High-speed video microscopy of respiratory cells from individuals OP-2905 II1 and OP-1226 II1 revealed motile cilia with normal ciliary beat frequency (approx. 7 Hz at 25°C). However, we found abnormal beat pattern with reduced bending of the distal ciliary axoneme (Videos S1, S2, S3, and S4 and Figure 3A) when compared to controls (Video S5 and Figure 3A) consistent with DNAH9 localization and function in distal respiratory ciliary axonemes. The very subtle beating defect is also consistent with the observed mild respiratory symptoms in DNAH9 mutant individuals. This ciliary beating defect appears to be more subtle when compared to defects caused by DNAH11 and DNAH5 mutations (Figure 3A and videos S6 and S7).
Figure 3 Schematics Depicting the Respective Aberrant Beating Patterns Caused by Recessive DNAH9, DNAH11, and DNAH5 Mutations and Localization of the Axonemal Outer Dynein Arm β-Heavy Chains (ODA HC) DNAH9 and Paralogous DNAH11 and the ODA γ-HC DNAH5 in Respiratory Cilia
(A) Schematic of representative respiratory ciliary beating defects caused by loss-of-function mutations in DNAH9, DNAH11, and DNAH5. Whereas ciliary beating in healthy individuals demonstrate effective beating (gray) and recovery (black) strokes to move mucus in the airways (arrow), DNAH9 and DNAH11 mutant cilia show reduced bending in the distal and proximal ciliary regions, respectively. DNAH5 mutant cilia are immotile with residual flickering movements.
(B and C) The ODA β-HC DNAH9 (green) localizes to the distal part of the ciliary axoneme and the ODA γ-HC DNAH5 (red) localizes to the whole ciliary axoneme. In contrast, the ODA β-HC DNAH11 (magenta) localizes to the proximal part of the ciliary axoneme. The overlapping front (white) results from the bending of the cilia. Scale bars represent 10 μm.
(D) Profiles of immunofluorescence intensity show an increase in the magenta signal in the proximal ciliary axoneme and an increase of the green signal in the distal ciliary axoneme. Both signals do not overlap indicating a distinct distal and proximal localization of β-heavy chain paralogs DNAH9 (green) and DNAH11 (magenta), respectively.
(E) ODA type 1 (DNAH5 and DNAH11) and ODA type 2 (DNAH5 and DNAH9) complexes define distinct compartments in respiratory cilia.