DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes

Abstract

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Keywords: immunofluorescence microscopy; left–right dynein; normal ciliary ultrastructure; primary ciliary dyskinesia; transmission electron microscopy.

Figure 1.

Diagnostic transmission electron microscopy (TEM) is not useful in approximately 30% of primary ciliary dyskinesia (PCD) cases. (A) Schematic of respiratory cell cilia and its cross-section highlighting the “9 + 2” organization of the ciliary axoneme and associated structures, as visualized by TEM. (B) TEM is diagnostic for loss of outer dynein arms (ODAs; e.g., DNAH5 mutations), microtubular disorganization with nexin-link/dynein regulatory complex (N-DRC) and inner dynein arm (IDA) defects (e.g., CCDC39 mutations), and loss of both ODAs and IDAs (e.g., DNAAF3 mutations). (C) TEM is not diagnostic for DNAH11 mutations, as they are indistinguishable from healthy control axonemes. Blue arrows, ODAs; orange arrows, absent ODAs; blue circles, IDAs; orange circles, absent IDAs. Scale bar: 100 nm. Individuals with PCD OI-16 II2 (DNAH5; p.Gln1464* hom.) and OP-1482 (CCDC39, c.610-2A>G hom.) were identified by whole-exome sequencing; the DNAH5, p.Gln1464* variant is novel and not present in the 1,000 Genomes and ExAC databases (sequencing data not shown); the CCDC39, c.610–2A>G variant was previously described (40). Individuals with PCD OP-98 II1 (DNAH11 p.Trp2604*, p.Ile4445Asnfs*3 het.) and OP-235 II2 (DNAH11 [b] p.Gln4233*, p.Leu4327Ser het.) as well as OP-989 II2 (DNAAF3, p.Leu108Pro hom.) were reported previously (14, 26).

Figure 2.

DNAH11 localizes exclusively to the proximal region of respiratory cilia. (A) Schematic of DNAH11 protein domains that are conserved among other DNAH proteins per the Conserved Domain Database. The P-loop region contains several ATPase domains as well as the microtubule binding (MTB) domain. DHC-N, dynein heavy chain, N-terminal region; DYN, dynein heavy chain/region D6 of dynein motor. Red highlight, the epitope of DNAH11 spanning amino acids 69–196. (B) Anti-DNAH11Western blotting of porcine axonemal extracts indicates immunoreactive bands of approximately 500 kD. M, marker lane; 1, silver stain of lysate; 2, anti-DNAH11. (C) Immunofluorescence microscopy (IFM) of human control respiratory cells shows that native DNAH11 (green) localizes to the proximal region of ciliary axonemes; α/β-tubulin (red) was used as counterstain to detect ciliary axonemes. D, distal; P, proximal; scale bar: 10 µm. (D) IFM of mouse respiratory cells expressing green fluorescent protein (GFP)-DNAH11 (GFP–left–right dynein [lrd]) demonstrates that GFP-DNAH11 localizes to the proximal region of ciliary axonemes, similar to the pattern of human DNAH11 localization. Acetylated α-tubulin was used as counterstain to detect ciliary axonemes (red). Scale bar: 10 μm. (E) In contrast to mouse respiratory cilia, GFP-DNAH11 localizes throughout the entire axoneme in motile monocilia of the left–right organizer (I) in early mouse embryos but is not detectable in nonmotile cilia of the node or primary cilia of endodermal cells (II). Body axis is specified as right (r), left (l), anterior (a), and posterior (p). Scale bars in (I) and (II): 10 μm. ac., acetylated.

Figure 3.

DNAH11 is absent in respiratory cilia of individuals with PCD with a homozygous DNAH11 splice-site mutation. (A) Pedigree of the consanguineous PCD family OP-327. PCD siblings are shaded black, and the unaffected sibling is shaded white. *Situs inversus totalis. (B) Genome-wide linkage analysis of family OP-327 indicates chromosome 7 (x-axis represents genomic position in centimorgans or cM) as the only significant logarithm of odds (LOD) score (y-axis); this genomic interval contains DNAH11 (red arrow). (C) Sanger sequencing confirmed a homozygous splice donor variant in DNAH11 (intron 73, c.11739 + 1G>A) in four PCD siblings (OP-327 II1–II4). The unaffected parent (OP-327 I2) and sibling (OP-327 II5) are heterozygous (A/G) for this variant, whereas wild-type DNA shows the G nucleotide at this position. The triplet nucleotide sequence (color) is shown above the translated amino acid sequence (single letters). (D) IFM shows that, in contrast to healthy human respiratory cells, DNAH11 (red) is absent in PCD siblings OP-327 II1–II4. α/β-tubulin (green) is used as counterstain to detect ciliary axonemes. Scale bar: 10 µm.

Figure 4.

DNAH11 is absent in respiratory cilia of individuals with PCD with both homozygous nonsense and homozygous or compound heterozygous missense DNAH11 mutations. We confirmed additional novel DNAH11 loss-of-function mutations (see Figures E3–E7 and Tables E2 and E3) and determined that, in contrast to healthy control (A), these cilia lack DNAH11 (B–F). α/β-tubulin (green) is used as counterstain to detect ciliary axonemes. Scale bars: 10 µm.

Figure 5.

DNAH11 and DNAH9 distinctly localize to proximal and distal regions of respiratory cilia, respectively. (A) IFM shows that DNAH9 (green) localizes to the distal region of respiratory cilia; DNAH5 (red) localizes throughout the entire cilium. Scale bar: 10 µm. (B) IFM shows that DNAH11 (purple) localizes distinctly in the proximal region of respiratory cilia in contrast to DNAH9 (green), which localizes distinctly in the distal region of respiratory cilia. Scale bar: 10 µm. (C) A schematic shows that DNAH11 and DNAH9 localize to distinct regions in respiratory cilia, thus defining distinct proximal and distal ODA subtypes. (D) Model of hyperkinetic ciliary beating. Whereas healthy respiratory cilia display an average beat frequency of approximately 6 Hz, cilia from individuals with DNAH11 mutations have an average beat frequency above 9 Hz, owing to reduced bending in the proximal ciliary region (see Figure E10 and Videos E1–E8). Blue shades indicate forward effective stroke; gray shades indicate recovery stroke. wt, wild type.

Figure 6.

DNAH11 localizes to the proximal region of respiratory cilia in individuals with PCD with mutations affecting ODA and IDA assembly. Similar to healthy control respiratory cells (A), DNAH11 localizes to the proximal region of respiratory cilia in individuals with PCD harboring homozygous nonsense mutations in DNAH5 (B) and DNAI2 (C) that lack ODA structures, as well as individuals with PCD harboring homozygous loss-of-function mutations in DNAAF1 (D) and DNAAF3 (E) that lack both ODA and IDA structures. Anti-DNAH11 (green) is costained with α/β-tubulin (red) to detect ciliary axonemes. Scale bar: 10 µm. Individuals with PCD OP-254 II1, OP-250 II1 and OP-989 II1 were reported previously (5, 25, 26). TEM of OI-16 II2 and OP-989 II1 are shown in Figure 1B. DIC, differential interference contrast microscopy.

Figure 7.

TEM tomography detects a partial reduction of ODAs in the proximal, but not distal, region of DNAH11-deficient cilia. Tomographic reconstruction of ciliary cross-sections from (A) wild-type (WT) and (B and C) DNAH11-deficient cilia (OP-1250 II1; see also Figure 4B and Figures E3 and E14, and Videos E1–E4) identify a partial reduction of ODAs (white arrowheads) in the proximal (B), but not distal (C), region of DNAH11-deficient cilia. Black arrowheads, structured ODAs; black arrows in B indicate the electron-dense material sometimes visible between microtubule doublets in the proximal ciliary region, approximately 400 nm distal to the transition zone and 100 nm distal to the base of the central pair microtubules. Tomographic reconstructions of ciliary longitudinal sections from (D) WT and (E and F) DNAH11-deficient cilia (OP-1250 II1) also identify a partial reduction of ODAs (white arrowheads) in the proximal (E), but not distal (F), region of DNAH11-deficient cilia. Scale bars: 50 nm.