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. 2013 Mar;45(3):262-8.
doi: 10.1038/ng.2533. Epub 2013 Jan 27.

The Nexin-Dynein Regulatory Complex Subunit DRC1 Is Essential for Motile Cilia Function in Algae and Humans

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Free PMC article

The Nexin-Dynein Regulatory Complex Subunit DRC1 Is Essential for Motile Cilia Function in Algae and Humans

Maureen Wirschell et al. Nat Genet. .
Free PMC article

Abstract

Primary ciliary dyskinesia (PCD) is characterized by dysfunction of respiratory cilia and sperm flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the nexin-dynein regulatory complex (N-DRC), an axonemal structure critical for the regulation of dynein motors, and show that mutations in the gene encoding DRC1, CCDC164, are involved in PCD pathogenesis. Loss-of-function mutations disrupting DRC1 result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas reinhardtii and humans. Our results highlight a role for N-DRC integrity in regulating ciliary beating and provide the first direct evidence that mutations in DRC genes cause human disease.

Conflict of interest statement

Author information: Chlamydomonas DRC1 sequences have been deposited into GenBank with the following accession numbers: Chlamydomonas DRC1 cDNA; JX311620; Chlamydomonas DRC1 protein; accession # AFU81554. The Human DRC1 protein (also known as C2orf39 and CCDC164) is NP_659475.2. Reprints and permissions information is available at www.nature.com/reprints. The authors claim no competing financial interests.

Figures

Figure 1
Figure 1. Localisation of mutations within Chlamydomonas and humans
Diagram of the DRC1 subunit in Chlamydomonas (top) and its human orthologue CCDC164 (bottom). The coiled coil motifs are drawn in dark grey, the positions of the mutations identified in algae and human are indicated with arrows.
Figure 2
Figure 2. The Chlamydomonas pf3 mutant is defective in DRC1 resulting in disruption of N-DRC integrity
(a) Western blot analysis of WT and Chlamydomonas mutant axonemes defective in the outer arm (pf28, sup-pf-2), inner arms (pf9, ida4) and N-DRC (pf2 (DRC4), pf3 (DRC1), sup-pf-3, sup-pf-4 (DRC5)) reveal that DRC1 is specifically missing in the drc- mutant pf3. Note, longer exposures of pf3 mutant axonemes reveals a very faint band that is missing in wild-type (not shown) suggesting that re-initiation of translation at a downstream methionine may occur at very low levels in the pf3 mutant. (b) Analysis of ciliary extracts reveal that DRC1 is extracted from the axoneme using 0.4–0.6 M NaI in WT, along with other DRC subunits (DRC4 shown). In the drc mutants, pf2 and pf3, the residual N-DRC structure is extracted more readily as shown by its release from the axoneme using high salt buffers (0.6 M NaCl) or lower molarity NaI buffers (0.2 M NaI). (c) Velocity sedimentation of NaI extracts from wild-type (PF2-HA) on sucrose density gradients reveal that the N-DRC (DRC1 and DRC4) sediments as a very large complex (>19S). Sedimentation of RSP16 of the radial spokes is shown as a control. In contrast, in the drc- mutants, pf2 and pf3, the residual N-DRC structure remaining in these mutant axonemes is readily disrupted. DRC1 sediments at ~19S in pf2, and DRC4 sediments at ~2S in pf3. (d) The drc1 mutation in pf3 results in altered levels of inner arm components (p44, p38, p28, and centrin), tektin and the CSC (CaM-IP3). Antibodies to DHC5, DHC9 and DHC11 suggest that these inner arm heavy chains are not significantly reduced in the pf3 mutant. There are no observable defects in the levels of the radial spokes (RSP16), dynein f (IC140), CCDC39 or Rib72.
Figure 3
Figure 3. Mutations in the CCDC164 gene in humans result in defects in the N-DRC links
(a) Schematic diagram of the 9+2 cilium and the N-DRC structure (modified after Lin et al. 2011) enlarged to show the predicted locations of N-DRC subunits. Transmission electron microscopy of respiratory cilia shows normal axonemal structure in the control (b) and normal tubular organization in CCDC164-mutant cilia (c, d). N-DRC links connecting outer doublets are depicted with arrows in the control (b). In patients OP-26II1 and OP-59II2 with homozygous nonsense CCDC164 mutations (c, d) the N-DRC links are missing (N = 12). Black scale bars (a–c) represent 0.2μm.
Figure 4
Figure 4. The CCDC164- mutations result in defective N-DRC assembly in respiratory cilia
High-resolution immuno-fluorescence analysis of the subcellular localization of GAS11 (DRC4) and LRRC48 (DRC3) in respiratory cells from controls (a and d) and PCD patients OP-26II1 (b), OP-39II1 (c and e) and OP-59II2 (f) carrying mutations in the CCDC164 gene. Axoneme-specific antibodies against α/β-tubulin (red, a, b and c) and acetylated α-tubulin (green, d, e and f) were used as axonemal control. Nuclei were stained with Hoechst 33342 (blue). In respiratory epithelial cells from controls, GAS11 (green, a) and LRRC48 (red, d) localize to the entire length of the axonemes. In respiratory epithelial cells from the patients carrying CCDC164-mutations, GAS11 (shown for patients OP-26II1 (b) and OP-39II1 (c) and LRRC48 (shown for patients OP-39II1 (e) and OP-59II2 (f) were completely absent from the ciliary axonemes. The yellow co-staining within the ciliary axoneme (a and d) indicates that both proteins co-localize within respiratory cilia. White scale bars indicate 10μm.
Figure 5
Figure 5. Comparison of axonemal bending patterns in Chlamydomonas and human respiratory cilia
(a) The beating pattern of pf3 mutant cilia (defective in DRC1) exhibits reduced amplitude and bending. (b) Respiratory cilia from individuals with mutations in the CCDC164 gene (orthologue of DRC1) are also reduced in the amplitude (grey) and appear stiff when compared to wild type. CCDC164 mutant cilia show a less severe phenotype compared to CCDC39 or CCDC40 mutants, which are characterized by strongly reduced amplitude and stiff and rigid cilia. (Black = effective stroke, Grey = recovery stroke). Illustrations in (a) are adapted and modified from Brokaw and Kamiya, 1987.

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