Proteomic composition and mutual assembly of the C2a projection in vertebrate motile cilia

Reviewer #1 (Public review):

The central pair apparatus of motile cilia consists of two singlet microtubules, termed C1 and C2, each of which is associated with a set of projections, referred to as the C1 and C2 projections. Each projection comprises multiple distinct structural domains, designated a, b, c, and so on. Biochemical studies combined with genetic analyses in Chlamydomonas identified three proteins as the major components of the C2a projection, and subsequent cryo-EM studies confirmed these findings.

In this paper, the authors aim to study the homologues of these three proteins-CCDC108/CFAP65, CFAP70, and MYCBPAP/CFAP147-using knockout mouse models. Biochemical and cell biological analyses demonstrate that, as in Chlamydomonas, these proteins are components of the C2 projection and form a complex that depends on the presence of each other. In addition, the authors use affinity purification to identify two previously uncharacterized proteins and show that they are central pair apparatus proteins that associate with the aforementioned complex. Knockout mice lacking any of the three core proteins exhibit phenotypes consistent with primary ciliary dyskinesia (PCD).

Overall, the manuscript is clearly written, and the data are convincing and support the authors' conclusions. However, given the previous findings in Chlamydomonas, this work provides limited conceptual advances to the field. Nonetheless, it represents a useful and well-documented resource for understanding the conserved organization of the central pair apparatus in motile cilia. It will be of interest to cell and developmental biologists, biochemists, and clinicians studying and treating human ciliopathies.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the protein composition and functional role of the C2a projection of the central apparatus (CA) in vertebrate motile cilia. Using three knockout mouse models (Ccdc108, Mycbpap, and Cfap70), the authors demonstrate that these genes - homologs of Chlamydomonas FAP65, FAP147, and FAP70 - are required for normal motile cilia function in ependymal and tracheal multiciliated cells. Specifically, the authors show that:

(1) Knockout mice for each gene exhibit primary ciliary dyskinesia phenotypes (hydrocephalus and sinusitis), accompanied by abnormal ciliary motion and reduced ciliary beat frequency.

(2) CCDC108, MYCBPAP, and CFAP70 physically interact and localize to the axonemal central lumen, consistent with the C2a projection.

(3) Loss of any one of these proteins destabilizes the others and disrupts CA integrity in a tissue-specific manner.

(4) ARMC3 and MYCBP are C2a-associated proteins.

Strengths:

(1) Clarity: the results are presented in a coherent sequence that facilitates understanding of both the rationale and conclusions.

(2) Genetic rigor: three independent knockout mouse lines that exhibit consistent motile cilia phenotypes provide in vivo support for the proposed role of these proteins.

(3) Integration of structural and functional analyses: combination of ultrastructural (TEM) and immunofluorescence data with CBF measurements provides convincing correlation between structural defects and impaired ciliary function.

(4) Mutual dependency model: reciprocal destabilization of CCDC108, MYCBPAP, and CFAP70 supports their interdependence in the C2a assembly.

(5) Expansion of the vertebrate C2a proteome: the identification of ARMC3 and MYCBP as C2a-associated proteins provides a foundation for future mechanistic studies.

Weaknesses:

(1) Mechanistic depth: the data show a convincing correlation between C2a and ciliary function, but the cell type-specificity of CCDC108, MYCBPAP, and CFAP70 knockout effects is underdeveloped. This is an interesting observation that raises mechanistic/structural questions not addressed in the study, such as what is the role of C2a in CP nucleation, maintenance, or mechanical stabilization? Is C2a composition different in different cell types?

(2) Cell model choice: co-immunoprecipitation was performed using mouse testis lysates. While this is a reasonable source of CA proteins from flagellated cells, the functional analyses in this study focus on ependymal and tracheal multiciliated cells. It would therefore be helpful for the authors to clarify the extent to which these interactions are expected to be conserved across ciliated cell types, and to discuss potential tissue-specific differences in CA assembly.

(3) Statistical analysis: the manuscript states "Statistical significance was defined as P < 0.5", which is likely a typo, but should be P < 0.05. In general, the statistical methods require more clarification. In several figures (e.g., 2B, 2D, 5J, 5K), multiple knockout genotypes are compared with WT, yet unpaired t-tests are reported. When more than two groups are analyzed, multiple pairwise t-tests inflate Type I error unless appropriately corrected; a one-way ANOVA with post hoc comparisons (e.g., Dunnett's test for WT-referenced comparisons) would be more appropriate. Furthermore, the analysis of ciliary movement modes (Figure 2D) involves categorical data, for which a t-test is not statistically appropriate. These comparisons could instead be evaluated using chi-square or Fisher's exact tests. Addressing these issues is important to ensure accurate statistical inference.

(4) Methods section: does not sufficiently describe how image-based quantifications were performed. For example, the criteria used to define cilia number, basal body number, and rotational beating are not specified, nor is how CBF measurements were analyzed. The authors should also provide details regarding analysis software and imaging parameters used (and whether they were kept constant across genotypes).

Author response:

Public Reviews:

Reviewer #1 (Public review):

The central pair apparatus of motile cilia consists of two singlet microtubules, termed C1 and C2, each of which is associated with a set of projections, referred to as the C1 and C2 projections. Each projection comprises multiple distinct structural domains, designated a, b, c, and so on. Biochemical studies combined with genetic analyses in Chlamydomonas identified three proteins as the major components of the C2a projection, and subsequent cryo-EM studies confirmed these findings.

In this paper, the authors aim to study the homologues of these three proteins-CCDC108/CFAP65, CFAP70, and MYCBPAP/CFAP147-using knockout mouse models. Biochemical and cell biological analyses demonstrate that, as in Chlamydomonas, these proteins are components of the C2 projection and form a complex that depends on the presence of each other. In addition, the authors use affinity purification to identify two previously uncharacterized proteins and show that they are central pair apparatus proteins that associate with the aforementioned complex. Knockout mice lacking any of the three core proteins exhibit phenotypes consistent with primary ciliary dyskinesia (PCD).

Overall, the manuscript is clearly written, and the data are convincing and support the authors' conclusions. However, given the previous findings in Chlamydomonas, this work provides limited conceptual advances to the field. Nonetheless, it represents a useful and well-documented resource for understanding the conserved organization of the central pair apparatus in motile cilia. It will be of interest to cell and developmental biologists, biochemists, and clinicians studying and treating human ciliopathies.

We thank the reviewer for their positive comments on our work.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the protein composition and functional role of the C2a projection of the central apparatus (CA) in vertebrate motile cilia. Using three knockout mouse models (Ccdc108, Mycbpap, and Cfap70), the authors demonstrate that these genes - homologs of Chlamydomonas FAP65, FAP147, and FAP70 - are required for normal motile cilia function in ependymal and tracheal multiciliated cells. Specifically, the authors show that:

(1) Knockout mice for each gene exhibit primary ciliary dyskinesia phenotypes (hydrocephalus and sinusitis), accompanied by abnormal ciliary motion and reduced ciliary beat frequency.

(2) CCDC108, MYCBPAP, and CFAP70 physically interact and localize to the axonemal central lumen, consistent with the C2a projection.

(3) Loss of any one of these proteins destabilizes the others and disrupts CA integrity in a tissue-specific manner.

(4) ARMC3 and MYCBP are C2a-associated proteins.

Strengths:

(1) Clarity: the results are presented in a coherent sequence that facilitates understanding of both the rationale and conclusions.

(2) Genetic rigor: three independent knockout mouse lines that exhibit consistent motile cilia phenotypes provide in vivo support for the proposed role of these proteins.

(3) Integration of structural and functional analyses: combination of ultrastructural (TEM) and immunofluorescence data with CBF measurements provides convincing correlation between structural defects and impaired ciliary function.

(4) Mutual dependency model: reciprocal destabilization of CCDC108, MYCBPAP, and CFAP70 supports their interdependence in the C2a assembly.

(5) Expansion of the vertebrate C2a proteome: the identification of ARMC3 and MYCBP as C2a-associated proteins provides a foundation for future mechanistic studies.

We appreciate our reviewer's positive comments.

Weaknesses:

(1) Mechanistic depth: the data show a convincing correlation between C2a and ciliary function, but the cell type-specificity of CCDC108, MYCBPAP, and CFAP70 knockout effects is underdeveloped. This is an interesting observation that raises mechanistic/structural questions not addressed in the study, such as what is the role of C2a in CP nucleation, maintenance, or mechanical stabilization? Is C2a composition different in different cell types?

We agree with our reviewer and value their insightful comments. Indeed, CP-MT defects, including the loss of one or both CP-MTs, were only observed in a subset of mouse ependymal cells (mEPCs) at day 10 post-serum starvation, and were rare in tracheal multiciliated cells, although the C2a projections were severely damaged in these tracheal cells. Based on these observations, we hypothesize that the loss of CP-MTs is probably a secondary effect caused by mechanical stress during ciliary movement. To investigate the role of C2a in CP-MT nucleation, maintenance, or mechanical stabilization, we plan to examine the axoneme structures of mEPCs at day 5 post-serum starvation using TEM. By comparing axoneme defects in these cells at days 5 and 10, we hope to gain insights into this question. Based on our findings and previous findings in Chlamydomonas, we speculate that the core components (CCDC108/FAP65, MYCBPAP/FAP147, and CFAP70/FAP70) of the C2a projection are highly conserved across species, but the peripheral associated C2a proteins may vary among different cell types. Therefore, we will perform co-immunoprecipitation using mEPCs and mouse tracheal epithelial cells to investigate potential cell-type-specific differences and expand the related discussion.

(2) Cell model choice: co-immunoprecipitation was performed using mouse testis lysates. While this is a reasonable source of CA proteins from flagellated cells, the functional analyses in this study focus on ependymal and tracheal multiciliated cells. It would therefore be helpful for the authors to clarify the extent to which these interactions are expected to be conserved across ciliated cell types, and to discuss potential tissue-specific differences in CA assembly.

We appreciate our reviewer's insightful comments. We will follow their suggestion and perform co-immunoprecipitation using mEPCs and mouse tracheal epithelial cells to investigate potential cell-type-specific differences and expand the related discussion.

(3) Statistical analysis: the manuscript states "Statistical significance was defined as P < 0.5", which is likely a typo, but should be P < 0.05. In general, the statistical methods require more clarification. In several figures (e.g., 2B, 2D, 5J, 5K), multiple knockout genotypes are compared with WT, yet unpaired t-tests are reported. When more than two groups are analyzed, multiple pairwise t-tests inflate Type I error unless appropriately corrected; a one-way ANOVA with post hoc comparisons (e.g., Dunnett's test for WT-referenced comparisons) would be more appropriate. Furthermore, the analysis of ciliary movement modes (Figure 2D) involves categorical data, for which a t-test is not statistically appropriate. These comparisons could instead be evaluated using chi-square or Fisher's exact tests. Addressing these issues is important to ensure accurate statistical inference.

We thank our reviewer for pointing out these errors. We will double-check our statistical results and perform new analyses following their suggestion.

(4) Methods section: does not sufficiently describe how image-based quantifications were performed. For example, the criteria used to define cilia number, basal body number, and rotational beating are not specified, nor is how CBF measurements were analyzed. The authors should also provide details regarding analysis software and imaging parameters used (and whether they were kept constant across genotypes).

We apologize for overlooking these method details. We will expand the relevant method section to include this information.