CFAP61 (Cilia- and flagella-associated protein 61; formerly C20orf26) is a structural scaffold protein and conserved component of the calmodulin- and radial spoke-associated complex (CSC) in motile cilia and flagella. The CSC, composed of CFAP61, CFAP91/MAATS1, and CFAP251/WDR66, connects the radial spokes to the nexin-dynein regulatory complex (N-DRC), mediating mechanochemical signal transduction that coordinates dynein motor activity and ciliary/flagellar beating. CFAP61 is specifically required for the stable assembly of radial spoke 3 (RS3) and contributes to RS2 base formation. In sperm, loss of CFAP61 causes collapse of axonemal architecture with loss of central pair, radial spokes, and inner dynein arms, resulting in multiple morphological abnormalities of the flagella (MMAF) and male infertility (SPGF84). Despite containing a Rossmann-like FAD/NAD(P)-binding superfamily fold (residues 665-998), CFAP61 has no enzymatic activity; this domain serves as a structural scaffold for protein-protein interactions within the axoneme. The protein also has a CFAP61-specific N-terminal domain and a C-terminal dimerisation domain. CFAP61 is expressed in all tissues with motile cilia, including respiratory epithelium, fallopian tubes, and testis, though disease phenotypes from human mutations are restricted to male infertility without classical primary ciliary dyskinesia symptoms.
Definition: A protein complex associated with the radial spokes of motile cilia and flagella, composed of CFAP61, CFAP91/MAATS1, and CFAP251/WDR66 (mammalian nomenclature; FAP61/CaM-IP3, FAP91/CaM-IP2, FAP251/CaM-IP4 in Chlamydomonas). The CSC connects radial spoke 2 (RS2), radial spoke 3 (RS3), and the nexin-dynein regulatory complex (N-DRC), mediating mechanochemical signal transduction for coordinated ciliary beating.
Justification: The CSC is a well-characterized multi-protein complex with a defined composition and function in motile cilia across eukaryotes. It has been the subject of numerous studies since its identification in Chlamydomonas (PMID:21613541). No GO CC term currently exists for this complex. Having a term would allow annotation of CFAP61, CFAP91, and CFAP251 as part_of the CSC.
Parent term: axoneme
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005930 axoneme | IDA PMID:28282151 Quantitative Proteomic Analysis of Human Airway Cilia Identi... | ACCEPT | Summary: CFAP61 was identified in the axoneme by quantitative proteomic analysis of human airway cilia. This is direct experimental evidence for axonemal localization in human respiratory epithelial cilia. Reason: Direct proteomic detection in purified human airway ciliary axonemes provides strong evidence for axonemal localization. This is consistent with CFAP61's known role as a structural component of the calmodulin- and spoke-associated complex (CSC), which is an integral axonemal complex. Supporting Evidence: PMID:28282151 ...Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance... PMID:37258679 ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and CFAP251... |
| GO:0005930 axoneme | IDA PMID:36659204 Absence of murine CFAP61 causes male infertility due to mult... | ACCEPT | Summary: Cfap61 was detected in the axoneme of mouse sperm by immunofluorescence/immunohistochemistry, initially at the neck of elongating spermatids, later at the midpiece of mature sperm. Reason: Direct immunolocalization in mouse sperm flagella axoneme. Transferred to human by MGI annotation. Consistent with human proteomic data (PMID:28282151) and the structural role of CFAP61 in the CSC complex. Supporting Evidence: PMID:36659204 ...Cfap61 is initially localized at the neck of sperm, where it potentially functions in flagellum formation, and is later localized to the midpiece of the sperm... |
| GO:0036126 sperm flagellum | IDA PMID:36659204 Absence of murine CFAP61 causes male infertility due to mult... | ACCEPT | Summary: Cfap61 protein was detected in sperm flagella by immunofluorescence in mouse, and separately confirmed in human sperm flagella by immunofluorescence with anti-CFAP61 antibodies. Reason: Direct immunolocalization data in both mouse (PMID:36659204) and human (PMID:35174165) sperm. CFAP61 localizes along the full length of human sperm flagella (in contrast to mouse where it is more restricted to midpiece in mature sperm). Core localization for this protein. Supporting Evidence: PMID:36659204 ...Cfap61 is initially localized at the neck of sperm, where it potentially functions in flagellum formation, and is later localized to the midpiece of the sperm... PMID:35174165 ...The specific signals of CFAP61, co-localizing with alpha-Tubulin, were detected on the sperm tails from a fertile control... |
| GO:0120316 sperm flagellum assembly | IMP PMID:35174165 Biallelic Variants in CFAP61 Cause Multiple Morphological Ab... | ACCEPT | Summary: Biallelic loss-of-function variants in CFAP61 (frameshift and nonsense) cause MMAF with severely disorganized axonemal ultrastructures in human sperm, including missing central pair, radial spokes, and inner dynein arms. Reason: Multiple independent human families with biallelic CFAP61 variants show MMAF phenotype. TEM revealed severely disorganized axonemes. Both CFAP61 and CFAP251 signals were absent from patient sperm tails. This is strong IMP evidence that CFAP61 is required for normal sperm flagellum assembly. Supporting Evidence: PMID:35174165 ...variants, c.451_452del (p.I151Nfs*4) in family 1 and c.847C > T (p.R283*) in family 2 and 3, were identified recessively co-segregating with the MMAF phenotype. Transmission electron microscopy analyses revealed severe disorganized axonemal ultrastructures... |
| GO:0030317 flagellated sperm motility | IMP PMID:34792097 CFAP61 is required for sperm flagellum formation and male fe... | ACCEPT | Summary: Cfap61 knockout mice show severely impaired sperm motility. Human patients with CFAP61 splice variant also show asthenozoospermia. The protein is required for axoneme stability during spermiogenesis, and loss leads to axoneme scattering and disrupted IFT protein distribution. Reason: Strong genetic evidence from both mouse knockout and human mutation that CFAP61 is required for flagellated sperm motility. The mechanism is indirect -- CFAP61 is needed for radial spoke assembly, and without intact radial spokes the axoneme becomes unstable and motility is lost. The qualifier acts_upstream_of_or_within (in GOA) is appropriate since CFAP61's role is structural rather than directly in the motility machinery. Supporting Evidence: PMID:34792097 ...CFAP61 is required for sperm flagellum formation and male fertility in human and mouse... the assembly of radial spoke components is impaired. As spermiogenesis progresses, the axoneme in Cfap61-/- cells becomes unstable and scatters... |
| GO:0120316 sperm flagellum assembly | IMP PMID:34792097 CFAP61 is required for sperm flagellum formation and male fe... | ACCEPT | Summary: Cfap61 knockout mice show MMAF phenotype with impaired radial spoke assembly during early spermatid development, progressing to axoneme instability and scattering. Reason: Complementary to the human IMP evidence from PMID:35174165. The mouse knockout provides detailed mechanistic insight: CFAP61 interacts with the CSC, radial spoke stalk and head, and its absence impairs radial spoke assembly early in spermiogenesis. Supporting Evidence: PMID:34792097 ...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... |
| GO:0036126 sperm flagellum | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically inferred sperm flagellum localization based on PANTHER tree analysis. This is now independently confirmed by direct experimental evidence (IDA from PMID:36659204 and IF data from PMID:35174165). Reason: The IBA annotation is correct and consistent with the IDA evidence. The phylogenetic inference is sound given the highly conserved CSC function across species, and is independently confirmed by direct experimental evidence. |
| GO:0120316 sperm flagellum assembly | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically inferred involvement in sperm flagellum assembly. Confirmed by direct IMP evidence from human and mouse studies. Reason: Correct inference, independently confirmed by direct experimental evidence (IMP from PMID:34792097 and PMID:35174165). Sperm flagellum assembly is a core function of CFAP61. |
| GO:0005856 cytoskeleton | IEA GO_REF:0000044 | MODIFY | Summary: Electronic annotation mapping from UniProt subcellular location. CFAP61 is indeed a cytoskeletal protein as a component of the axoneme. Reason: While technically correct, 'cytoskeleton' is too general. CFAP61 specifically localizes to the axoneme, which is already annotated with stronger evidence. The more specific term 'axoneme' (GO:0005930) is preferred and has IDA evidence. Proposed replacements: axoneme |
| GO:0005929 cilium | IEA GO_REF:0000044 | MODIFY | Summary: Electronic annotation from UniProt subcellular location. CFAP61 is present in cilia. Reason: CFAP61 is specifically a component of motile cilia (not primary/immotile cilia). The term 'motile cilium' (GO:0031514) would be more accurate and is already present as an ISS annotation. Proposed replacements: motile cilium |
| GO:0005930 axoneme | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation of axoneme localization from UniProt. Correct and supported by direct experimental evidence. Reason: Correct annotation, consistent with IDA evidence from PMID:28282151 and PMID:36659204. Axoneme localization is a core aspect of CFAP61 function as a CSC component. |
| GO:0003341 cilium movement | ISS GO_REF:0000024 | ACCEPT | Summary: Transferred from Tetrahymena FAP61 ortholog (UniProtKB:A8IF44). In Tetrahymena, loss of FAP61 reduces cell swimming and affects ciliary waveform. In Chlamydomonas, CSC disruption impairs flagellar motility. Reason: Well-supported ISS annotation. The role of CFAP61 in cilium movement is clearly established in multiple organisms through the CSC complex function. While direct evidence in human respiratory cilia is not yet available, the conserved mechanism of radial spoke-mediated motility regulation makes this a strong inference. Patients with CFAP61 mutations notably do not present PCD symptoms, suggesting possible redundancy in respiratory cilia, but the protein is present in airway cilia (PMID:28282151). Supporting Evidence: PMID:25694453 ...loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected... PMID:21613541 ...analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity... |
| GO:0031514 motile cilium | ISS GO_REF:0000024 | ACCEPT | Summary: Transferred from Tetrahymena ortholog. CFAP61 is a component of motile cilia, confirmed by its proteomic identification in human airway cilia. Reason: Correct and well-supported. CFAP61 was directly identified in human airway motile cilia by proteomics (PMID:28282151), confirming the ISS transfer. Motile cilium is the appropriate specific CC term for this protein. Supporting Evidence: PMID:28282151 ...Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance... |
| GO:0044782 cilium organization | ISS GO_REF:0000024 | MODIFY | Summary: Transferred from Chlamydomonas (Q23F13) and Tetrahymena (Q24DE2) orthologs. CFAP61 is involved in the structural organization of cilia through its role in radial spoke assembly. Reason: While CFAP61 does contribute to cilium organization, the term is very broad. The more specific term 'radial spoke assembly' (GO:0062177) better captures the actual function of CFAP61 within the CSC. CFAP61 is specifically required for RS3 assembly and contributes to RS2 base formation. Proposed replacements: radial spoke assembly |
| GO:0001536 radial spoke stalk | ISS GO_REF:0000024 | ACCEPT | Summary: Transferred from Tetrahymena FAP61 ortholog. The CSC localizes at the base of radial spokes and CFAP61 specifically contributes to RS3 stem structure. In Liu et al. 2021, CFAP61 was shown to interact with radial spoke stalk and head. Reason: The qualifier 'colocalizes_with' is appropriate for this ISS annotation. CFAP61 as a CSC component is located at the base of radial spokes and interacts with stalk proteins. The colocalizes_with qualifier correctly indicates proximity/association rather than being part_of the radial spoke stalk per se. Supporting Evidence: PMID:25694453 ...FAP61-null cilia lack an adjacent portion of the RS3 stem region... PMID:34792097 ...CFAP61 interacts with the CSC, radial spoke stalk and head... PMID:37258679 ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and CFAP251... then links the RS3 subunits CFAP251 and CFAP61... |
| GO:0005930 axoneme | ISS GO_REF:0000024 | ACCEPT | Summary: Transferred from Tetrahymena ortholog. Axonemal localization is confirmed by multiple lines of evidence in human and mouse, including direct cryo-EM resolution of CFAP61 within the human respiratory-cilia axoneme (PMID:37258679, PDB 8J07). Reason: Correct annotation, consistent with IDA evidence from PMID:28282151 and PMID:36659204. Axoneme localization is a core aspect of CFAP61 function. The cryo-EM structure of the human axoneme (PMID:37258679) directly resolves CFAP61 as part of the RS3 base, providing structural confirmation of axonemal localization and architecture. Supporting Evidence: PMID:37258679 ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and CFAP251... |
| GO:0005200 structural constituent of cytoskeleton | ISS PMID:25694453 The CSC proteins FAP61 and FAP251 build the basal substructu... | NEW | Summary: CFAP61 functions as a structural scaffold within the axonemal CSC complex. It has no known enzymatic activity despite containing a Rossmann-like fold domain. Its loss specifically disrupts the structural integrity of radial spoke 3 in Tetrahymena and the entire axoneme in mammalian sperm. This is a molecular function annotation that is currently missing from GOA. Reason: CFAP61 has no molecular function annotation in GOA. Its role as a structural scaffold in the axoneme CSC is well established. The protein contains a Rossmann-like FAD/NAD(P)-binding fold that functions as a scaffolding domain rather than an enzyme. GO:0005200 (structural constituent of cytoskeleton) is the most appropriate MF term for a non-enzymatic structural component of the axoneme. Supporting Evidence: PMID:25694453 ...FAP61-null cilia lack an adjacent portion of the RS3 stem region... PMID:34792097 ...CFAP61 is a conserved component of the calmodulin- and radial spoke-associated complex (CSC) of cilia... CFAP61 interacts with the CSC, radial spoke stalk and head... file:human/CFAP61/CFAP61-deep-research-falcon.md CFAP61 is best supported as a structural/assembly and stabilization factor for the sperm flagellar axoneme, acting through the CSC-radial spoke system. |
| GO:0062177 radial spoke assembly | ISS PMID:25694453 The CSC proteins FAP61 and FAP251 build the basal substructu... | NEW | Summary: CFAP61 is required for radial spoke 3 assembly in Tetrahymena and for radial spoke component assembly during spermiogenesis in mouse. This process annotation is not currently in GOA but is a core function of the protein. Reason: Radial spoke assembly is the primary biological process function of CFAP61 within the CSC. In Tetrahymena, FAP61 loss specifically ablates RS3 stem structure. In mouse spermatids, CFAP61 loss impairs radial spoke component assembly during early spermiogenesis. This is more specific and informative than the existing 'cilium organization' annotation. Supporting Evidence: PMID:25694453 ...loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected... PMID:34792097 ...During early stages of Cfap61-/- spermatid development, the assembly of radial spoke components is impaired... PMID:37258679 ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and CFAP251... file:human/CFAP61/CFAP61-deep-research-falcon.md Its pathogenic loss disrupts assembly and/or maintenance of radial spokes and associated axonemal complexes, producing axoneme instability and disorganization. |
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Download this section (compressed HTML)Q: Why do human patients with biallelic CFAP61 loss-of-function mutations not present with primary ciliary dyskinesia (PCD) symptoms affecting respiratory cilia, despite CFAP61 being present in airway cilia? Is there functional redundancy in respiratory cilia that compensates for CFAP61 loss?
Q: What is the functional significance of the Rossmann-like FAD/NAD(P)-binding fold in CFAP61? Does it bind any cofactor or ligand, or is it purely a structural scaffold? Has anyone tested for oxidoreductase or other enzymatic activity?
Q: How does CFAP61 interact with intraflagellar transport (IFT) proteins WDR35, IFT22, and IFT81? Is CFAP61 itself an IFT cargo, or does it play a role in organizing IFT within the axoneme?
Experiment: Express and purify the isolated Rossmann-like domain (residues 665-998) of CFAP61 and test for FAD/NAD(P) binding by isothermal titration calorimetry or thermal shift assay. Test for oxidoreductase activity using standard spectrophotometric assays. If no cofactor binding or activity is detected, use the domain for pull-down experiments to identify its binding partners within the CSC/radial spoke machinery.
Hypothesis: The Rossmann-like domain of CFAP61 has no catalytic activity and functions purely as a protein-protein interaction scaffold.
Type: biochemical assay
Experiment: Generate conditional Cfap61 knockout in mouse airway epithelial cells. Analyze ciliary beating frequency and waveform by high-speed video microscopy. Perform cryo-ET of respiratory cilia to determine whether RS3 is affected. Compare with Cfap91 and Cfap251 knockouts to test whether CSC component redundancy differs between tissues.
Hypothesis: CFAP61 function in respiratory cilia is compensated by redundant mechanisms, explaining the absence of PCD in patients with CFAP61 mutations.
Type: mouse genetics / ciliary physiology
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