CCDC151 Localizes to Respiratory Ciliary Axonemes and CCDC151 Mutations Are Associated with Loss of the Ciliary Outer Dynein Arms and Ciliary Immotility in Humans
To further explore the role of CCDC151 in human disease, we next examined protein localization in respiratory ciliated cells. We first screened protein lysates isolated from human nasal respiratory epithelial cells using commercially available rabbit polyclonal antibodies directed against CCDC151. Immunoblot analysis showed that the antibodies specifically recognize CCDC151, detecting a single protein band of the predicted molecular weight (∼64 kDa) (Figure 4A). We then used this antibody to analyze the subcellular localization of the protein in human motile respiratory cilia. IF showed that CCDC151 localizes to the axonemes of wild-type human respiratory epithelial cells, overlapping with an acetylated α-tubulin marker of ciliary axonemes. However, the protein was undetectable in the respiratory cilia of individuals OP-675 and OP-1255, consistent with the predicted loss-of-function consequences of the CCDC151 nonsense mutations they carry (Figure 4B). We used high-speed videomicroscopy to analyze respiratory ciliary beating in individuals with CCDC151 mutations. Both individuals OP-675 and OP-1255 had completely immotile cilia compared to the coordinated synchronous beating of cilia from unaffected control individuals, recapitulating the functional defects of the Ccdc151Snbl mice (Movies S10, S11, and S12).
Ultrastructural analysis by TEM of respiratory ciliary axonemes from individuals carrying CCDC151 mutations showed a loss of the outer dynein arms (mean of ODAs detected: 0.8–0.9) from ciliary axonemes compared to those of unaffected control individuals (ODA mean: 7.5–9) (Figure 5C). These results are consistent with the ultrastructural ciliary phenotype of ccdc151ts272a (flanders) mutants, ccdc151 RNAi planarians, and Ccdc151Snbl mice (Figures 2H, S4E, and 3C). We further examined this defect at the molecular level by immunofluorescence staining of the respiratory cells of individuals OP-675 and OP-1255 using antibodies directed against two established markers of human dynein arm integrity, the ODA marker DNAH5 and IDA marker DNALI1 (which is a light intermediate dynein associated with some IDAs). DNAH5 was undetectable in the axonemes of CCDC151 mutant individuals, suggesting that CCDC151 deficiency likely causes a disruption of axonemal ODA assembly (Figure 5A). In contrast, DNALI1 correctly localized to the axonemes of both individuals’ respiratory cells. This marker showed a similar distribution to that of control individual’s cilia (Figure 5B), suggesting that CCDC151 mutations do not alter assembly of IDA proteins.
Together, the TEM and IF data indicate that the axonemes of CCDC151-deficient cilia have ODA defects but that DNALI1-related IDA assembly is undisturbed. We also examined the integrity of the ciliary nexin dynein regulatory complexes (N-DRC) in CCDC151 mutant cilia by immunolocalization using antibodies directed against an integral N-DRC component, GAS8 (human DRC4).33–35 Similarly to DNALI1, GAS8 correctly localized to mutant ciliary axonemes, indicating that N-DRC assembly is not affected (Figure 5B).