CFAP61 is required for sperm flagellum formation and male fertility in human and mouse
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Evaluation Summary:
This manuscript describes a specific role for CFAP61 - a known component of axonemal radial spokes - in formation and function of sperm flagella in the mouse, and identifies CFAP61 as a disease gene linked to male infertility in a human patient. Furthermore, the authors show that CFAP61 interacts with several radial spoke components, including head and stalk regions, as well as with intraflagellar transport proteins. Overall, the quality of the data is high and the mouse work is consistent with a previously published report. The study underscores the physiological importance of CFAP61 in male fertility and will be of interest to cell and structural biologist studying flagella and motile cilia function, as well as to clinicians involved in fertility genetics. The study can serve a starting point to revealing the precise mechanism by which CFAP61 regulates sperm flagella formation and function and for further analysis human patient data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Defects in the structure or motility of cilia and flagella may lead to severe diseases such as primary ciliary dyskinesia (PCD), a multisystemic disorder with heterogeneous manifestations affecting primarily respiratory and reproductive functions. We report that CFAP61 is a conserved component of the calmodulin- and radial spoke-associated complex (CSC) of cilia. We find that a CFAP61 splice variant, c.143+5G>A, causes exon skipping/intron retention in human, inducing a multiple morphological abnormalities of the flagella (MMAF) phenotype. We generated Cfap61 knockout mice that recapitulate the infertility phenotype of the human CFAP61 mutation, but without other symptoms usually observed in PCD. We find that CFAP61 interacts with the CSC, radial spoke stalk and head. During early stages of Cfap61−/− spermatid development, the assembly of radial spoke components is impaired. As spermiogenesis progresses, the axoneme in Cfap61−/− cells becomes unstable and scatters, and the distribution of intraflagellar transport proteins is disrupted. This study reveals an organ-specific mechanism of axoneme stabilization that is related to male infertility.
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Evaluation Summary:
This manuscript describes a specific role for CFAP61 - a known component of axonemal radial spokes - in formation and function of sperm flagella in the mouse, and identifies CFAP61 as a disease gene linked to male infertility in a human patient. Furthermore, the authors show that CFAP61 interacts with several radial spoke components, including head and stalk regions, as well as with intraflagellar transport proteins. Overall, the quality of the data is high and the mouse work is consistent with a previously published report. The study underscores the physiological importance of CFAP61 in male fertility and will be of interest to cell and structural biologist studying flagella and motile cilia function, as well as to clinicians involved in fertility genetics. The study can serve a starting point to revealing the …
Evaluation Summary:
This manuscript describes a specific role for CFAP61 - a known component of axonemal radial spokes - in formation and function of sperm flagella in the mouse, and identifies CFAP61 as a disease gene linked to male infertility in a human patient. Furthermore, the authors show that CFAP61 interacts with several radial spoke components, including head and stalk regions, as well as with intraflagellar transport proteins. Overall, the quality of the data is high and the mouse work is consistent with a previously published report. The study underscores the physiological importance of CFAP61 in male fertility and will be of interest to cell and structural biologist studying flagella and motile cilia function, as well as to clinicians involved in fertility genetics. The study can serve a starting point to revealing the precise mechanism by which CFAP61 regulates sperm flagella formation and function and for further analysis human patient data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #3 (Public Review):
In this manuscript Liu and colleagues describe an essential and specific function of CFAP61 in sperm flagella formation and function in human and mouse. CFAP61 (or its homolog in other organisms) was previously shown by several groups to be part of the Calmodulin and radial Spoke associated Complex (CSC), which is located at the base of radial spokes (RS) and interacts with the Dynein Regulatory Complex (DRC) and inner dynein arm of motile cilia/flagella. Here, the authors first re-investigate exomes from a previously described cohort of patients with a multiple morphological abnormalities of the flagella (MMAF) phenotype, and identify one patient with a homozygous intronic variant in CFAP61 that they show induces skipping of CFAP61 exon 2. Next, they generate a Cfap61 mutant mouse, and using various …
Reviewer #3 (Public Review):
In this manuscript Liu and colleagues describe an essential and specific function of CFAP61 in sperm flagella formation and function in human and mouse. CFAP61 (or its homolog in other organisms) was previously shown by several groups to be part of the Calmodulin and radial Spoke associated Complex (CSC), which is located at the base of radial spokes (RS) and interacts with the Dynein Regulatory Complex (DRC) and inner dynein arm of motile cilia/flagella. Here, the authors first re-investigate exomes from a previously described cohort of patients with a multiple morphological abnormalities of the flagella (MMAF) phenotype, and identify one patient with a homozygous intronic variant in CFAP61 that they show induces skipping of CFAP61 exon 2. Next, they generate a Cfap61 mutant mouse, and using various approaches they convincingly demonstrate that loss of CFAP61 severely affects spermatogenesis whereas other ciliated tissues such as trachea appear normal. Consistently, Cfap61 mutant males are infertile but do not display additional phenotypes indicative of ciliary dysfunction. The authors also present immunoprecipitation data suggesting that CFAP61 and its Chlamydomonas homolog, FAP61, interact with a range of axonemal proteins, including known components of the CSC but also other RS components, as well as some axonemal dynein arm and IFT proteins.
The overall quality of the data is good and the manuscript is nicely presented. Most of the phenotypes described for the Cfap61 mutant mice are reminiscent of those previously reported for another Cfap61 mutant mouse model (Huang et al., 2020, Science Bulletin vol. 65 p. 854-864). Furthermore, while the biochemical data largely confirms previously published work done in model organisms like Chlamydomonas and Tetrahymena, additional work will be needed to fully understand the precise mechanism by which CFAP61 interacts with various axonemal components to regulate sperm flagella formation and function.
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Reviewer #2 (Public Review):
Through analysis of exome sequencing of a patients cohort affected by infertility, Liu et al discovered a patient whose genome contains a missense mutation (C143+5G>A) in CFAP61, the human homologue of FAP61 radial spoke protein in Chlamydomonas. The objective of the authors is to confirm that the mutation in CFAP61 causes the infertility phenotype at the organism level and to detail defects in sperm flagella at the molecular level caused by the lack of CFAP61.
The authors first confirm that the mutation found in the patient changes a splice site, which in turn leads to exon skipping of CFAP61 using a minigene assay. Subsequently, they show that sperm flagella from the patient and obtained from a Cfap61 -/- CRISPR-Cas9 KO model show multiple morphological abnormalities of the sperm flagella (MMFA phenotype). …
Reviewer #2 (Public Review):
Through analysis of exome sequencing of a patients cohort affected by infertility, Liu et al discovered a patient whose genome contains a missense mutation (C143+5G>A) in CFAP61, the human homologue of FAP61 radial spoke protein in Chlamydomonas. The objective of the authors is to confirm that the mutation in CFAP61 causes the infertility phenotype at the organism level and to detail defects in sperm flagella at the molecular level caused by the lack of CFAP61.
The authors first confirm that the mutation found in the patient changes a splice site, which in turn leads to exon skipping of CFAP61 using a minigene assay. Subsequently, they show that sperm flagella from the patient and obtained from a Cfap61 -/- CRISPR-Cas9 KO model show multiple morphological abnormalities of the sperm flagella (MMFA phenotype). This observations are consistent with previous analysis of a Cfap61 -/- mouse KO model also generated by CRISPR-Cas9 by Huang et al in Science Bulletin article published previously. In particular they show that CFAP61 is critical for the late stages of Radial Spoke assembly in elongated spermatids and spermatozoa and contribute to flagella stability, while it does not appear to impact round spermatids organization.
The manuscript is overall interesting, well executed and the interpretation is largely consistent with the data. Most of the results are centered on the characterization of sperm flagella phenotype in the mouse Cfap61 -/- KO, which largely mirrors published work.
A well executed systematic analysis of protein-protein interactions in pull down assays with human and Chlamydomonas Radial spoke proteins suggest that CFAP61 is an elongated protein part of the Calmodulin and Spoke associated complex (CSC).
Analysis of airway multiciliated cells from Cfap61 -/- mice show that CFAP61 plays an important role only in flagella, but not in airway motile formation or function.
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Reviewer #1 (Public Review):
This work reported an MMAF patient carrying CFAP61 splicing variant. The author then make a Cfap61-knockout mouse using CRISPR/CAS9 to examine the function of Cfap61 in vivo. Beyond the phenotype of MMAF, the author tried to study the mechanism. They found that CFAP61 can interact with many RS proteins and other proteins relating to the sperm flagellum in mice. These results together with IF results in several markers in CSC, RS and other proteins in sperm flagella suggested the failure of flagella axoneme assembly in Cfap61-/- mice.
Since the function of Cfap61 in mice has been reported before, some results in the first half has been uncovered. While the second half of the mechanism research is complete and the data support the conclusions, but there are still some concerns.1. To conclude that CFAP61 is …
Reviewer #1 (Public Review):
This work reported an MMAF patient carrying CFAP61 splicing variant. The author then make a Cfap61-knockout mouse using CRISPR/CAS9 to examine the function of Cfap61 in vivo. Beyond the phenotype of MMAF, the author tried to study the mechanism. They found that CFAP61 can interact with many RS proteins and other proteins relating to the sperm flagellum in mice. These results together with IF results in several markers in CSC, RS and other proteins in sperm flagella suggested the failure of flagella axoneme assembly in Cfap61-/- mice.
Since the function of Cfap61 in mice has been reported before, some results in the first half has been uncovered. While the second half of the mechanism research is complete and the data support the conclusions, but there are still some concerns.1. To conclude that CFAP61 is required for sperm flagellum formation for human, more evidence from human experiments would be needed. The human protein interaction and IF experiments in human smear slides could be useful to support the claim.
2. IF results in Cfap61+/+ and Cfap61-/- for mature spermatozoa using anti-CFAP61 antibody would be needed.
3. According to previous reports and the in vitro results from this paper, CFAP61 should have a strong interaction with CFAP91 (MAATS1)/ CFAP251 (WDR66) since they are all CSC members. It is unclear why the IP-MS results of anti-CFAP61 antibody show neither of these two proteins (Figure 6-figure supplement 1).
4. According to the diagram given by the authors, CFAP61 is located in the RS stalk and neck part, but it's an inference from IP results in vitro. A more direct evidence for this would make the paper stronger.
5. It is a little confusing that CFAP61 can also interact with IFT proteins. It would have been helpful if the authors could provide more evidence that the IFT retention is directly caused by deletion of CFAP61 but not a consequence.
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