Mislocalization of DNAH5 and DNAH9 in respiratory cells from patients with primary ciliary dyskinesia

Abstract

Rationale: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by recurrent infections of the airways and situs inversus in half of the affected offspring. The most frequent genetic defects comprise recessive mutations of DNAH5 and DNAI1, which encode outer dynein arm (ODA) components. Diagnosis of PCD usually relies on electron microscopy, which is technically demanding and sometimes difficult to interpret.

Methods: Using specific antibodies, we determined the subcellular localization of the ODA heavy chains DNAH5 and DNAH9 in human respiratory epithelial and sperm cells of patients with PCD and control subjects by high-resolution immunofluorescence imaging. We also assessed cilia and sperm tail function by high-speed video microscopy.

Results: In normal ciliated airway epithelium, DNAH5 and DNAH9 show a specific regional distribution along the ciliary axoneme, indicating the existence of at least two distinct ODA types. DNAH5 was completely or only distally absent from the respiratory ciliary axoneme in patients with PCD with DNAH5- (n = 3) or DNAI1- (n = 1) mutations, respectively, and instead accumulated at the microtubule-organizing centers. In contrast to respiratory cilia, sperm tails from a patient with DNAH5 mutations had normal ODA heavy chain distribution, suggesting different modes of ODA generation in these cell types. Blinded investigation of a large cohort of patients with PCD and control subjects identified DNAH5 mislocalization in all patients diagnosed with ODA defects by electron microscopy (n = 16). Cilia with complete axonemal DNAH5 deficiency were immotile, whereas cilia with distal DNAH5 deficiency showed residual motility.

Conclusions: Immunofluorescence staining can detect ODA defects, which will possibly aid PCD diagnosis.

Figure 1.

Specific antibodies localize the human axonemal outer dynein arm heavy chain DNAH5 to the ciliary axoneme, the microtubule-organizing centers, and cytosolic compartments of respiratory epithelial cells. (A) Silver staining (left) and Western blot analysis (right) of axonemal protein extracts from pig trachea. High molecular mass bands (about 500 kD) represent the axonemal dynein heavy chains. DNAH5 antibodies specifically detect a single band with the predicted size. (B–D) Immunofluorescence staining of human respiratory epithelial cells with DNAH5-specific antibodies (red). Costaining with antibodies against axoneme-specific acetylated α-tubulin (C) and γ-tubulin, which stains the microtubule-organizing centers in the vicinity of the basal body (D), was used as control (green). Nuclei were stained with Hoechst 33342 (blue). Overlays and bright-field images are shown on the right in (C) and (D).

Figure 2.

DNAH9 localizes exclusively to distal parts of respiratory ciliary axonemes: costaining of human respiratory epithelial cells with antibodies against axonemal ODA heavy chains DNAH9 (green) and DNAH5 (red). DNAH5 localizes along the entire length of the axonemes, within the microtubule-organizing centers, and specific cytosolic compartments. Nuclei were stained with Hoechst 33342 (blue). (A) DNAH9 staining; (B) DNAH5 staining; (C) overlay; and (D) bright-field image.

Figure 3.

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.

Figure 4.

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.