PMC

(A) Representative motile cilia area map from Dnah5^–/– mice indicates robust ciliary motion but at a lower CBF than demonstrated in B. (B) Representative ciliary motile area map from Dnah5^+/+ littermate control mice (n = 5). Colored pixels indicate areas of active cilia movement at varied CBFs. (C) Quantification of the percentage of motile ciliated areas in Dnah5^–/– mice compared with Dnah5^+/+ littermate control mice. P = NS. (D and E) Representative waveform plots from Dnah5^+/+ and Dnah5^–/– mice, respectively, indicating diminished ciliary activity at the ROI examined (n = 5 mice). “Slice” refers to the ROI of ciliary displacement. (F) Cilia demonstrate normal arc angle. P = NS. (G) CBF is reduced in Dnah5^–/– mice compared with Dnah5^+/+ littermate control mice. ****P < 0.0001. (H) Reduced CBF results in diminished ESR in Dnah5^–/– mice. (I and J) Reduced fluid transport is observed in Dnah5^–/– mice (I) compared with controls (J). (K) As a result of reduced CBF but not reduced ciliated area, Dnah5^–/– mice demonstrate impaired mucociliary clearance. *P < 0.05. n = 6 Dnah5^+/+ animals and 5 Dnah5^–/– animals; 4 ROIs per trachea. All data are represented as mean (line) with minimum and maximum boundaries displayed. Statistical significance determined by Mann-Whitney t test. Scale bar: 10 microns (two dimensions, A and B); 10 micron by 10-second images (I and J).

Mislocalization of ODA components in respiratory epithelial cells from patients with PCD with DNAH5 and DNAI1 mutations: overlay images of respiratory epithelial cells from patients with PCD carrying the DNAH5 mutations (A) 1855NfsX5, (B) 2814fsX1, and (C) IVS74-1G→C, costained with antibodies against DNAH5 (red) and acetylated α-tubulin (green). Nuclei were stained with Hoechst 33342 (blue). Mutant DNAH5 (A–C) is expressed and localizes correctly to the ODA preassembly site at the microtubule-organizing centers but is unable to enter the ciliary compartment. Note that the mutant protein accumulates within the cytosolic DNAH5 compartments including the proximal perinuclear region and the expanded microtubule-organizing centers. (D) Colocalization of DNAH5 (red) and γ-tubulin (green) in respiratory epithelial cells from a patient with PCD with mutant DNAH5 (1855NfsX5), demonstrating expansion of the microtubule-organizing centers. The overlay (yellow) is shown at the top. (E) Overlay image of respiratory cells from a patient with the DNAH5 mutation 1855NfsX5. DNAH9 (green) is absent from the ciliary axonemes. Only weak and diffuse DNAH9 staining is detected within the cytoplasm. DNAH5 (red) is also absent within the ciliary axoneme but accumulates in the microtubule-organizing centers. (F) Overlay image of a respiratory cell from a patient with a mutation of the ODA intermediate chain gene DNAI1 (219+3insT/W568X). The ciliary axoneme is stained with acetylated α-tubulin (green). DNAH5 (red) is undetectable in the distal part of the axonemes but accumulates in the cytoplasmic compartments. Yellow within the proximal ciliary axoneme indicates that both proteins are present in these regions.

The spatial distribution of ODA heavy chains DNAH5 and DNAH9 in sperm tails remains unchanged in a patient with PCD carrying a homozygous DNAH5 mutation. (A and B) Spermatozoa were costained with antibodies against DNAH5 (red) and acetylated α-tubulin (green). (C and D) Costaining of DNAH5 (red) and DNAH9 (green). (A and C) Sperm cells from a healthy donor. (B and D) Sperm cells from PCD patient F373-II3. Overlay and bright-field images are shown on the right. DNAH5 localizes exclusively to the proximal part of the sperm tail, whereas DNAH9 localizes to the entire sperm axoneme in both wild-type and DNAH5 mutant sperm. In contrast to respiratory cilia, the distribution of ODA heavy chains in sperm axonemes is not influenced by the DNAH5 mutation in F373-II3.

Mis-localization of mutant DNAH5 in respiratory epithelial cells from patients with primary ciliary dyskinesia carrying DNAH5 mutations. Confocal immunofluorescence analysis of human respiratory epithelial cells with anti-DNAH5 antibodies (red) and with antibodies against axoneme-specific acetylated α-tubulin (green) as control (). Nuclei were stained with Hoechst 33342 (blue). (A) In respiratory epithelial cells from healthy probands, DNAH5 localizes along the entire length of the axonemes and at the microtubule organizing centers. (B) In respiratory epithelial cells from patient OP40 II1, carrying the compound heterozygous DNAH5 mutations R1716L and 4487NfsX1, mutant DNAH5 is expressed and correctly targeted to the ciliary base where it accumulates. In contrast to healthy control subjects, we observed complete absence of mutant DNAH5 from the ciliary axonemes. (C) In Patient F718 II1, who carries compound heterozygous DNAH5 mutations affecting splicing at the 3′-end of the gene, mutant DNAH5 is absent from the distal part of the ciliary axonemes but is still detectable within the proximal part.