Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Steven L Brody; Jiehong Pan; Tao Huang; Jian Xu; Huihui Xu; Jeffrey Koenitizer; Steven K Brennan; Rashmi Nanjundappa; Thomas G Saba; Andrew Berical; Finn J Hawkins; Xiangli Wang; Rui Zhang; Moe R Mahjoub; Amjad Horani; Susan K Dutcher

doi:10.1101/2024.03.20.585965

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It has not yet been peer reviewed by a journal.

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[Preprint]. 2024 Mar 21:2024.03.20.585965. [Version 1] doi: 10.1101/2024.03.20.585965

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Steven L Brody ^1,^*, Jiehong Pan ¹, Tao Huang ¹, Jian Xu ¹, Huihui Xu ², Jeffrey Koenitizer ¹, Steven K Brennan ², Rashmi Nanjundappa ¹, Thomas G Saba ³, Andrew Berical ⁴, Finn J Hawkins ⁴, Xiangli Wang ⁵, Rui Zhang ⁵, Moe R Mahjoub ^1,⁶, Amjad Horani ^3,⁶, Susan K Dutcher ^6,^7,^**

PMCID: PMC10983967 PMID: 38562900

Summary

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

Keywords: cilia, primary ciliary dyskinesia, airway, dynein, proteomics, periciliary barrier, Notch-signaling

Introduction

Fluid propelled by motile cilia is an essential function during development at the embryonic node, and flow in the brain, reproductive, and respiratory tracts^1–3. These tasks are accomplished by molecular motors with companion regulatory complexes docked along the length of the cilia’s microtubule skeleton, the axoneme⁴. Seminal studies of cilia from Chlamydomonas reinhardtii using freeze-fracture with transmission electronic microscopy (EM) uncovered a 96-nm repeating pattern of motors and other structures on the outer circumference of the nine doublet microtubules (DMT)^5,6 (Figure 1A). Recent single particle cryo-EM of cilia provides exquisite detail that allows the identification of proteins and their position along the ciliary axoneme^7–14,15. In parallel, advances in proteomics and genomics provide a catalog of motile ciliary components and pathogenic gene variants ^16–21. These complementary discovery tools moved the understanding of cilia biology and human disease forward. However, a major gap exists in understanding how the ciliary proteins are placed in a precise, repeating pattern and how those processes are disrupted in disease variants.

Insufficient cilia motor function in humans results in altered organ laterality, infertility, and respiratory tract infection, which are hallmarks of the genetic disease primary ciliary dyskinesia (PCD)²². Pathogenic variants in nearly 60 genes cause PCD^23,24; the phenotypes provide powerful tools for uncovering cilia biology^25,26. Variants of cilia genes code for dynein arms, the radial spokes (RS), nexin dynein regulatory complex (N-DRC) or central apparatus^27–32 (Figure 1A, 1B). Paired with model organism genetics, these variants provide structure-function relationships, but do not inform the ciliary assembly process. A common cause of PCD includes pathologic variants in CCDC39 and CCDC40^33–35. For unknown reasons, patients with CCDC39 or CCDC40 variants exhibit worse disease as indicated by decreased lung function and increased mucus plugging of airways, compared to individuals with variants in 20 other PCD genes^36–39. Current dogma suggests that PCD is caused simply by ciliary dysmotility, however this concept does not explain the range of patient phenotypes. Thus, a second gap in understanding is how variants in different genes impact multiciliated cells.

CCDC39 and CCDC40 are alpha-helical proteins that form a heterodimer (CCDC39/40) repeating every 96-nm along the length of the DMT¹⁰ (Figure 1B). The dimer directly contacts most of the structures within the repeating unit^7,9. These include proteins of the N-DRC and inner dynein arms (IDAs) that are missing in patients with CCDC39 and CCDC40 variants^33,34. CCDC39/40 was suggested to be a molecular ruler for positioning of axonemal structures⁴⁰. The ruler model proposed that CCDC39/40 measures a specific distance along the microtubule to direct the placement of IDAs and N-DRC⁴⁰. An alternative model is that CCDC39/40 provides specific sites or “addresses” to dock ciliary complexes^7,41.

We aim to identify how CCDC39/40 provides addresses and how its loss leads to severe lung disease. The variant cilia show reduced motility and are short. Proteomics of cilia from CCDC39 human variants show a devastating loss of 90 ciliary proteins, which defines a large “connectome” dependent on CCDC39/40 for anchoring. Loss of this connectome leads to the absence of major ciliary structures, impaired ciliary integrity, and an unexpected cytoplasmic burden of unanchored proteins, leading to cell stress. The findings provide insight into the cilia-independent impact of pathologic genetic variants on patients with motile ciliopathies.

Results

Loss of ciliary integrity in CCDC39 and CCDC40 variants

To understand the function of the human CCDC39/40 heterodimer, we obtained freshly isolated, primary airway epithelial cells from the respiratory tract of normal individuals and those with biallelic pathologic variants in CCDC39 or CCDC40. Most variants have frameshift mutations likely resulting in nonsense mediated decay of the RNA (Table S1). To avoid secondary insults on cilia that are caused by the inflamed airway environment in PCD, we isolated and expanded the airway basal progenitor cells in cultures with medium supplemented with antimicrobial drugs, then differentiated cells using the air-liquid interface condition (ALI) to generate multiciliated cells (Figure 1C). The onset of CCDC39 and CCDC40 expression during ALI differentiation coincides with the expression of the cilia transcription factor FOXJ1 (Figure 1D). The specificity of antibodies to CCDC39 and CCDC40 used for these studies was validated in cultured cells obtained from individuals with pathologic CCDC39 or CCDC40 variants by immunofluorescence and immunoblot analysis (Figure 1E, 1F). In some human variants we found that the unaffected partner protein is absent and in others, present by immunoblot analysis and is likely retained in the cytoplasm (Figure 1F, Figure S1A, S1D). CCDC39 protein is absent in the cytoplasm of the Chlamydomonas ccdc40 mutant (Figure 1G), which suggests that formation of the heterodimer enhances stability. Functional analysis of cilia in CCDC39 and CCDC40 variant human cultures show most are immotile or flicker (Figure 1H and Videos V1, V2). The surface transport of microbeads in variants is markedly decreased as expected (Figure 1I; Figure S1B, S1C; Videos V3-5).

We previously observed that cilia of Chlamydomonas with null alleles in ccdc39 and ccdc40 were short with ciliary splaying⁴². Cilia in human CCDC39 and CCDC40 variants are approximately half the length of normal, when measured intact on the surface of cultured cells or following treatment of cells with a detergent-containing buffer that removes cilia from cells (Figure 1J, 1K). Immunostaining using an anti-tubulin antibody shows splaying in some cilia isolated from variants, likely caused by the loss of the nexin-dynein regulatory complex that links the DMT pairs. TEM of human variant axonemes in cross-section show the loss of organization of the nine DMTs as reported previously^33,34,43. Notably, imaging the length of the cilia showed splayed microtubules (Figure 1L), which was not previously described in PCD patients. Analysis of TEM images of Chlamydomonas ccdc39 and ccdc40 identify other ultrastructural defects including misplaced DMTs and open B-tubules at the inner junction (Figure 1M, 1N), indicating the extensive loss of DMT integrity.

To determine how loss of CCDC39 or CCDC40 in the variants may lead to ciliary splaying and therefore instability, we used single particle cryo-EM of cilia from wild-type Chlamydomonas cells. We previously reported that CCDC39/40 is restricted to the groove between protofilaments A02 and A03, adjacent to IDAs, RS, and the N-DRC (Figure 1O, 1P)¹⁰. Previously, we were unable to resolve the C-terminus. With improved cryo-EM data processing and 3D classification, the last ~30 residues of the C-termini of CCDC39/40 are now resolved near the inner junction (Figure 1P). They extend as a hook from the A- to the B-tubule, forming a 4-helices bundle that agrees well with the AlphaFold prediction (Figure 1Q). The importance of the A-to-B tubule extension was illustrated by analysis of a CCDC39 variant (WU152), which carries a frameshift that is predicted to truncate the last 32 amino acids and shows persistence of CCDC39 and CCDC40 protein (Figure S1D). This individual has clinically severe PCD; cilia show lowered expression of CCD39 and CCDC40 and beat frequency (Figure S1E, S1F), suggesting an essential role for the CCDC39 C-terminal structure. A temperature-sensitive mutation in ccdc39 (L845P) is also within this hook region (Figure 1Q) and causes loss of the heterodimer at the restrictive temperature but reduced function at intermediate temperatures⁴². Taken together, the loss of CCDC39/40 function accounts for a unique defect in ciliary structure.

CCDC39/40 is required for assembly of a connectome of ciliary proteins via CARPs

Prior analysis of Chlamydomonas cilia in ccdc39 and ccdc40 mutants identified deficiencies in IDAs, N-DRC and tektin^40,42. Other proteins were preserved, although studies were limited by antibody availability⁴². Proteomics of isolated cilia would provide a comprehensive assessment of the role of CCDC39/40. Cilia obtained from ALI preparations of cells from four normal donors identified 440 proteins for comparison to cilia from a CCDC39 variant individual (Figure 2A, 2B).

Proteomics of cilia from the CCDC39 variant shows extensive loss of at least 90 proteins (Figure 2C, Table S2). Outer dynein arms are retained, but many of the known structural proteins are lost or reduced. All the IDA proteins are missing, as are 9 of the 12 N-DRC proteins; DRC5, DRC6, DRC12 were not detected in the normal cilia. CFAP73 and CFAP100, which regulate the normal waveform via the two-headed IDA⁴⁴, are missing. Many radial spoke (RS) proteins are missing or reduced, as are several proteins within the central apparatus (CA), consistent with the loss of this structure in the cross-sections of the ccdc39 or 40 mutants (Figure 1L–1N). Many proteins with unknown roles and locations are also depleted or reduced (Figure 2C). Inspection of single particle cryo-EM reconstructions shows that at least 13 of the missing proteins are in direct contact with the CCDC39/40 heterodimer (Figure 2C)^{7–12,14,45–55}. Each of these 13 missing proteins, plus MORN3, which is not present in our proteomics, is linked to a major cilia complex (Figure 2D). Due to this special arrangement, we refer to these 14 proteins as ciliary address recognition proteins (CARPs). The CARPs are diagrammed to indicate their position relative to ciliary structures (Figure 2E).

Ciliary radial spokes are differentially depleted in CCDC39/40 variants

RS act as a mechanotransducer to control ciliary waveform^9,56. Chlamydomonas has two full radial spokes (RS1, RS2) and a shorter third RS (RS3) structure while humans have three full radial spokes within each 96-nm repeat unit (Figure 3A)^7,9,15,57,58. Each RS contains unique proteins, as well as shared head and stalk proteins.

Figure 3. — **(A)** Diagram of the radial spokes (RS) in *Chlamydomonas* and human in a 96-nm repeat along the DMT. *Chlamydomonas* has two complete and one partial RS. The human has three complete RS proteins at the bases. RSPH3 is shared by all RS.

**(B)** Immunofluorescent (IF) detection of Ace-TUB, CCDC39, and RS proteins in normal and *CCDC39* variant airways. Arrow shows indicated protein. Bar=25 μm.

**(C)** IF detection of Ace-TUB, CCDC39, and radial spoke (RS) proteins in normal cells transduced with non-targeted or *CCDC96*-specific shRNA. Arrow shows indicated protein. Bar=25 μm.

**(D)** TEM of isolated *Chlamydomonas* wild-type and *ccdc39* axonemes. Repeating distances of radial spokes within a 96-nm repeat (32 nm) and between repeats (96 nm) are indicated.

**(E)** Quantification of distance from panel D.

**(F)** Immunoblot detection of RS head protein RSP16 shared by all RS in *Chlamydomonas* strains.

We found that radial spokes are differentially depleted in the CCDC39/40 variants. Cryo-EM analysis shows that each of the three radial spokes is anchored to CCDC39/40 by different CARPs^7,9. The loss of RS2 and RS3 proteins were investigated in CCDC39 and CCDC40 variants based on the availability of antibodies (Figure 3B, Figure S2A). By proteomic analysis the RS2 and RS3 CARP, CFAP91^7,52 is absent. Likewise, RS3-specific CARP, CCDC96, is absent and confirmed by immunostaining (Figure 2B, Figure S2A). Immunostaining confirms the reduction of RS2 stalk protein LRRC23, RS3-specific proteins CFAP61 and LRCC34. RSPH3, a shared RS protein, is present in the cilia of normal and variant cells by immunostaining, consistent with the assembly of some radial spokes. We used depletion of the RS3 CARP, CCDC96 in human cells to ask about its role for RS3. CCDC96 shRNA resulted in loss of CCDC96 and decrease in the surface transport of beads (Figure S2B-S2D). CCDC39 and RSPH3 were preserved (Figure 3C). There was loss of both LRRC34 and LRRC23, suggesting that CCDC96 plays a role in both RS2 and RS3 (Figure 3C).

Additionally, analysis shows that RS1 CARP (IQUB) is diminished. Chlamydomonas was used to confirm the retention of RS1 and loss of RS2 in the ccdc39 mutant. Analysis of longitudinal TEM images of ccdc39 show loss of the periodicity and rigidity of RS (Figure 3D and 3E). RSP16, a shared radial spoke head protein, is present in cilia isolated from both ccdc39 and fap253 (CARP and IQUB ortholog) by immunoblot analysis, confirming the presence of RS (Figure 3F). However, immunoblots of cilia from the ccdc39; fap253 double mutant lacks RSP16, indicating the complete loss of RS. The finding suggests that in the absence of CCDC39/40, RS1 still binds the microtubule lattice without address information but with variable positioning. Together, our observations suggest that CCDC39/40 provide unique addresses for the attachment of all RS via designated ciliary address recognition proteins (CARP).

Altered microtubule inner proteins in CCDC39/40 variants

Microtubule inner proteins (MIPs) are a network of proteins within the lumen of the A and B tubules that show 8-, 16-, and 48-nm periodicities^10,12,13,59. Some MIPs directly contact outer surface proteins. In our proteomics of CCDC39 variant cilia, three of the four tektin proteins (TEKT2,3,4) are decreased compared to normal cilia (Figure 4A). TEK3 does not contact CCDC39/40 but was absent in variant cilia by proteomics and verified by immunolocalization (Figure 4A, 4B). Decreased TEK2 and TEK4 may result from an inability to form the TEK3-TEK2 and TEK3-TEK4 heterodimers⁷. Additionally, CFAP90 (inner junction) and CFAP141 (outer junction) are absent in our proteomics (Figure 4A). CFAP90 is localized at the inner junction¹⁰, near the hook of the CCDC39/40 heterodimer (Figure 1Q). There was no difference in normal and CCDC39 in the total number of peptides of other MIPS (Figure 4A). The 48-nm MIPS are present in the cilia by immunostaining (Figure 4C).

By cryo-EM data of Chlamydomonas cilia, the 48-nm MIPs are disrupted in the ccdc39 mutant compared to wild-type (Figure 4D). However, the MIPs with 8-nm and 16-nm periodicity are largely preserved (Figure 4D). The disorganization of Chlamydomonas MIPs contrasts with the structural analysis that only found the loss of tektin in human CCDC39/40 variants⁷. Like in our human proteomics, tektin was diminished in ccdc39⁴² and in N-DRC mutants (DRC1 and DRC2)⁶⁰ (Figure 4D). We predict that missing and misplaced MIPs in the variants contribute to abnormal ciliary function.

CCDC39/40 connectome localization during cilia assembly

Our data suggest that the CCDC39/40 heterodimer creates a set of addresses for anchoring the CARPs on the ciliary axoneme. By this hypothesis, we predict a temporal pattern of trafficking, whereby the heterodimer would enter the cilia early, followed by the CARPs and finally those complexes that preassemble in the cytoplasm⁶¹. Guided by stages of ciliogenesis (Figure 5A), we examined trafficking of CCDC39/40 in normal cells. During early stages of multiciliated cell differentiation marked by centriole amplification, CCDC39 and 40 are expressed present in the cytoplasm, but do not directly colocalize with centrioles (Figure 5B). Just prior to cilia assembly, CCDC39 and CCDC40 are enriched at the apical domain but infrequently colocalize (Figure 5C). Subsequently, CCDC39/40 colocalize in short cilia as they begin to grow (Figure 5C).

We then tested the temporal pattern of trafficking of CCDC39/40, the CARPs, and the connectome during normal cilia assembly. The RS3 CARP (CCDC96) and IDA CARP (CFAP57) are localized with CCDC40 in very short cilia (Figure 5D). In contrast, non-CARPs (CFAP100, CFAP61), remain within the cytoplasm, near the basal bodies (Figure 5E, 5F). Localization of these connectome proteins in normal multiciliated cells in early cilia assembly may identify a staging site for later transport into the cilia. In later stages of ciliogenesis, connectome proteins are in the cilia (Figure 5G). In normal cells, immunoprecipitation with CCDC40 pulls down connectome proteins CFAP100 and CFAP73 to a greater degree at late compared to early stages of cilia growth (Figure 5H).

In CCDC39/40 variants, connectome proteins remain within the apical domain (Figure 5G), a location similar to the assembly staging site in normal early cilia (Figures 5E). Ultimately, the unbound connectome members are degraded (Figure 5I). We suggest cilia assembly proceeds with CCDC39/40 binding the DMTs, followed by CARP binding to the heterodimer, and subsequently, the remaining connectome. We cannot rule out that CARPs are co-transported with CCDC39/40. In sum, there is an orderly assembly sequence, dependent on CCDC39/40 and in the absence of the heterodimer, the proteins remain at the staging site or degrade (Figure 5J).

Single cell RNA transcriptomics reveals increased proteostasis in CCDC39/40 variants

To interrogate multiciliated cell dysfunction from CCDC39/40 loss, we used single cell RNA sequencing (scRNAseq). Transcriptomes were compared from normal (n=6) and variant (n=3) cell cultures. Cell types were identified using unsupervised clustering and annotated with known airway epithelial markers (Figure 6A)¹⁷. CCDC39 and CCDC40 are primarily expressed in the cluster of mature multiciliated cells (Cil3) (Figure 3SA). In this cluster, over 1800 genes are differentially expressed in the variants compared to normal (Extended Data Table). Enrichment analysis of differential gene expression in multiciliated cells (Cil3), identified cilia structure and function, proteostasis, cell stress, oxidative phosphorylation, and Notch signaling pathways (Figure 6B, Figure S3B-S3F). Genes upregulated have roles in: (1) proteostasis, including components of the E3 ubiquitin ligase complex⁶², multiple proteasome subunits, heat shock chaperone proteins that handle unfolded proteins and aggregates⁶³ (Figure S3B, S3E); (2) cell stress response genes that include SAA1, SAA2, and SAA4, which are secreted acute phase reactants in sterile inflammation^64,65 (Figure 6B), (3) mitochondrial complex I subunits of NADH ubiquinone oxidoreductase and complex V subunits of ATP synthase indicating upregulation of ATP production (Figure S3C); and (4) HES1, a target of Notch signaling that increases secretory cell differentiation and airway secretory cell proteins^66–68 (Figure 6B). Together, these data indicate the presence of cellular stress in the variant cells, possibly due to the need to degrade the large burden of undocked connectome proteins. We examined the proteasome in variant cells using an antibody against PSMB6, a component of the 20S core subunit (Figure 6C). PSMB6 is located throughout in the cytoplasm of normal cells. In contrast, PSMB6 is localized just beneath the cilia in the variant cells. The pattern is coordinate with the location of undocked connectome members in variant cells (Figure 5H), suggesting a site-specific requirement for proteostasis. Consistent with our finding, increased proteostasis leads to increased ATP demand and oxidant production. These factors likely cause cell stress that may drive Notch-signaling^69–72.

Figure 6. — **(A)** UMAP reductions of normal (n=6) and *CCDC39/40* variant (*CCDC39*, WU182 and WU 157; CCDC40 WU146) differentiated airway epithelial cells showing clusters for basal (bas); secretory (sec); multiciliated (cil) cells.

**(B)** Dotplot shows manually annotated, differentially expressed genes in each cluster that show significantly increased expression in the variant compared to multiciliated cells. Ciliated cells are in purple.

**(C)** Immunofluorescent (IF) detection of Ace-TUB and PSMB6 in normal and *CCDC39* (WU182) variant cells. Box identifies an example of PSMB6 in multiciliated cells. Arrow indicates the location of PSMB6 in the cytoplasm.

**(D)** Quantitation of cell numbers in each cluster from A. Comparison of Sec2 normal vs. *CCDC39*/*CCDC40* (39/40) variant, p=0.0025.

**(E)** Immunohistochemistry for detection of PAS-positive mucous cells in the airway from a lung of a normal donor and a *CCDC39* variant.

**(F)** IF detection of α-tubulin (α-TUB) and MUC5AC in cell cultures from unique normal donor (n=4), unique *CCDC39/40* variants (n=4), and PCD variants *DNAAF5*, *DNAH5*, *DNAI1* (n=1 per genotype).

**(G)** Quantitation of normal cells with genotypes *CCDC39*, *CCDC40* and other PCD variants from Panels F and L. *CCDC39* variant cells expressing control (Cont) and the *CCDC39* Transgene (TG) are compared.

**(H)** IF detection of α-TUB and MUC5AC in *CCDC39* variant cells. Cil, multiciliated cell; Muc, MUC5AC cell; MC, Multiciliated-MUC5AC cell.

**(I)** IF detection of MUC5AC and Ace-TUB in normal cells transduced with non-targeted *CCDC39* or *CCDC40* shRNA.

**(J)** Quantitation of panel I. Normal cells transduced with non-targeted, *CCDC39*, or *CCDC40* shRNA are compared.

**(K)** IF detection of α-TUB and MUC5AC in iPSC cells that are cells derived from the control original iPSC from a normal donor, or CRISPR-Cas9 mediated deficiency of *DNAH5* or *CCDC40* in the normal iPSC. Bar=25 μm

**(L)** Quantitation of panel K. Each point represents a microscope field of cells from the control original iPSC line, *DNAH5* CRISPR knockout, or *CCDC40* CRISPR knockout. The control cells are compared to the knockout lines.

**(M)** IF detection of α-TUB and MUC5AC in transgene (TG) rescued variant cells transduced with a control (*Cont*) or *FOXJ1-CCDC39* transgene. Quantitation of M is in panel G.

**(N)** IF detection of α-TUB and MUC5AC in unique normal and *CCDC39* and *CCDC40* variant cultures (ALI >28) day then treated with vehicle or NOTCH inhibitor DAPT for two weeks.

**(O)** Quantitation of panel N. Vehicle cells are compared to DAPT-treated for each group. Each point is the mean of fields of each condition in at least three independent experiments. Normal (n=3); *CCDC39* (WU182, n=2); *CCDC40* (WU146, n=2).

The bar indicates the medium. Differences between groups were determined using Mann-Whitney in D, G, O or Kruskal Wallis with Dunn’s Multiple comparison test in J and L; ns=non-significant, *p<0.05, **p<0.01, ***p<0.001.

Bar=5 m in C, H; Bar=25 μm in E, F, K, I, M, N.

CCDC39/40 variants switch fates in airway epithelial cells

Comparing variant and normal cells, scRNAseq analysis shows an increased number of secretory cells (Sec1 and Sec2) (Figure 3D), suggesting a shift in differentiation. The balance of airway multiciliated versus secretory cell differentiation is controlled by Notch^66,73. In vivo, chronic airway inflammation is found in patients with asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD). These airways have increased mucous cells (also called goblet cells), marked by increased MUC5AC, a gel-forming, secreted mucin⁷⁴. There is also an abundance of mucous cells in tissue samples obtained from an airway of a patient with a CCDC39 variant who underwent lung transplantation (Figure 6D). We examined the production of MUC5AC in ALI cultured cells. Control cultures show a high percentage of multiciliated cells, with few MUC5AC-expressing cells. In contrast, CCDC39/40 variant cultures have significantly more MUC5AC-expressing cells at the expense of multiciliated cells (Figure 6F, 6G). Cultured cells from PCD patients with variants in DNAAF5, DNAI1, and DNAH5 show significantly fewer MUC5AC-staining cells than the CCDC39/40 cultures (Figure 6F, 6G). A small number of cells expressing both MUC5AC and cilia proteins are present in the CCDC39/40 cultures and represent a transition state in cell differentiation⁷⁵ (Figure 6H).

It is possible that the chronic inflammatory environment in the airways of CCDC39 and CCDC40 patients account for the increased mucous cells in vitro, as observed in cultures of cells from COPD subjects^76,77. Accordingly, we depleted CCDC39 or CCDC40 from normal donor airway cells using shRNA (Figure 6I, 6J, Figure S4A-D). CCDC39 or CCDC40 depleted cultures show increased numbers of MUC5AC-expressing cells and fewer multiciliated cells than controls. Thus, the shift in the differentiation phenotype is unlikely to be attributable to effects of a chronically infected, inflamed PCD airway. To exclude the possibility that the MUC5AC phenotype was the result of exposure of cells to an airway environment, we assessed cultures of inducible pluripotent stem cells (iPSC) that underwent CCDC40 or DNAH5 deletion by CRISPR-CAS9 editing⁷⁸. Cells were differentiated to airway basal cells, then cultured using ALI conditions. CCDC40 deficient cells show more MUC5AC-expressing cells compared to those with a DNAH5 deletion or the control iPS cells (Figure 6K, 6L; Figure S4E). These findings suggest that the CCDC39 or CCDC40 patient cells or their airway basal cells are not altered by the environment. Finally, lentivirus-mediated delivery of a transgene expressing CCDC39 under control of the FOXJ1 promotor (Transgene, TG) in the CCDC39 variant cells showed rescue compared to a control lentivirus (Cont) (Figures 6M, 6G, Figure S5A-S5E). We observed a decrease in the percentage of MUC5AC cells and an increased percentage of ciliated cells (Figure 6G, 6L). Thus, the shift in cell fate likely occurs by the transition of multiciliated to secretory cells via a genetics loss of CCDC39/40 and is consistent with increased mucus plugging of airways observed in computed tomography imaging of patients with CCDC39/40 variants³⁹.

To ask if the shift in differentiation from multiciliated to mucous cells in the CCDC39/40 cultures was mediated by Notch, we inhibited Notch signaling with the γ-secretase inhibitor DAPT⁷⁹. We delayed treatment of the variant cultures with vehicle or DAPT until after ALI day 28, when MUC5AC cells were increased. In cells treated with DAPT, the number of MUC5AC-expressing cells was markedly decreased and acetylated α-tubulin expressing cells was increased compared to a persistence of MUC5AC-expressing cells in vehicle-treated variant cells (Figure 6N, 6O). We conclude that Notch signaling is required to shift the differentiation of the multiciliated variant cells to mucous cells and that continuous inhibition of Notch signaling blocks the switch from multiciliated to mucous cells. As expected, the CCDC39 and CCDC40 variants remain immotile after treatment (Figure S4F). Taken together, CCDC39/40 deficiency results in loss of ciliary proteins, increased intracellular stress, activated Notch activity, and altered maintenance of multiciliated cell differentiation.

CCDC39/40 variants disrupt the periciliary barrier

We defined the intracellular impact of a failed connectome, but the effect of the altered cilia integrity remains unaddressed. In addition to ciliary motility, the cilia are an intrinsic component of the periciliary barrier. Seminal studies defined the periciliary barrier as containing a brush-like polymer of tethered mucins (MUC1, MUC4, MUC16) that surround the cilia and act as a selective filter to particle entry^74,80. One striking feature of the CCDC39/40 variants is ciliary splaying (Figure 1L-N). To better visualize the DMT splaying, we used scanning EM. Cells were untreated or treated with detergent-containing buffer that disrupts the ciliary membrane (Figure 7A). Normal cilia exhibit intact DMTs in both conditions. CCDC39/40 variants show DMT splaying, particularly at the distal ends in the untreated condition and further unraveling of the DMT with detergent treatment (Figure 7A-C). Normal isolated cilia show little splaying regardless of treatment. In contrast, at least 15% of variant isolated cilia show splaying with no treatment, while about 50% splay when the membrane is removed. Splaying, as well as cilia length and cilia beat frequency were rescued using a CCDC39 transgene (Figures 7F-J, Supplement S5A-F; Video V6).

To determine the effect of the respiratory tract environment, we examined the extent of cilia splaying from freshly obtained samples (Figure 7D). Using non-detergent conditions, the median number of splayed cilia in freshly obtained and cultured samples were similar (Figure 7C, 7E). Surprisingly, untreated, fresh samples were significantly more variable in their splaying phenotype (F=0.03) (Figure 7D, 7E), suggesting that the respiratory tract environment destabilizes the ciliary membrane.

The periciliary barrier relies on both intact cilia and inter-ciliary tethered mucins^74,80. MUC4 is present within the ciliary region of normal and DNAH5 variant cells but absent in the periciliary layer of cells with CCDC39 variants (Figure 7K). Its presence in the periciliary layer of the CCDC39 variant can be rescued by the transgene (Figure 7K), suggesting that the periciliary barrier is abnormal due to the variant. While it is difficult to visualize the polymer-like structure of this barrier, it can be functionally defined as being impervious to particles that are greater or equal to 40 nm in diameter⁸⁰. To test the barrier function of variant multiciliated cells, we labeled the cilia of live cells using a fluorescent reporter, SPY-tubulin. Fluorescent-coated spherical particles (100 nm or 40 nm) were added to the apical surface (Figures 7L). The particles penetrate the periciliary space of the CCDC39 and CCDC40 variant cells, but not those of control cells (Figures 7M, 7O). Likewise, there is no detectable entry of microparticles in cells from individuals with PCD due to DNAI1 or DNAAF5 variants, which also have non-beating cilia. The periciliary space function in CCDC39 cells was restored with the transgene (Figures 7N, 7O). The findings suggest that there is an incompetent periciliary barrier in CCDC39 and CCDC40 variant cells, increasing susceptibility to injury and infection that is not related to ciliary beating, contributing to severe lung disease in affected patients.

Discussion

The CCDC39/40 heterodimer appears by cryo-EM as a ribbon-like structure that repeats every 96-nm along the length of the outer doublet microtubules^7,10. We propose that the heterodimer guides multiple structural complexes to their correct positions by providing addresses for their docking. CCDC39/40 anchors an extensive connectome of over 90 proteins that is much greater than suggested by earlier studies^33,34,40,42. The severe lung disease in CCDC39/40 patients may be explained by the large burden of the undocked connectome. This proteotoxic load is postulated to cause alterations in the multiciliated cell transcriptional and differentiation programs. In addition to the intracellular effects, we suggest that the microtubular disorganization results in a change in the periciliary barrier from signals in the extracellular environment.

Ciliary address recognition proteins (CARP) identify addresses on CCDC39/40 for docking major ciliary complexes.

Cilia of the CCDC39 variant were missing 50 proteins while 40 other proteins were significantly decreased. Notably, 14 of the missing proteins directly contact CCD39/40 and are likely to be the ciliary address recognition proteins (CARPs) for IDA, the N-DRC, and the RSs. CARPs attach to specific amino acid sequences of CCDC39/40 (the addresses) (Figure 2D, 2E)^{7–12,14,45–55}. During assembly, CARPs enter cilia coincident with CCDC39/40. The IDA and RS are prefabricated in the cytoplasm into megadalton complexes but do not include the CARPs^61,81–83. The entry and docking of these structures follow the CARPs (Figure 5C-E). Therefore, the loss of CCDC39/40, and thus the addresses for CARPs in the variants result in the failure of docking of entire complexes (Figure 3B, Figure 5H). Our findings are relevant as variants in several CARPs are diseases-causing. CFAP57, DRC1, DRC2 and DRC4 variants result in PCD associated with the loss of the respective IDA or N-DRC complex^47,84–86. Variants in CCDC146 and ZYMND12 result in human male sterility. We do not know the extent of protein loss in these variants, or their impact on multiciliated cell function.

CCDC39/40-dependent ciliary proteins with unknown localization are missing or reduced

There are approximately 32 proteins with unknown roles that are missing or reduced in cilia from the CCDC39 variant; most of these are transcribed primarily in multiciliated tissues or testes (Figure 2C). VCP and UBXN11 are reduced; UBXN11 is reported to interact with the proteosome quality control AAA-ATPase, VCP⁸⁷. A related protein, UBXN10 along with VCP, controls primary cilia length. The reduction of UBXN11 may contribute to the short cilia of the CCDC39/40 variants⁸⁷. Other proteins that are absent may belong to RS3, as current structural maps are incomplete. In future studies it will be important to understand the interaction of these uncharacterized proteins with CCDC39/40 using high resolution cryo-EM and proteomics.

CCDC39/40 variants have an impaired periciliary barrier

Two striking features of the CCDC39/40 variant cilia is their short length and ciliary splaying via the loss of N-DRC (Figure 2C)^8,41,88. Loss of N-DRC proteins also causes microtubule misalignment in PCD patients with DRC4 variants, but splaying was not investigated. Our SEM of the CCDC39/40 variants in cultured cells shows that removal the ciliary membranes increased splaying substantially (Figure 7A-C). Interestingly, the degree of cilia splaying observed in fresh nasal biopsies of individuals with CCDC39/40 variants was more variable (Figure 7E). We suggest that this significant variability in splaying occurs from inflammatory environmental insults on the ciliary membrane, which normally functions to support ciliary integrity.

The tethered mucin MUC4 was decreased in the periciliary layer (Figure 7K), suggesting a role of the ciliary membrane to establish or maintain the interconnected polymer structure. The relationship between ciliary integrity and a functional periciliary barrier has not been explored beforehand. The periciliary space plays an important role in separating the mucus layer and its contents from the airway epithelia^74,80. We observed that fluorescent-labeled microbeads enter the periciliary space of variant cells but not in normal cells, which suggests that pathogens and foreign particles may more easily perturb multiciliary cells in the variants. Short cilia could also impact the function of the periciliary barrier. Disruption of the periciliary layer would further impair airway host defense and contribute to severe lung disease in these patients.

CCDC39/40 variants have mucous cell metaplasia in vitro

We were surprised to find that in vitro differentiation of CCDC39/40 variant cells led to mucous cell metaplasia (Figure 6F, 6G). These findings are consistent with increased airway mucus plugging observed by x-ray imaging lungs of patients with CCDC39 and CCDC40 variants. Cultures of airway cells from individuals with COPD and cystic fibrosis show a similar phenotype that is proposed to arise from altered basal cell differentiation in response to a chronically inflamed environment^76,77,79,89. Four lines of evidence suggest that the altered cell fate switching observed in CCDC39/40 variants is associated with the genotype and not the environment. First, an increase in mucous cells was not observed in cultures of cells with other PCD variants that have immotile cilia and were isolated from an inflamed environment. Second, mucous cell metaplasia was observed in cells from normal individuals after depleting CCDC39 and CCDC40 by shRNA (Figure 6I, 6J). Third, an increased number of mucous cells was present in cultures of CCDC40 knockout iPS cells derived from peripheral blood cells of a healthy donor. Fourth, transgene rescue with a normal CCDC39 gene in the CCDC39 variant cells reversed the mucous cell phenotype (Figure 6K, 6L). These lines of evidence strongly suggest that the loss of CCDC39/40 affects the shift in the cell fate of airway cells.

Single cell transcriptomics shows alterations in multiple pathways that may contribute to the mucous cell phenotype. Oxidant stress is reported to increase the number of mucous cells and expression of MUC5AC in airway epithelial cells and associated with intracellular ER stress or oxidants from neutrophilic inflammation, and cigarette smoke^71,72,74. SAA1, SAA2 and SAA4 are upregulated in the variant multiciliated cells (Figure 6B). These secreted proteins are known to respond to injury, and activate inflammatory pathways in neighboring cells that may provide Notch signaling to multiciliated cells⁶⁵. The observation that DAPT treatment decreased mucous cell number in the variant suggests inhibition of Notch signaling as an adjunctive therapy for patients with CCDC39/40 variants⁶⁶.

Increased transcription of proteosome subunits, heat shock chaperones, and E3 ligases is consistent with increased proteostatic stress⁶³. We estimate that proteins from the 6 inner dynein arms, on each ~6 μm long cilium, from an average of 200 cilia per cell will result in one billion kilodaltons of protein that must be continually degraded by each cell. We propose that the unbound connectome proteins are responsible for generating the proteotoxic stress. The dramatic re-localization of the proteasome protein PMSB6 to the apical region of the multiciliated cell is concurrent with accumulation of unbound connectome proteins at what we term the “assembly staging zone” (Figures 5H, 6C). This similar localization of the proteosome and unbound connectome may indicate a demand for local quality control. Proteostasis requires significant ATP, which is consistent with the increased transcription of mitochondrial genes (Figure S3C). CCDC39/40 variants share transcriptional pathways with neurodegenerative diseases (Alzheimer’s, Parkinson’s, and Huntington Disease), which have increased protein burden resulting in proteotoxicity and ATP demand^63,90 (Figure S3E). Of note, neurodegenerative disease and a growing number of other diseases are being treated using inhibitors of ubiquitin and proteosome pathway modulators⁹¹.

Limitations of the study

We report an extensive disruption the ciliary proteome from the CCDCC39 variant, but we have not examined the proteome from the CCDC40 variant. However, there is interdependence between CCDC39 and CCDC40 proteins as described in Chlamydomonas²², and we validated a similar loss of selected proteins in both CCDC39 and CCDC40 variant cells (e.g., Figure 3, Figure S3). Second, our proposal is that CCDC39/40 provides amino acid sequences as addresses for attachment of CARPs to CCDC39/40 as supported by multiple cryo-EM models and experimental data (Figure 2D). The mutagenesis of these addresses will be the focus of future studies. Finally, we do not know if other PCD variants will lead to similar proteostatic stress or ciliary barrier impairment by the disruption of ciliary membranes due to airway inflammation.

Supplementary Material

Supplement 1

media-1.pdf^{(1.1MB, pdf)}

Supplement 2

NIHPP2024.03.20.585965v1-supplement-2.pdf^{(5.4MB, pdf)}

Acknowledgements.

Transmission EM was performed with the assistance of Dr. Wandy Beatty in the Department of Molecular Microbiology at Washington University and scanning EM by Dr. Sanja Sviben in the Washington University Center for Cellular Imaging (WUCCI), which is supported by Washington University School of Medicine, The Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813), and the Foundation for Barnes-Jewish Hospital (3770). Proteomics was performed with the assistance of Dr. Shin-Cheng Tzeng in the Donald Danforth Plant Science Center Proteomic and Mass Spectrometry Core. We thank Greg Longmore (Washington University) for the UbiC-YFP lentivirus transfer plasmid and Mehmet Kesimer (University of North Carolina, Chapel Hill) for the anti-MUC4 antibody.

Funding:

National Institutes of Health: R01 HL146601 (SLB), R01 HL128370 (SLB, SKD, MRM); R35 GM131909 (SKD); K08 HL150223 (AH); R01 GM138854 (RZ); R01 HL139799 (F.J.H.). Cystic Fibrosis Foundation Harry Shwachman Award BERICA22Q0 (AB). Barnes Jewish Hospital Foundation (SLB)

Funding Statement

Footnotes

Declaration of Interest

The authors declare no competing interests

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This is a preprint.

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Steven L Brody

Jiehong Pan

Tao Huang

Jian Xu

Huihui Xu

Jeffrey Koenitizer

Steven K Brennan

Rashmi Nanjundappa

Thomas G Saba

Andrew Berical

Finn J Hawkins

Xiangli Wang

Rui Zhang

Moe R Mahjoub

Amjad Horani

Susan K Dutcher

Roles

Summary

Introduction

Figure 1. Loss of ciliary microtubular integrity of human multiciliated cells in CCDC39 and CCDC40 variants.

Results

Loss of ciliary integrity in CCDC39 and CCDC40 variants

CCDC39/40 is required for assembly of a connectome of ciliary proteins via CARPs

Figure 2. CCDC39/40 is required for assembly of a connectome of ciliary proteins.

Ciliary radial spokes are differentially depleted in CCDC39/40 variants

Figure 3. Ciliary radial spokes are differentially depleted in CCDC39/40 variants.

Altered microtubule inner proteins in CCDC39/40 variants

Figure 4. Disorganization of microtubule inner proteins (MIPs) in CCDC39/40 variants.

CCDC39/40 connectome localization during cilia assembly

Figure 5. CCDC39/40 traffics independently of connectome proteins.

Single cell RNA transcriptomics reveals increased proteostasis in CCDC39/40 variants

Figure 6. CCDC39/40 variant affect differentiation via Notch signaling.

CCDC39/40 variants switch fates in airway epithelial cells

CCDC39/40 variants disrupt the periciliary barrier

Figure 7. CCDC39/40 variants have disrupted periciliary barrier.

Discussion

Ciliary address recognition proteins (CARP) identify addresses on CCDC39/40 for docking major ciliary complexes.

CCDC39/40-dependent ciliary proteins with unknown localization are missing or reduced

CCDC39/40 variants have an impaired periciliary barrier

CCDC39/40 variants have mucous cell metaplasia in vitro

Limitations of the study

Supplementary Material

Acknowledgements.

Funding:

Funding Statement

Footnotes

References

Associated Data

Supplementary Materials

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