Figure 1.
Family pedigree and genetic analyses.
(A) Pedigree of subject with PCD from an Ashkenazi-Jewish family. The closed symbol represents the affected individual and symbols with central closed circles represent heterozygous individuals. (B) Computerized x-ray tomography of the chest of the affected individual showing bronchiectasis. (C) Transmission electron microscopy of cilia from an affected individual failed to show structural defects. (D) Chromatogram showing the nucleotide sequence of the CCDC65 exon 6 adjacent to the mutation site, at position c.876_877delAT (ch12:47598804-5), which is predicted to result in frameshift leading to a premature termination codon.
Figure 2.
Expression of CCDC65 and its relationship with Foxj1 and ciliogenesis.
(A) CCDC65 expression, relative to Foxj1 measured using RT-PCR, increased following the onset of ciliogenesis on air-liquid interface culture days 10 to 14 (p<0.0001; ANOVA). (B) CCDC65 expression determined by RNA sequencing at the indicated times after C. reinhardtii deflagellation, which resulted in marked increase in DRC2 expression, the algal CCDC65 orthologue.
Figure 3.
CCDC65 localization in ciliated airway epithelial cells.
(A) Schematic of a ciliary axoneme cross section, with the N-DRC enlarged (adapted from [30]). (B) Photomicrographs of CCDC65 immunofluorescent staining in nasal epithelial cells revealing cytoplasmic and axonemal localization of the protein in normal cells compared to decreased expression in epithelial cells collected from the PCD subject (CCDC65, red; acetylated α-tubulin, green; DAP, blue), Scale bar = 10 µm.
Figure 4.
The effect of CCDC65 shRNA on cilia beat frequency of airway epithelial cells.
(A) Mean (± standard deviation) of cilia beat frequency in nasal cells from non-PCD (NL) and PCD subject (PCD). (B) Mean cilia beat frequency in cells transduced with different targeted CCDC65 shRNA sequences (shown are the mean ± standard deviation of n = 50 fields, p<0.0001; ANOVA).
Figure 5.
CCDC65 is involved in nexin-dynein regulatory complex (DRC) assembly.
Photomicrographs of normal airway epithelial cells following immunofluorescent staining for GAS8, a component of the N-DRC, which revealed ciliary localization of the protein but markedly reduced levels in nasal epithelial cells from the PCD subject (GAS8, red; acetylated α-tubulin, green; DAPI, blue). Scale bar = 10 µm.