ZMYND10 stabilizes intermediate chain proteins in the cytoplasmic pre-assembly of dynein arms

Abstract

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.

Fig 1. Zmynd10^−/− mice exhibited phenotypes consistent with cilia motility defects.

(A, B) Unlike Zmynd10^+/+ mice (A), Zmynd10^−/− mice (B) exhibited characteristic head deformation resulting from severe hydrocephalus. (C, D) The coronal brains sections showed enlarged ventricular cavity and reduced cortical thickness in Zmynd10^−/− mice. (E, F) Hematoxylin and eosin (H&E) staining of the coronal brain sections showed expanded ventricles in Zmynd10^−/− mice. Scale bars, 1 mm. (G, H) Alcian Blue staining of the paranasal cavities showed mucus congestion along the nasal epithelium in Zmynd10^−/− mice. Scale bars, 1 mm. (I–L) H&E staining and Masson’s trichrome staining of Zmynd10^+/+ (I, K) and Zmynd10^−/− (J, L) mouse lung sections at P29 showed marked interstitial widening due to inflammatory infiltrates and edema. Original magnification, 40× (scale bar, 500 μm), 100×, and 200× (scale bar, 100 μm).

Fig 2. Zmynd10^−/− mice exhibited ODA and IDA defects.

(A–D) TEM analysis of Zmynd10^+/+ and Zmynd10^−/− multiciliated cells from the trachea of P14 mice. The number and morphology of cilia and basal bodies were similar between Zmynd10^−/− (B, D) and Zmynd10^+/+ (A, C) mice. Scale bars, 500 μm. (E, F) TEM micrographs of the motile cilia axonemes showing normal 9+2 microtubule structures in both Zmynd10^+/+ and Zmynd10^−/− mice. Scale bars, 500 μm. (G, H) ODAs and IDAs (white arrows) were observed in the peripheral microtubule doublets of motile cilia in Zmynd10^+/+ mice (G); however, ODAs and IDAs were absent in ciliary axonemes of Zmynd10^−/− mice (H, black arrows). Scale bars, 500 μm. (I, J) Cilia (I) and basal bodies (J) were counted in TEM images of tracheas of Zmynd10^+/+ and Zmynd10^−/− mice. (K, L) Immunofluorescence analysis of acetylated α-tubulin (Ac-α-tub, green) and DNAI2 (red) expression in mTEC cultures at ALI day 14. DNAI2 was significantly decreased and did not colocalize with acetylated α-tubulin in Zmynd10^−/− mTECs, suggesting that motile cilia lacked ODAs (J). Scale bars, 10 μm.

Fig 3. ZMYND10 did not regulate mRNA expression of dynein arm components.

(A–C) Graphs comparing reads per kilobase of transcript per million mapped reads (RPKM) of ODA and IDA components. Total RNA was isolated from the P21 testis (A), P14 lung (B), and P14 brain (C) tissues from Zmynd10^+/+ and Zmynd10^−/− mice. The expression of most dynein arm components did not differ between wild-type and mutant mice.

Fig 4. ZMYND10 formed a cytoplasmic protein complex.

(A) GST pulldown assays of the purified MYND domain of ZMYND10 or the full-length protein. DNAI1, IQUB, REPTIN, LRRC6, and C21ORF59 in mTECs were pulled down by full-length ZMYND10, but not by the MYND domain alone. (B) Interactors of ZMYND10. Dotted lines are interactions identified in this study, whereas solid lines are from previous studies. (C, D) Immunofluorescence analysis of DNAI2 (green) or acetylated α-tubulin (Ac-α-tub, green) and C21ORF59 (red) in mTECs. At ALI day 14, C21ORF59 was observed in the cytoplasm whereas DNAI2 was localized in both cytoplasm and cilia in Zmynd10^+/+ mTECs. DNAI2 and C21ORF59 were downregulated in Zmynd10-deficient mTECs. Scale bars, 10 μm. (E) Immunofluorescence analysis of Ac-α-tub (green) and REPTIN (red) in mTECs. REPTIN was localized in the cytoplasm of Zmynd10^+/+ mTECs, but was absent in Zmynd10^−/− cells. Scale bars, 10 μm.

Fig 5. Dynein arm subunits and interaction partners of ZMYND10 were downregulated in Zmynd10^−/− mice.

(A) Representative immunoblot analyses of DNAH7, DNAI2, DNAI1, IQUB, LRRC6, C21ORF59, and ZMYND10 in the testis extracts from Zmynd10^+/+ and Zmynd10^−/− mice. (B) Bar graphs represent band intensities of the blot shown in panel A, and data represent the mean ± SD of more than three independent experiments. Band intensities were normalized to β-actin. *P < 0.05; **P < 0.005; t-test. (C–E) Immunofluorescence analysis of tracheal multiciliated epithelia in Zmynd10^+/+ and Zmynd10^−/− mice. (C) DNAH5 (red), an ODA heavy chain protein, localized to both cilia and cytoplasm of the Zmynd10^+/+ trachea, but was almost completely absent from the Zmynd10^−/− trachea. DNAI2 (red) was present in both the cilia and cytoplasm in the Zmynd10^+/+ trachea and colocalized with acetylated α-tubulin (Ac-α-tub) along cilia. However, DNAI2 expression was negligible in the Zmynd10^−/− trachea (D). IQUB (red), a cytoplasmic protein and ZMYND10 interaction partner, was almost undetectable in the Zmynd10^−/− trachea (E). Scale bars, 10 μm. (F–H) Graphs comparing reads per kilobase of transcript per million mapped reads (RPKM) of dynein arm assembly factors and ZMYND10-interacting proteins in the testis (F), lung (G), and brain (H) tissues of Zmynd10^+/+ and Zmynd10^−/− mice.

Fig 6. ZMYND10 stabilized LRRC6 and intermediate chain proteins of ODA.

The stability of LRRC6, DNAI1, and DNAI2 proteins was examined using protein stability assays. Protein samples were harvested at the indicated times after treatment with cycloheximide (100 μg/mL).(A, B) Representative immunoblots of LRRC6 stability assays (A). The stability of LRRC6 was increased upon co-expression of ZMYND10 (B). (C, D) Representative immunoblots of DNAI1 stability assays (C). DNAI1 stability was increased by co-expression of ZMYND10 (D). (E, F) Representative immunoblots of DNAI2 stability assays (E). DNAI2 was stabilized by co-expression of both DNAI1 and ZMYND10 (F). Data are representative of at least three independent experiments, and band intensities were normalized to β-actin. *P < 0.05; **P < 0.005; t-test.