{"gene":"CFAP65","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2019,"finding":"Biallelic loss-of-function mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF), including absent, short, coiled, bent, and irregular-calibre flagella with severely decreased motility, as demonstrated in both human subjects and Cfap65 CRISPR-Cas9 knockout mice.","method":"Whole-exome sequencing in human MMAF cohort; CRISPR-Cas9 knockout mouse model with phenotypic characterization","journal":"Journal of medical genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal human genetic and mouse knockout evidence, replicated across two independent publications (PMID:31413122 and PMID:31501240) with multiple families and orthogonal methods","pmids":["31413122","31501240"],"is_preprint":false},{"year":2019,"finding":"CFAP65 mutations cause severe defects in sperm morphology including acrosome hypoplasia, disruption of the mitochondrial sheath, and absence of the central pair complex, as shown by ultrastructural and immunostaining analyses of patient spermatozoa.","method":"Transmission electron microscopy and immunostaining of spermatozoa from patients with CFAP65 mutations","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ultrastructural analysis in human patients, single lab but two orthogonal methods (TEM + immunostaining)","pmids":["31413122"],"is_preprint":false},{"year":2021,"finding":"CFAP65 is required for acrosome biogenesis (maturation phase), manchette development, and mitochondrial sheath assembly during spermiogenesis. In Cfap65-knockout mice, hyper-constricted sperm heads appear at step 9 spermatids with abnormal manchette, and flagellar elongation is severely impaired.","method":"Cfap65 knockout mouse model with histological, immunofluorescence, and electron microscopy analysis of spermatids","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotype and multiple orthogonal methods (histology, IF, EM, proteomics)","pmids":["34231842"],"is_preprint":false},{"year":2021,"finding":"CFAP65 forms a cytoplasmic protein network with MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1 during spermiogenesis, as demonstrated by endogenous immunoprecipitation and immunostaining in mouse testes.","method":"Endogenous co-immunoprecipitation and immunostaining in mouse testis","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and immunostaining from single lab, multiple binding partners identified but limited orthogonal validation","pmids":["34231842"],"is_preprint":false},{"year":2021,"finding":"In Chlamydomonas, FAP65 (the CFAP65 ortholog) is a component of the C2a projection of the ciliary central apparatus. Loss of FAP70 results in loss of FAP65 from the axoneme, and FAP65 co-immunoprecipitates with HA-tagged FAP70, placing CFAP65 within the C2a projection complex alongside FAP70 and FAP147 (MYCBPAP).","method":"Cryo-electron microscopy, mass spectrometry of fap70 mutant axonemes, and co-immunoprecipitation with HA-FAP70 in Chlamydomonas","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural localization combined with MS and co-IP in a well-established model organism for cilia research","pmids":["33988244"],"is_preprint":false},{"year":2022,"finding":"Ccdc108 (CFAP65 ortholog) is required for basal body migration and docking to the apical membrane in multiciliated cells (MCCs) of Xenopus laevis embryos. Ccdc108 interacts with the IFT-B complex, and both Ccdc108 and IFT-B proteins localize to migrating centrioles, basal bodies, and cilia. Ccdc108 governs centriolar recruitment of IFT proteins, while IFT licenses targeting of Ccdc108 to the cilium. Ccdc108 is also required for centriolar recruitment of Drg1 and activated RhoA to establish the apical actin network in MCCs.","method":"Xenopus laevis morpholino knockdown, co-immunoprecipitation with IFT-B complex, live imaging, immunofluorescence localization","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction (Co-IP with IFT-B), loss-of-function with defined cellular phenotype, multiple orthogonal methods (imaging, IP, localization with functional consequence)","pmids":["35201641"],"is_preprint":false},{"year":2024,"finding":"MYCBPAP interactome analysis in transgenic mice revealed that CFAP65 is a binding partner of MYCBPAP, and together with CFAP70 they constitute the C2a projection of the sperm flagellar central apparatus.","method":"Endogenous immunoprecipitation combined with mass spectrometry in MYCBPAP transgenic mice","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS in transgenic mouse model, single lab, identifies CFAP65 as part of the C2a complex consistent with Chlamydomonas data","pmids":["39092789"],"is_preprint":false},{"year":2024,"finding":"CFAP65 physically interacts with MYCBPAP as shown by endogenous immunoprecipitation with mass spectrometry in Mycbpap-knockout and control mouse testes, further supporting CFAP65's role in the central apparatus of the sperm axoneme.","method":"Endogenous immunoprecipitation and mass spectrometry in mouse testes","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS from single lab, replicates the CFAP65-MYCBPAP interaction found in PMID:39092789","pmids":["39704931"],"is_preprint":false},{"year":2017,"finding":"CFAP65 (as CCDC108) acts downstream of mitochondrial DNA depletion in a TFAM-mtDNA-calcium-CFAP65-PCK1 axis mediating mitochondrial retrograde signaling. Knockdown of CFAP65 rescued the effects of TFAM depletion on cancer cell morphology and proliferation, and PCK1 was found to act downstream of CFAP65 in this calcium-mediated retrograde signaling pathway.","method":"siRNA knockdown of CFAP65 in MKN45 gastric cancer cells; epistasis analysis with TFAM knockdown; calcium signaling pathway analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis by knockdown rescue, single lab, pathway placement with specific downstream effector identified","pmids":["29259235"],"is_preprint":false},{"year":2012,"finding":"Disruption of CCDC108 (CFAP65) at an inversion breakpoint in Rose-comb chickens is postulated to cause poor sperm motility in homozygous males, as CCDC108 contains an MSP (major sperm protein) domain and is expressed in testis; the inversion disrupts the gene at one breakpoint.","method":"Genetic mapping of chromosomal inversion breakpoint in chickens; expression analysis in testis","journal":"PLoS genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect genetic association (breakpoint disruption), no direct functional experiment on CFAP65/CCDC108 protein activity; postulated role only","pmids":["22761584"],"is_preprint":false},{"year":2021,"finding":"CFAP65 is expressed at all levels of mouse germ cells during spermatogenesis, as demonstrated by cellular immunofluorescence assay in mouse testes.","method":"Immunofluorescence in mouse testis","journal":"Zhonghua nan ke xue = National journal of andrology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single immunofluorescence localization experiment, single lab, no functional consequence directly linked","pmids":["34914225"],"is_preprint":false},{"year":2023,"finding":"CFAP47 regulates the expression of CFAP65 through physical interaction, as suggested by mechanism analysis in spermatozoa from patients with CFAP47 mutations showing reduced CFAP65 levels.","method":"Immunofluorescence and western blotting of patient spermatozoa; interaction analysis","journal":"Frontiers in endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single immunofluorescence/WB observation in patient material, no direct biochemical demonstration of physical interaction for CFAP65 specifically","pmids":["37424856"],"is_preprint":false}],"current_model":"CFAP65 is a conserved cilia and flagella-associated protein that forms part of the C2a projection of the ciliary/flagellar central apparatus (together with FAP70/CFAP70 and FAP147/MYCBPAP), is required during spermiogenesis for acrosome biogenesis, manchette organization, and mitochondrial sheath assembly, interacts with IFT-B complex components to regulate basal body migration and docking in multiciliated cells, and participates in a cytoplasmic protein network (with MNS1, RSPH1, TPPP2, ZPBP1, SPACA1) whose loss—through biallelic mutations—causes male infertility with multiple morphological abnormalities of the sperm flagella (MMAF) in humans and mice."},"narrative":{"mechanistic_narrative":"CFAP65 is a conserved cilia- and flagella-associated protein that functions as a structural component of the C2a projection of the ciliary/flagellar central apparatus and is essential for sperm flagellar assembly [PMID:33988244, PMID:39092789]. In the Chlamydomonas axoneme, the CFAP65 ortholog FAP65 localizes to the C2a projection together with FAP70 (CFAP70) and FAP147 (MYCBPAP), with FAP70 required for its retention in the axoneme [PMID:33988244]; the CFAP65–MYCBPAP–CFAP70 association reconstitutes this C2a complex in the mammalian sperm central apparatus [PMID:39092789, PMID:39704931]. During spermiogenesis CFAP65 organizes a cytoplasmic protein network with MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1 and is required for acrosome biogenesis, manchette development, and mitochondrial sheath assembly, such that its loss produces hyper-constricted spermatid heads, abnormal manchette, and impaired flagellar elongation [PMID:34231842]. In multiciliated cells, the ortholog Ccdc108 interacts with the IFT-B complex to control reciprocal centriolar recruitment and ciliary targeting, governing basal body migration and docking and establishment of the apical actin network via Drg1 and activated RhoA [PMID:35201641]. Biallelic loss-of-function mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF), with absent central pair, acrosome hypoplasia, and disrupted mitochondrial sheath, recapitulated in knockout mice [PMID:31413122, PMID:31501240].","teleology":[{"year":2012,"claim":"An early genetic clue placed CCDC108/CFAP65 in male reproductive function, raising the question of whether it acts in sperm motility.","evidence":"Mapping of a chromosomal inversion breakpoint disrupting CCDC108 in Rose-comb chickens, with testis expression and an MSP domain","pmids":["22761584"],"confidence":"Low","gaps":["Indirect breakpoint association only, no direct functional test of the protein","Postulated motility role not demonstrated mechanistically"]},{"year":2017,"claim":"A non-ciliary role was probed by asking whether CFAP65 participates in mitochondrial retrograde signaling in somatic cells.","evidence":"siRNA knockdown and epistasis analysis in MKN45 gastric cancer cells placing CFAP65 in a TFAM-mtDNA-calcium-CFAP65-PCK1 axis","pmids":["29259235"],"confidence":"Medium","gaps":["Mechanism of how CFAP65 transduces calcium signaling to PCK1 unresolved","Relationship to the ciliary/flagellar function unclear","Single cell line, single lab"]},{"year":2019,"claim":"Whether CFAP65 loss causes human disease was answered by establishing it as a cause of MMAF-associated male infertility with reciprocal mouse evidence.","evidence":"Whole-exome sequencing of an MMAF cohort plus a CRISPR-Cas9 knockout mouse, with TEM and immunostaining of patient spermatozoa","pmids":["31413122","31501240"],"confidence":"High","gaps":["Molecular function underlying flagellar defects not yet defined at this stage","Genotype-phenotype variability across mutations not characterized"]},{"year":2021,"claim":"The cell-biological basis of the sperm phenotype was defined by showing CFAP65 is required for acrosome biogenesis, manchette development, and mitochondrial sheath assembly, acting within a cytoplasmic partner network.","evidence":"Cfap65 knockout mouse with histology, IF and EM of spermatids, plus endogenous co-IP and immunostaining identifying MNS1, RSPH1, TPPP2, ZPBP1, SPACA1","pmids":["34231842"],"confidence":"High","gaps":["Whether the partner network is a single complex or transient associations unknown","Direct biochemical contacts within the network not mapped"]},{"year":2021,"claim":"The structural identity of CFAP65 was established by placing its Chlamydomonas ortholog in the C2a projection of the central apparatus.","evidence":"Cryo-EM, mass spectrometry of fap70 mutant axonemes, and co-IP with HA-FAP70 in Chlamydomonas","pmids":["33988244"],"confidence":"High","gaps":["High-resolution position of CFAP65 within C2a not resolved at the residue level","Whether the mammalian assembly mirrors Chlamydomonas not directly shown here"]},{"year":2022,"claim":"A distinct multiciliated-cell function was revealed by showing the ortholog Ccdc108 mediates basal body migration and docking through reciprocal regulation with the IFT-B complex.","evidence":"Xenopus morpholino knockdown, co-IP with IFT-B, live imaging and immunofluorescence localization","pmids":["35201641"],"confidence":"High","gaps":["Direct IFT-B subunit contacted by CFAP65 not pinpointed","How a central-apparatus protein also acts at basal bodies mechanistically unclear","Morpholino-based, not validated by genetic knockout"]},{"year":2024,"claim":"The mammalian C2a complex was confirmed by demonstrating CFAP65 physically binds MYCBPAP alongside CFAP70.","evidence":"Endogenous IP-MS in MYCBPAP transgenic mice and in Mycbpap-knockout/control mouse testes","pmids":["39092789","39704931"],"confidence":"Medium","gaps":["Stoichiometry and assembly order of the CFAP65-CFAP70-MYCBPAP complex unknown","Direct binary interfaces not mapped"]},{"year":null,"claim":"How CFAP65's roles as a structural central-apparatus subunit, an IFT-B-interacting basal body regulator, and a putative mitochondrial retrograde signaling factor are mechanistically reconciled remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model defining CFAP65's binding interfaces","No biochemical reconstitution of the C2a or cytoplasmic network","Connection between somatic signaling and ciliary roles unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[4,6]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[4,5]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[5]}],"complexes":["C2a projection of the ciliary/flagellar central apparatus"],"partners":["CFAP70","MYCBPAP","MNS1","RSPH1","TPPP2","ZPBP1","SPACA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6ZU64","full_name":"Cilia- and flagella-associated protein 65","aliases":["Coiled-coil domain-containing protein 108"],"length_aa":1925,"mass_kda":217.2,"function":"Plays a role in flagellar formation and sperm motility (PubMed:33472045). Essential for acrosome formation, manchette organization, spermatid head morphogenesis and mitochondrial sheath assembly within the sperm flagellum during spermiogenesis (By similarity). Also required for proper assembly and stabilization of the C2a projection, a structural subunit of the central pair apparatus within the axoneme of motile cilia and flagella (By similarity)","subcellular_location":"Cell projection, cilium, flagellum membrane; Cytoplasmic vesicle, secretory vesicle, acrosome membrane; Cytoplasm; Cell projection, cilium, flagellum; Cytoplasm, cytoskeleton, cilium axoneme; Cytoplasm, cytoskeleton, flagellum axoneme","url":"https://www.uniprot.org/uniprotkb/Q6ZU64/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CFAP65","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CFAP65","total_profiled":1310},"omim":[{"mim_id":"618664","title":"SPERMATOGENIC FAILURE 40; SPGF40","url":"https://www.omim.org/entry/618664"},{"mim_id":"617593","title":"SPERMATOGENIC FAILURE 20; SPGF20","url":"https://www.omim.org/entry/617593"},{"mim_id":"617592","title":"SPERMATOGENIC FAILURE 19; SPGF19","url":"https://www.omim.org/entry/617592"},{"mim_id":"614270","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 65; CFAP65","url":"https://www.omim.org/entry/614270"},{"mim_id":"301057","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 47; CFAP47","url":"https://www.omim.org/entry/301057"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":8.9},{"tissue":"fallopian tube","ntpm":24.2},{"tissue":"retina","ntpm":8.5},{"tissue":"testis","ntpm":8.8}],"url":"https://www.proteinatlas.org/search/CFAP65"},"hgnc":{"alias_symbol":["DKFZp434O0527","MGC35338"],"prev_symbol":["CCDC108"]},"alphafold":{"accession":"Q6ZU64","domains":[{"cath_id":"2.60.40.10","chopping":"140-238","consensus_level":"high","plddt":82.33,"start":140,"end":238},{"cath_id":"2.60.40.10","chopping":"245-341","consensus_level":"high","plddt":87.8153,"start":245,"end":341},{"cath_id":"2.60.40.10","chopping":"344-462","consensus_level":"medium","plddt":79.1051,"start":344,"end":462},{"cath_id":"2.60.40.10","chopping":"573-605_641-771","consensus_level":"medium","plddt":88.0728,"start":573,"end":771},{"cath_id":"2.60.40.10","chopping":"774-875","consensus_level":"medium","plddt":86.8066,"start":774,"end":875},{"cath_id":"2.60.40.10","chopping":"878-990","consensus_level":"medium","plddt":83.2768,"start":878,"end":990},{"cath_id":"2.60.40.10","chopping":"994-1105","consensus_level":"high","plddt":82.4606,"start":994,"end":1105},{"cath_id":"2.60.40.10","chopping":"1110-1212_1225-1292","consensus_level":"medium","plddt":82.5379,"start":1110,"end":1292},{"cath_id":"2.60.40.10","chopping":"1429-1543_1607-1640","consensus_level":"medium","plddt":79.1266,"start":1429,"end":1640}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZU64","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZU64-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZU64-F1-predicted_aligned_error_v6.png","plddt_mean":70.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CFAP65","jax_strain_url":"https://www.jax.org/strain/search?query=CFAP65"},"sequence":{"accession":"Q6ZU64","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6ZU64.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6ZU64/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZU64"}},"corpus_meta":[{"pmid":"28552195","id":"PMC_28552195","title":"Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella.","date":"2017","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28552195","citation_count":226,"is_preprint":false},{"pmid":"33472045","id":"PMC_33472045","title":"Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility.","date":"2021","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33472045","citation_count":105,"is_preprint":false},{"pmid":"22761584","id":"PMC_22761584","title":"The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility.","date":"2012","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22761584","citation_count":97,"is_preprint":false},{"pmid":"31413122","id":"PMC_31413122","title":"Biallelic mutations in CFAP65 lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations.","date":"2019","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31413122","citation_count":66,"is_preprint":false},{"pmid":"31501240","id":"PMC_31501240","title":"Biallelic mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella in humans and mice.","date":"2019","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31501240","citation_count":63,"is_preprint":false},{"pmid":"29259235","id":"PMC_29259235","title":"Transcriptomic analysis of mitochondrial TFAM depletion changing cell morphology and proliferation.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29259235","citation_count":37,"is_preprint":false},{"pmid":"34231842","id":"PMC_34231842","title":"CFAP65 is required in the acrosome biogenesis and mitochondrial sheath assembly during 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In Cfap65-knockout mice, hyper-constricted sperm heads appear at step 9 spermatids with abnormal manchette, and flagellar elongation is severely impaired.\",\n      \"method\": \"Cfap65 knockout mouse model with histological, immunofluorescence, and electron microscopy analysis of spermatids\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotype and multiple orthogonal methods (histology, IF, EM, proteomics)\",\n      \"pmids\": [\"34231842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CFAP65 forms a cytoplasmic protein network with MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1 during spermiogenesis, as demonstrated by endogenous immunoprecipitation and immunostaining in mouse testes.\",\n      \"method\": \"Endogenous co-immunoprecipitation and immunostaining in mouse testis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and immunostaining from single lab, multiple binding partners identified but limited orthogonal validation\",\n      \"pmids\": [\"34231842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In Chlamydomonas, FAP65 (the CFAP65 ortholog) is a component of the C2a projection of the ciliary central apparatus. Loss of FAP70 results in loss of FAP65 from the axoneme, and FAP65 co-immunoprecipitates with HA-tagged FAP70, placing CFAP65 within the C2a projection complex alongside FAP70 and FAP147 (MYCBPAP).\",\n      \"method\": \"Cryo-electron microscopy, mass spectrometry of fap70 mutant axonemes, and co-immunoprecipitation with HA-FAP70 in Chlamydomonas\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structural localization combined with MS and co-IP in a well-established model organism for cilia research\",\n      \"pmids\": [\"33988244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ccdc108 (CFAP65 ortholog) is required for basal body migration and docking to the apical membrane in multiciliated cells (MCCs) of Xenopus laevis embryos. Ccdc108 interacts with the IFT-B complex, and both Ccdc108 and IFT-B proteins localize to migrating centrioles, basal bodies, and cilia. Ccdc108 governs centriolar recruitment of IFT proteins, while IFT licenses targeting of Ccdc108 to the cilium. Ccdc108 is also required for centriolar recruitment of Drg1 and activated RhoA to establish the apical actin network in MCCs.\",\n      \"method\": \"Xenopus laevis morpholino knockdown, co-immunoprecipitation with IFT-B complex, live imaging, immunofluorescence localization\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction (Co-IP with IFT-B), loss-of-function with defined cellular phenotype, multiple orthogonal methods (imaging, IP, localization with functional consequence)\",\n      \"pmids\": [\"35201641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MYCBPAP interactome analysis in transgenic mice revealed that CFAP65 is a binding partner of MYCBPAP, and together with CFAP70 they constitute the C2a projection of the sperm flagellar central apparatus.\",\n      \"method\": \"Endogenous immunoprecipitation combined with mass spectrometry in MYCBPAP transgenic mice\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS in transgenic mouse model, single lab, identifies CFAP65 as part of the C2a complex consistent with Chlamydomonas data\",\n      \"pmids\": [\"39092789\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CFAP65 physically interacts with MYCBPAP as shown by endogenous immunoprecipitation with mass spectrometry in Mycbpap-knockout and control mouse testes, further supporting CFAP65's role in the central apparatus of the sperm axoneme.\",\n      \"method\": \"Endogenous immunoprecipitation and mass spectrometry in mouse testes\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS from single lab, replicates the CFAP65-MYCBPAP interaction found in PMID:39092789\",\n      \"pmids\": [\"39704931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CFAP65 (as CCDC108) acts downstream of mitochondrial DNA depletion in a TFAM-mtDNA-calcium-CFAP65-PCK1 axis mediating mitochondrial retrograde signaling. Knockdown of CFAP65 rescued the effects of TFAM depletion on cancer cell morphology and proliferation, and PCK1 was found to act downstream of CFAP65 in this calcium-mediated retrograde signaling pathway.\",\n      \"method\": \"siRNA knockdown of CFAP65 in MKN45 gastric cancer cells; epistasis analysis with TFAM knockdown; calcium signaling pathway analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis by knockdown rescue, single lab, pathway placement with specific downstream effector identified\",\n      \"pmids\": [\"29259235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Disruption of CCDC108 (CFAP65) at an inversion breakpoint in Rose-comb chickens is postulated to cause poor sperm motility in homozygous males, as CCDC108 contains an MSP (major sperm protein) domain and is expressed in testis; the inversion disrupts the gene at one breakpoint.\",\n      \"method\": \"Genetic mapping of chromosomal inversion breakpoint in chickens; expression analysis in testis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect genetic association (breakpoint disruption), no direct functional experiment on CFAP65/CCDC108 protein activity; postulated role only\",\n      \"pmids\": [\"22761584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CFAP65 is expressed at all levels of mouse germ cells during spermatogenesis, as demonstrated by cellular immunofluorescence assay in mouse testes.\",\n      \"method\": \"Immunofluorescence in mouse testis\",\n      \"journal\": \"Zhonghua nan ke xue = National journal of andrology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single immunofluorescence localization experiment, single lab, no functional consequence directly linked\",\n      \"pmids\": [\"34914225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CFAP47 regulates the expression of CFAP65 through physical interaction, as suggested by mechanism analysis in spermatozoa from patients with CFAP47 mutations showing reduced CFAP65 levels.\",\n      \"method\": \"Immunofluorescence and western blotting of patient spermatozoa; interaction analysis\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single immunofluorescence/WB observation in patient material, no direct biochemical demonstration of physical interaction for CFAP65 specifically\",\n      \"pmids\": [\"37424856\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CFAP65 is a conserved cilia and flagella-associated protein that forms part of the C2a projection of the ciliary/flagellar central apparatus (together with FAP70/CFAP70 and FAP147/MYCBPAP), is required during spermiogenesis for acrosome biogenesis, manchette organization, and mitochondrial sheath assembly, interacts with IFT-B complex components to regulate basal body migration and docking in multiciliated cells, and participates in a cytoplasmic protein network (with MNS1, RSPH1, TPPP2, ZPBP1, SPACA1) whose loss—through biallelic mutations—causes male infertility with multiple morphological abnormalities of the sperm flagella (MMAF) in humans and mice.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CFAP65 is a conserved cilia- and flagella-associated protein that functions as a structural component of the C2a projection of the ciliary/flagellar central apparatus and is essential for sperm flagellar assembly [#4, #6]. In the Chlamydomonas axoneme, the CFAP65 ortholog FAP65 localizes to the C2a projection together with FAP70 (CFAP70) and FAP147 (MYCBPAP), with FAP70 required for its retention in the axoneme [#4]; the CFAP65–MYCBPAP–CFAP70 association reconstitutes this C2a complex in the mammalian sperm central apparatus [#6, #7]. During spermiogenesis CFAP65 organizes a cytoplasmic protein network with MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1 and is required for acrosome biogenesis, manchette development, and mitochondrial sheath assembly, such that its loss produces hyper-constricted spermatid heads, abnormal manchette, and impaired flagellar elongation [#2, #3]. In multiciliated cells, the ortholog Ccdc108 interacts with the IFT-B complex to control reciprocal centriolar recruitment and ciliary targeting, governing basal body migration and docking and establishment of the apical actin network via Drg1 and activated RhoA [#5]. Biallelic loss-of-function mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF), with absent central pair, acrosome hypoplasia, and disrupted mitochondrial sheath, recapitulated in knockout mice [#0, #1].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"An early genetic clue placed CCDC108/CFAP65 in male reproductive function, raising the question of whether it acts in sperm motility.\",\n      \"evidence\": \"Mapping of a chromosomal inversion breakpoint disrupting CCDC108 in Rose-comb chickens, with testis expression and an MSP domain\",\n      \"pmids\": [\"22761584\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Indirect breakpoint association only, no direct functional test of the protein\", \"Postulated motility role not demonstrated mechanistically\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A non-ciliary role was probed by asking whether CFAP65 participates in mitochondrial retrograde signaling in somatic cells.\",\n      \"evidence\": \"siRNA knockdown and epistasis analysis in MKN45 gastric cancer cells placing CFAP65 in a TFAM-mtDNA-calcium-CFAP65-PCK1 axis\",\n      \"pmids\": [\"29259235\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of how CFAP65 transduces calcium signaling to PCK1 unresolved\", \"Relationship to the ciliary/flagellar function unclear\", \"Single cell line, single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Whether CFAP65 loss causes human disease was answered by establishing it as a cause of MMAF-associated male infertility with reciprocal mouse evidence.\",\n      \"evidence\": \"Whole-exome sequencing of an MMAF cohort plus a CRISPR-Cas9 knockout mouse, with TEM and immunostaining of patient spermatozoa\",\n      \"pmids\": [\"31413122\", \"31501240\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function underlying flagellar defects not yet defined at this stage\", \"Genotype-phenotype variability across mutations not characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The cell-biological basis of the sperm phenotype was defined by showing CFAP65 is required for acrosome biogenesis, manchette development, and mitochondrial sheath assembly, acting within a cytoplasmic partner network.\",\n      \"evidence\": \"Cfap65 knockout mouse with histology, IF and EM of spermatids, plus endogenous co-IP and immunostaining identifying MNS1, RSPH1, TPPP2, ZPBP1, SPACA1\",\n      \"pmids\": [\"34231842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the partner network is a single complex or transient associations unknown\", \"Direct biochemical contacts within the network not mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The structural identity of CFAP65 was established by placing its Chlamydomonas ortholog in the C2a projection of the central apparatus.\",\n      \"evidence\": \"Cryo-EM, mass spectrometry of fap70 mutant axonemes, and co-IP with HA-FAP70 in Chlamydomonas\",\n      \"pmids\": [\"33988244\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution position of CFAP65 within C2a not resolved at the residue level\", \"Whether the mammalian assembly mirrors Chlamydomonas not directly shown here\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A distinct multiciliated-cell function was revealed by showing the ortholog Ccdc108 mediates basal body migration and docking through reciprocal regulation with the IFT-B complex.\",\n      \"evidence\": \"Xenopus morpholino knockdown, co-IP with IFT-B, live imaging and immunofluorescence localization\",\n      \"pmids\": [\"35201641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct IFT-B subunit contacted by CFAP65 not pinpointed\", \"How a central-apparatus protein also acts at basal bodies mechanistically unclear\", \"Morpholino-based, not validated by genetic knockout\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The mammalian C2a complex was confirmed by demonstrating CFAP65 physically binds MYCBPAP alongside CFAP70.\",\n      \"evidence\": \"Endogenous IP-MS in MYCBPAP transgenic mice and in Mycbpap-knockout/control mouse testes\",\n      \"pmids\": [\"39092789\", \"39704931\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and assembly order of the CFAP65-CFAP70-MYCBPAP complex unknown\", \"Direct binary interfaces not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CFAP65's roles as a structural central-apparatus subunit, an IFT-B-interacting basal body regulator, and a putative mitochondrial retrograde signaling factor are mechanistically reconciled remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model defining CFAP65's binding interfaces\", \"No biochemical reconstitution of the C2a or cytoplasmic network\", \"Connection between somatic signaling and ciliary roles unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\n      \"C2a projection of the ciliary/flagellar central apparatus\"\n    ],\n    \"partners\": [\n      \"CFAP70\",\n      \"MYCBPAP\",\n      \"MNS1\",\n      \"RSPH1\",\n      \"TPPP2\",\n      \"ZPBP1\",\n      \"SPACA1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}