAH9 was absent from the distal ciliary axonemes of DNAH5, DNAI1, and DNAI2 mutant cilia (Figure 5), indicating that the ODA proteins DNAH5, DNAH9, DNAI1, and DNAI2 are components of the multimeric ODA type 2 in the distal ciliary axonemes and also essential for the assembly of distal ODAs type 2.\nFigure 4 DNAH9 Mutant Cilia Disrupt ODA Type 2 Complexes in the Distal Compartment of Respiratory Cilia\n(A) In contrast to healthy control subjects, DNAH9 (red) is absent or severely reduced in the distal compartment of DNAH9 mutant cilia. Ciliary axonemes are counterstained with anti-acetylated tubulin (green). Schematic (right side) highlights loss of DNAH9 (red) in the distal compartment of respiratory cilia.\n(B) In contrast to healthy control subjects, DNAH5 (green) is absent or severely reduced in the distal compartment of DNAH9 mutant cilia. DNAH5 retains localization in the proximal compartment of DNAH9 mutant cilia. The ODA docking complex protein CCDC114 (red) localizes along the entire ciliary axoneme in both control and DNAH9 mutant cilia. Schematic (right side) highlights loss of DNAH5 (green) in the distal compartment of respiratory cilia. Nuclei are stained with Hoechst33342 (blue). Scale bars represent 10 μm. For each individual, 15–20 cells were analyzed from two independent IF experiments.\nFigure 5 Mutations in DNAH5, DNAI1, DNAI2, and DNAH11 Affect Axonemal Localization of DNAH9\nRespiratory cilia double-labeled with antibodies directed against acetylated tubulin (green) and DNAH9 (red) show colocalization of DNAH9 with acetylated tubulin at the distal part of the cilia from an unaffected control (yellow). In contrast, DNAH9 is absent or severely reduced in DNAH5, DNAI1, and DNAI2 mutant ciliary axonemes. Interestingly, in DNAH11 mutant cilia the distal localization of DNAH9 shifts from distal to a panaxonemal localization. Nuclei were stained with Hoechst33342 (blue). Scale bars represent 10 μm. For each individual, 15–20 cells were analyzed."}