Undocking of an extensive ciliary network induces proteostasis and cell fate switching resulting in severe primary ciliary dyskinesia

Abstract

Primary ciliary dyskinesia is a rare monogenic syndrome that is associated with chronic respiratory disease, infertility, and laterality defects. Although more than 50 genes causative of primary ciliary dyskinesia have been identified, variants in the genes encoding coiled-coil domain-containing 39 (CCDC39) and CCDC40 in particular cause severe disease that is not explained by loss of ciliary motility alone. Here, we sought to understand the consequences of these variants on cellular functions beyond impaired motility. We used human cells with pathogenic variants in CCDC39 and CCDC40, Chlamydomonas reinhardtii genetics, cryo-electron microscopy, and proteomics to define perturbations in ciliary assembly and cilia stability, as well as multiple motility-independent pathways. Analysis of proteomics of cilia from patient cells identified that the absence of the axonemal CCDC39/CCDC40 heterodimer resulted in the loss of a network of more than 90 ciliary structural proteins, including 14 that were defined as ciliary address recognition proteins, which provide docking for the missing structures. The absence of the network impaired microtubule architecture, activated cell quality control pathways, switched multiciliated cell fate to mucus-producing cells and resulted in a defective periciliary barrier. In CCDC39 variant cells, these phenotypes were reversed through expression of a normal CCDC39 transgene. These findings indicate that the CCDC39/CCDC40 heterodimer functions as a scaffold to support the assembly of an extensive network of ciliary proteins, whose loss results in both motility-dependent and motility-independent phenotypes that may explain the severity of disease. Gene therapy might be a potential treatment option to be explored in future studies.

Fig. 1.. Cells with CCDC39 and CCDC40 variants show a loss of ciliary integrity

(A) Diagram of human multiciliated cell, motile cilium cross-section with nine microtubule doublets (DMT; A and B) linked to the major complexes, inner and outer dynein arms (IDA, ODA), radial spokes (RS), nexin dynein regulatory complex (N-DRC), central pair apparatus (CPA). The area within the dashed rectangle is shown in more detail in Fig. 1B. (B) Diagram of a 96-nanometer unit showing the CCDC39/CCDC40 heterodimer (shown as the yellow and orange ribbon) on the DMT with associated ciliary complexes as in Fig1A, including Radial spokes 1,2,3 (RS1, RS2, RS3) (C) Schematic showing the sources of primary airway epithelial cells and subsequent culture at air-liquid interface (ALI) to produce multiciliated cells. (D) Immunoblot of CCDC39, CCDC40, and FOXJ1 from normal primary airway epithelial cells obtained from a healthy donor and cultured at ALI. Actin served as the loading control. (E) Immunofluorescence images of CCDC39 (magenta) and CCDC40 (green) staining in normal and variant primary airway epithelial cells at ALI culture day 28. Nuclei are stained blue. Scale bar=10 μm (CCDC39-variant cells were from WU182 and CCDC40-variant cells were from WU146; these were also used in Fig. 1, E, F, and H to L). (F) Immunoblot of CCDC39 and CCDC40 comparing normal (lane 1) and variant cells (lanes 2, 3). (G) Immunoblot of CCDC39 in Chlamydomonas wild-type, ccdc39, and ccdc40 mutants (n=2). (H) Graph displaying cilia beat frequency in normal (n=1 normal donors),CCDC39 and CCDC40 variant cells. Each point represents an average of a single microscopic field at 10x magnification from 5 fields of 5 technical replicate samples. (I) Graph displaying cilia transport of microbeads, measured in μm, on the surface of well-differentiated normal (n=1), CCDC39 and CCDC40 variant cells. Each point represents one bead from technical replicates. (J) Immunofluorescence of acetylated α-tubulin (Ace-TUB) in cilia isolated from human normal and CCDC39 and CCDC40 variant cells. Detail shows splaying cilia in variants. Scale bar=10 μm (left) and 5 μm (right) (K) Quantification of cilia length from Fig. 1J; n=47–50 cilia measured from normal (n=3), CCDC39 and CCDC40 variant cells. Each point represents a cilium. (L) Transmission electron microscopy (TEM) of cilia from normal and CCDC39 and CCDC40 variant cells showing the microtubules in longitudinal (top) and cross-section (bottom). Bar=500 nm (top) and 100 nm (bottom). Yellow arrows indicate splayed microtubule doublets. (M) TEM of cross-sections of cilia isolated from Chlamydomonas wild-type, ccdc39, and ccdc40. Yellow asterisks indicate disorganized DMT. White arrows indicate an opening of the B-microtubule. (N) Quantification of open B-microtubules from Fig. 1M; n=200 cilia of each genotype. The total loss of the central apparatus in the ccdc39 and ccdc40 mutant strains is significantly different from wild-type (p<0.0001) by chi-squared and Fisher’s exact testing. (O) Diagram of a DMT cross-section showing the ciliary complexes and the location of the CCDC39/CCDC40 heterodimer. The area within the dashed rectangle is shown at 90⁰ in Fig. 1P. The abbreviations are defined above. (P) Three-dimensional structure of the Chlamydomonas axoneme resolved by cryo-EM using Chimera (v.1.13) showing the CCDC39/CCDC40, heterodimer and associated proteins. The N terminus of CCDC39/40 is labeled (N). The C-terminus (C) extends from the A- to the B-tubule. Bar=10 nm. (Q) Predicted conformation of CCDC39/40 C-terminus hook region generated in AlphaFold. The inner junction of DMT contains FAP20 and PARCG. L845 is the mutant residue in the temperature-sensitive ccdc39 Chlamydomonas mutant. Images in D, E, F, and J are representative of at least three independent experiments. Genotypes in E, F, and H to L: CCDC39, WU182; CCDC40; WU146. In H, I, and K, the bar indicates the median Differences between groups were determined using the Kruskal-Wallis test followed by Dunn’s multiple comparison test; **p<0.01, ***p<0.001.

Fig. 2.. The CCDC39/40 heterodimer is required for assembly of a ciliary protein network via CARPs.

(A) Schematic of cilia isolation from cells from normal (n=3 independent donors) and CCDC39 variant (n=1, WU182, with two independent preparations) donors. Cells were cultured under ALI conditions for over six weeks and the cilia were removed from the apical surface by detergent treatment. The cilia were isolated by differential centrifugation and analyzed by mass spectrometry. Peptide numbers were determined from samples of equivalent protein concentration and were normalized to account for the short axonemes in CCDC39 variants. (B) Immunofluorescence of Ace-TUB (green) and centrin (CETN1, magenta) in normal cells before and after treatment with detergent and cilia isolation. Nuclei are stained blue. Scale bar=10 μm. (C) Heat map of mean abundance of proteins normalized for tubulin in CCDC39 variant cilia compared to normal cilia. Proteins with direct CCDC39/40 contact noted by presence of the blue box to the right. The color key scales from 0–500 peptides (white to red). (D). Ciliary structures and their CCDC39/40 ciliary address recognition protein (CARP). The location of each CARP was identified in published structural data from cryo-electron microscopy. (E) Diagram showing the relationship between CCDC39/40, CARPs and ciliary structures.

Fig. 3.. Ciliary radial spokes are differentially depleted in CCDC39 versus CCDC40 variant cilia.

(A) Diagram of Chlamydomonas (top) and human (bottom) radial spokes (RS) in a 96-nm repeat along the DMT. Chlamydomonas has two complete and one partial radial spoke, whereas humans have three complete radial spokes in the 96-nm repeat. RSPH3 is shared by all three radial spokes. Chlamydomonas and human CARPs are shown along the CCDC39/40 heterodimer. (B) Immunofluorescence of Ace-TUB (green), CCDC39 (magenta, top row), and radial spoke proteins (magenta, lower rows) in normal and CCDC39 (WU182) and CCDC40 (WU146) variant cells. Nuclei are stained blue. scale bar=5 μm. (C) Schematic of lentivirus mediated transfection of normal human basal cells with non-targeted or CCDC96 shRNA. Transduced cells are selected and cultured at ALI for at least 28 days. (D) Immunofluorescence of Ace-TUB (green), CCDC39 (magenta, top right), and radial spoke proteins (magenta, lower panels) in normal cells transduced with non-targeted or CCDC96-specific shRNA. Nuclei are stained blue. scale bar=5 μm. (E) TEM of isolated Chlamydomonas wild-type and ccdc39 null mutant axonemes. Repeating distances of radial spokes within a 96-nm repeat (32 nm, short bracket) and between repeats (96 nm, large bracket) are indicated. (F) Quantification of distances in Fig. 3D. n=200 cross-sections from the wild-type strain and n=100 from each mutant meiotic product in 2 biologic replicates. (G) Immunoblot of radial spoke head protein RSP16 shared by both complete radial spokes in Chlamydomonas.

Fig. 4.. The CCDC39/40 network is disrupted during cilia assembly in CCDC39 and CCDC40 variants.

(A) Schematic showing the stages of normal motile ciliogenesis in air-liquid interface (ALI) cultures. (B) Superresolution microscopy of CETN1 (magenta), CCDC39 (green), and (E-cadherin (CDH1, white) during basal body amplification. Early centriologenesis is shown on the left, late centriologenesis is shown on the right. The top panel is maximal projection immunofluorescence, the middle panel is three-dimensional rendering using Imaris software, the bottom panel is a xz reconstruction of the top panel. Scale bar=5 μm. (C) Immunofluorescence of CCDC39 (magenta) and CCDC40 (green) at pre-cilia (top) and emerging cilia (bottom) stages are shown in normal cells. Ace-TUB is shown in yellow. The white arrow indicates co-localization in cilia. Scale bar=5 μm. (D) Immunofluorescence of Ace-TUB (blue), CCDC39 (cyan), and CCDC40 (red) in 4x expanded normal cells, showing mature and emerging cilia. The white arrow indicates the cilia, and the white arrowhead indicates the basal body. Scale bar=10μm; inset, 5 μm. (E) Schematic of CARPs CCDC96 and CFAP57 located at the base of RS3, binding to CCDC39/40. Immunofluorescence in normal cells of Ace-TUB (yellow), CCDC40 (green) and CARPs CCDC 96 (top, magenta) or CFAP57 (bottom, magenta) in cells with emerging cilia. White arrows indicate co-localization. Scale bar=5 μm. (F) Schematic of network proteins CFAP100 binding near an IDA and CFAP61 located at the stalk of RS3. Immunofluorescence in normal cells of Ace-TUB (yellow), CCDC40 (green), and network proteins CFAP100 (top, magenta) or CFAP61 (bottom, magenta) in cells with emerging cilia. White arrows indicate network proteins, located in the cytoplasm below cilia. Scale bar=5 μm. (G) Schematic of network protein CFAP100 at the staging site during early ciliogenesis relative to basal body protein CETN1. Immunofluorescence of CFAP100 (magenta) and CETN1 (green) during early cilia growth (days 10–21 in ALI) in normal cells. Scale bar=5 μm. (H) Immunofluorescence of Ace-TUB (green), network members CFAP73, CFAP100, and MLF1 (all magenta) in normal and CCDC39 variant tissues collected from airways. Nuclei are stained blue. White arrowheads point to co-localization of proteins in the cilia of normal cells and the network protein below the cilia in the variant. Scale bar=5 μm. (I) Immunoprecipitation with IgG and CCDC40 antibodies (left) and CCDC40 in normal cells from early (ALI 14) and late (ALI 40) stages (right). (J) Immunoblot of CFAP73 and CFAP100 in normal, CCDC39, and CCDC40 variant cells (ALI>28d). Short (S) and long (L) exposures are shown. For this experiment, the immunoblot from Fig. 1F was reprobed. Panels B to I used cells from 2–3 unique normal donors and two variant genotypes (CCDC39, WU182; CCDC40, WU146).

Fig. 5.. CCDC39 and CCDC40 variants alter the proteasome and cell fate.

(A) Uniform manifold approximation and projection (UMAP) reductions of normal (n=6 donors) and CCDC39 and CCDC40 variants (CCDC39, WU182 and WU 157, CCDC40 WU146) cultures at ALI day 35, showing clusters for basal (bas); secretory (sec); multiciliated (cil); ionocytes (iono); dividing cells (div). (B) Dotplot showing differentially expressed genes of normal and variant cells in each cluster in Fig. 5A that show significantly increased expression in Cil3 in cells from pooled data from CCDC39 and CCDC40 variants. Classes of differentially expressed genes on the left column were identified by enrichment analysis (Ox phos, oxidative phosphorylation; Notch sig, Notch signaling and secreted proteins). The scale from red-to-grey indicates the average expression (avg exp ) and the dot size the relative percent of cells expressing the gene within each cluster (pct exp) (C) Immunofluorescence of Ace-TUB (green) and PSMB6, PSME1, or PSME2 (all magenta) in normal (top row) and CCDC39 (WU182) variant (bottom row) cells. White arrows indicate the localization of PSMB6, PSME1, and PSME2 in normal and variant cells. Dashed line shows the cell boundaries in one example. Scale bar=5 μm. (Representative of at least 2 unique normal donors and 2 different genotypes) (D) Percentage of cells in each cluster shown in Fig. 5A. Comparison of Sec1 plus Sec2 normal vs. CCDC39/CCDC40 (39/40) variant, p=0.024 using an unpaired t test. (E) Periodic acid Schiff (PAS) staining of mucin-positive cells in a formalin fixed paraffin embedded lung section from a normal donor and an individual carrying a CCDC39 variant (WU182). Scale bar=25 μm. (F) Immunofluorescence of α-TUB (green) and MUC5AC (magenta) in normal donors (n=4), CCDC39 and CCDC40 variants (n=4), and PCD variants DNAAF5, DNAH5, DNAI1, and Sperm associated antigen 1 (SPAG1) (n=1 per genotype). Scale bar=25 μm. (F) Graphs quantifying the percentage surface area of α-TUB (left) and MUC5AC (right), from Fig. 5F. Circles represent the median surface area of both indicated proteins from photomicrographs of 3–5 fields per sample. (G) Immunofluorescence of α-TUB (green) and MUC5AC (magenta) in CCDC39 variant cells. Examples of two cells (MC) with both MUC5AC and ciliary α-Tubulin. Cil, multiciliated cell; Muc, MUC5AC cell; MC, mucociliary. Scale bar=5 μm. (H) Schematic for lentivirus-mediated shRNA transduction of normal primary culture airway cells. Basal cells are transduced with non-targeted, CCDC39 shRNA, or CCDC40 shRNA, then differentiated to ALI for at least 4 weeks. (I) Immunofluorescence of MUC5AC (magenta) and α-TUB (green) in normal cells transduced with non-targeted, CCDC39, or CCDC40 shRNA. Scale bar=25 μm. (J) Graphs quantifying the percentage surface area of α-TUB (left) and MUC5AC (right), from Fig. 5J. (K) Immunofluorescence of α-TUB (green) and MUC5AC (magenta) in iPSCs that were derived from a normal donor and iPSCs with CRISPR-Cas9-mediated knockout of DNAH5 or CCDC40. Scale bar=25 μm. (L) Graphs quantifying the percentage surface area of α-TUB (left) and MUC5AC (right), from Fig. 5L. (M) Immunofluorescence of α-TUB (green) and MUC5AC (magenta) in normal and CCDC39 variant cultures (ALI >28) day treated with vehicle or NOTCH inhibitor DAPT for two weeks. Normal (n=3); CCDC39 (WU182, n=2); CCDC40 (WU146, n=2). Scale bar=25 μm. (N) Graphs quantifying the percentage surface area of α-TUB (left) and MUC5AC (right), from Fig. 5N. Data in M are representative of at least n=3 technical replicates; each point represents quantitation from one microscopic field. Gene The bars indicate the median with differences across groups determined using the Kruskal-Wallis test followed by Dunn’s multiple comparison test in G, K, M and between groups determined using the Mann-Whitney U test in O; ns=non-significant, *p<0.05, **p<0.01, ***p<0.001 compared to the normal (G), non-targeted (J) or parental (L).

Fig. 6.. Loss of CCDC39 or CCDC40 disrupts the periciliary barrier.

(A) Scanning EM images of normal and variant (CCDC39, WU152; CCDC40, WU151) cilia from cultured cells, either untreated (top) or treated with detergent (below) to remove the ciliary membrane. White arrows indicate splaying. Enlargement of untreated cilia are shown at the bottom, with white arrows again indicating splaying. Scale bars=1 μm. (B) Immunofluorescence of Ace-TUB (green) in cilia from cultured normal, CCDC39 or CCDC40 variant cells, either untreated (top) or treated with detergent (bottom). White arrows indicate splaying in cilia. Scale bar=5 μm. (C) Graphs quantifying the percentage of splayed cilia seen in Fig. 6B (CCDC39, WU152 and WU182; CCDC40, WU146 and WU151). Each circle is the percentage of splayed cilia per microscopic field 63X; n=300–500 total cilia. (D) Immunofluorescence of Ace-TUB (green) in cilia from freshly obtained nasal brush biopsy from normal (n=2), CCDC39 (n=2; WU120, 152) or CCDC40 (n=2, WU149, 151) variant cells, all untreated with detergent. (E) Graph quantifying the percentage of splayed cilia seen in Fig. 6D, showing normal versus CCDC39/CCDC40 variant. Each circle is the percentage of splayed cilia in a 63X microscopic field, n=200–400 total cilia. (F) Representative immunofluorescence of MUC4 (magenta) and Ace-TUB (green) in the periciliary region of cultures from normal and CCDC39 and DNAH5 variant cells. Nuclei are stained blue. Scale bar=5 μm. (G) Graph quantifying MUC4 in normal and CCDC39 and DNAH5 variant periciliary regions. (n=2 normal; CCDC39, WU120, WU182; DNAH5, WU165). Each point represents the percentage area of MUC4 relative to Ace-TUB staining in the region of cilia (n=60–80, 20–50 μm regions per sample; n=2 samples per genotype). (H) Representative immunofluorescence of keratan sulfate (KS, magenta) and α-TUB (green) in the periciliary region of normal and CCDC39 and DNAH5 variant cultured cells. Nuclei are stained blue. Scale bar=5 μm. (I) Graph quantifying KS in the periciliary region of normal and CCDC39 and DNAH5 variant cultured cells (n=2 normal; CCDC39, WU120, WU182; DNAH5, WU165). Each point represents the percentage area of MUC4 relative to Ace-TUB staining in the region of cilia (n=60–80 20–50 μm regions per sample; n=2 samples per genotype). (J) Fluorescence images showing nanoparticle (magenta) location relative to cilia (marked by SiR tubulin, green) in normal and variant (CCDC39, WU182; CCDC40, WU146; DNAAF5, WU108; DNAI1, WU103) cultures. Nuclei are stained blue. Scale bar=5 μm. (K) Graph quantifying particle clusters in the periciliary layer shown in Fig. 6K. Each point represents the number of particle clusters within the cilia of at least 10, 20–50 μm regions in normal and variant cells. B and E have at least n=3 technical replicates. In C, E, G, I, L the bar indicates the median. In E, difference between groups was determined by the Mann-Whitney U test and in C, G, I, L by the Kruskal-Wallis test followed by Dunn’s multiple comparison test; ns=non-significant, ***p<0.001 compared to normal.

Figure 7.. Gene therapy corrects motility-dependent and -independent CCDC39 variant cellular phenotypes.

(A) Schematic of lentivirus-mediated delivery of control GFP (Cont) or FOXJ1-CCDC39 transgene (TG) to basal cells of CCDC39 variant cells (WU182, WU120). Transduced cells were selected and cultured at ALI for greater than 28 days. (B) Graph quantifying the cilia beat frequency in normal cells and CCDC39 variant cells transduced with lentivirus carrying either Cont or TG. Each point represents the mean of measures from 5 fields of cells from each normal, non-transduced or CCDC39 (WU182, WU120) transduced cell sample. (C) Immunofluorescence of Ace-TUB in cilia of cells scraped from cultures of non-transduced CCDC39 variant cells or those transduced with TG. Scale bar = 10 μm. (D) Graph quantifying the cilia length seen in Fig. 7C in non-transduced normal cells and CCDC39 (WU182, WU120) variant cells, and CCDC39 variant cells transduced with the TG, n=300–500 cilia per group. (E) Graph quantifying the cilia splaying seen in Fig. 7C in non-transduced normal cells and CCDC39 (WU182, WU120) variant cells, and CCDC39 variant cells transduced with the TG, n=300–500 cilia per group. Each circle is the percentage of splayed cilia per microscopic field 63X; n=300–500 cilia. (F) Immunofluorescence of MUC4 (magenta) and Ace-TUB (green) in the periciliary layer (PCL) of non-transduced CCDC39 variant cells or those transduced with the CCDC39 transgene. Nuclei are stained blue. Scale bar=5 μm. (G) Graph quantifying MUC4 in the periciliary layer of non-transduced normal, non-transduced CCDC39 variant cells and those transduced with the TG. (n=2 normal; CCDC39, WU120 and WU182). Each point represents the percentage area of MUC4 relative to Ace-TUB staining in the region of cilia (n=20–40, 20–50 μm periciliary regions per sample; n=2 samples per genotype). (H) Immunofluorescence of KS (magenta) and α-TUB (green) in the periciliary layer (PCL) of non-transduced CCDC39 variant cells or those transduced with the CCDC39 transgene. Nuclei are stained blue. Scale bar=5 μm. (I) Graph quantifying KS in the periciliary layer of non-transduced normal, non-transduced CCDC39 variant cells and those transduced with the TG (n=2 normal; CCDC39, WU120 and WU182). Each point represents the percentage area of KS relative to α-TUB staining in the region of cilia (n=20–40, 20–50 μm regions per sample; n=2 samples per genotype). (J) Fluorescence images showing nanoparticle (magenta) location relative to cilia (marked by SiR tubulin, green) in non-transduced CCDC39 variant cells or those transduced with TG. Scale bar=5 μm. (K) Graph quantifying particle clusters in the periciliary layer shown in non-transduced normal, non-transduced CCDC39 variant cells, or those transduced with the TG. Each point represents the number of particle clusters 20–50 μm regions; n=8–18 regions of normal (n=2) or CCDC39 variant cells (WU182, WU120). (L) Fluorescence images of MUC5AC (magenta) and multiciliated cells (marked by α-TUB, green) in non-transduced CCDC39 variant cells or those with the TG. Nuclei are stained blue. Scale bar=5 μm; (M) Graphs quantifying the percentage surface area of α-TUB (left) and MUC5AC (right), in non-transduced normal and CCDC39 variant cells and variant with TG. Each point represents the mean of a 63x microscopy field (10–12 fields per cell preparation) from normal (n=2) or CCDC39 variant cells (WU182, WU120). Difference between groups in B,D,E,G,I,K was determined by the Kruskal-Wallis test followed by Dunn’s multiple comparison test. ns=non-significant; *p<0.05, **p<0.01, ***p<0.001.