{"gene":"CCDC61","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2018,"finding":"Ccdc61 localizes to centrosomes and is required for spindle assembly and symmetry in mitosis; its depletion causes loss of intrinsic symmetry of microtubule tracks within the spindle and impairs the binding between Cep170 and TANK-binding kinase 1 (TBK1), an interaction required for microtubule stability.","method":"siRNA knockdown, microtubule tip-tracking experiments, co-immunoprecipitation, fluorescence microscopy","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction data plus live microtubule dynamics in KD cells, single lab, multiple orthogonal methods","pmids":["30354798"],"is_preprint":false},{"year":2019,"finding":"hVFL3/CCDC61 localizes to subdistal appendages (SAP) and proximal ends of the mother centriole, physically interacts with Cep170, is required for centrosome cohesion (depletion increases mother-daughter centriole distance and exacerbates centriole splitting when the rootletin/C-Nap1 linker is disrupted), is required for centrosome positioning in interphase cells, and directly binds microtubules.","method":"Immunofluorescence, co-immunoprecipitation (physical interaction with Cep170), siRNA depletion with phenotypic rescue, microtubule co-sedimentation assay","journal":"Biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, MT binding assay, KD epistasis with C-Nap1, positioning assay) in single lab, consistent with independent structural study","pmids":["31789463"],"is_preprint":false},{"year":2020,"finding":"CCDC61 is a structural paralog of SAS6; crystal structures reveal it contains two homodimerization interfaces homologous to SAS6 but that drive formation of linear filaments rather than rings. CCDC61 binds microtubules, and residues involved in microtubule binding are required for ciliary function in Chlamydomonas.","method":"X-ray crystallography, in vitro microtubule-binding assay, mutagenesis of microtubule-binding residues, Chlamydomonas complementation/ciliary function assay","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro reconstitution of MT binding plus mutagenesis with functional readout in a single rigorous study","pmids":["32375023"],"is_preprint":false},{"year":2022,"finding":"Ccdc61 encodes a basal body component localizing proximally at the junction with striated rootlets in Xenopus multiciliated cells; morpholino knockdown causes defects in basal body docking, spacing, and polarization, impairs the apical cytoskeleton, and reduces ciliary beating and cilia-powered fluid flow.","method":"Morpholino knockdown in Xenopus embryonic ciliated epidermis, immunofluorescence localization, fluid-flow assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotypes at multiple organizational scales, single lab, vertebrate model","pmids":["35067717"],"is_preprint":false},{"year":2023,"finding":"A synthetic peptide derived from the CCDC61 protein specifically captures intact centrosomes via affinity pull-down, enabling centrosome proteome isolation (CAPture-MS), demonstrating that CCDC61 provides a centrosome-specific molecular handle sufficient for one-step centrosome purification.","method":"Affinity capture using CCDC61-derived peptide coupled to beads, mass spectrometry, electron microscopy verification of intact centrosomes","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical demonstration of centrosome-specific binding activity of CCDC61 peptide, single study with multiple validation methods","pmids":["37852252"],"is_preprint":false},{"year":2017,"finding":"In Paramecium tetraurelia, VFL3-A (ortholog of CCDC61/VFL3) localizes transiently near basal bodies at early duplication, at the junction between the striated rootlet and the basal body, is required for recruitment of Centrin 3, and is required for rotational asymmetry of the basal body that specifies assembly sites for appendages guiding basal body movement to the cell surface; depletion results in unanchored basal bodies with disorganized rootlet distribution.","method":"RNAi depletion in Paramecium, immunofluorescence localization, phenotypic analysis of basal body docking and rootlet organization","journal":"Cilia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined molecular (Centrin 3 recruitment) and structural phenotype, single lab, ciliate ortholog","pmids":["28367320"],"is_preprint":false},{"year":2015,"finding":"Using the vfl3 mutant of Chlamydomonas (which lacks normal striated fibers connecting flagella), hydrodynamic synchronization experiments demonstrated that flagellar synchronization requires intracellular mechanical coupling through internal fibers rather than hydrodynamic forces; vfl3 cells with impaired mechanical connections between flagella cannot synchronize under physiological hydrodynamic forces.","method":"Controlled external flow hydrodynamics experiments, high-speed imaging of vfl3 mutant flagella","journal":"Physical review letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct mechanical perturbation experiment using vfl3 mutant as genetic tool, single study with clear functional readout","pmids":["26684142"],"is_preprint":false},{"year":2001,"finding":"In Chlamydomonas, a mutation in the VFL3 gene abolishes the templated centriole duplication pathway without eliminating de novo centriole assembly, establishing that VFL3 is specifically required for the templated (preexisting-centriole-dependent) pathway of centriole duplication.","method":"Genetic analysis of vfl3 mutant, quantification of de novo vs templated centriole assembly in centrioleless cells","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in defined mutant background with quantitative assembly assay, single lab, Chlamydomonas ortholog","pmids":["11267867"],"is_preprint":false},{"year":1984,"finding":"In Chlamydomonas vfl-3 mutant, which lacks normal striated fibers and microtubular rootlets, flagella beat vigorously but display variable rotational orientation of basal bodies, establishing that striated fibers and/or associated structures are required for establishing or maintaining correct rotational orientation of basal bodies rather than for flagellar beating per se.","method":"High-speed cinephotomicrography, structural polarity marker analysis in vfl-3 mutant","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined mutant with clear structural and functional phenotype, single lab, foundational Chlamydomonas ortholog study","pmids":["6699086"],"is_preprint":false}],"current_model":"CCDC61/hVFL3 is a centrosomal protein — a structural paralog of SAS6 that forms linear filaments via two homodimerization interfaces — which localizes to subdistal appendages and proximal ends of the mother centriole, directly binds microtubules (with MT-binding residues required for ciliary function), physically interacts with Cep170 to support TBK1-mediated microtubule stability, is required for centrosome cohesion and positioning in interphase, and is required for spindle assembly and symmetry in mitosis; in unicellular eukaryotes its orthologs establish basal body rotational asymmetry and anchor striated rootlets/cytoskeletal fibers to enable proper ciliary orientation, templated centriole duplication, and flagellar synchronization through mechanical coupling."},"narrative":{"mechanistic_narrative":"CCDC61 (hVFL3) is a centriolar/centrosomal structural protein that organizes the geometry and connectivity of basal bodies and centrioles across eukaryotes [PMID:31789463, PMID:32375023, PMID:6699086]. Crystal structures establish it as a structural paralog of SAS6: it retains two homodimerization interfaces homologous to those of SAL6 but uses them to polymerize into linear filaments rather than rings, and it directly binds microtubules through residues required for ciliary function [PMID:32375023]. In human cells it localizes to subdistal appendages and proximal ends of the mother centriole, where it directly binds microtubules, physically interacts with Cep170, and is required for centrosome cohesion and interphase centrosome positioning; loss of CCDC61 increases mother-daughter centriole distance and exacerbates splitting when the rootletin/C-Nap1 linker is disrupted [PMID:31789463]. The Cep170 interaction supports the Cep170-TBK1 association required for microtubule stability, and CCDC61 depletion disrupts intrinsic spindle symmetry and mitotic spindle assembly [PMID:30354798]. Across unicellular eukaryotes and Xenopus, its orthologs localize at the junction between basal bodies and striated rootlets and are required for basal body rotational asymmetry, docking, spacing and polarization, templated centriole duplication, and mechanical coupling that synchronizes flagellar beating [PMID:35067717, PMID:28367320, PMID:26684142, PMID:11267867, PMID:6699086]. Its centrosome-specific binding is sufficiently selective that a CCDC61-derived peptide captures intact centrosomes for proteome isolation [PMID:37852252].","teleology":[{"year":1984,"claim":"Established the founding phenotype linking this gene to ciliary architecture by asking whether basal body orientation depends on associated fibers rather than on beating machinery.","evidence":"High-speed cinephotomicrography and polarity marker analysis of the Chlamydomonas vfl-3 mutant lacking striated fibers and microtubular rootlets","pmids":["6699086"],"confidence":"Medium","gaps":["Gene product not molecularly identified","Mechanism connecting fibers to rotational orientation unknown"]},{"year":2001,"claim":"Defined a specific requirement in centriole biogenesis by distinguishing templated from de novo assembly pathways.","evidence":"Genetic analysis of vfl3 mutant with quantification of de novo vs templated centriole assembly in centrioleless Chlamydomonas","pmids":["11267867"],"confidence":"Medium","gaps":["Molecular role in templating unresolved","No biochemical activity assigned"]},{"year":2015,"claim":"Showed how the structures depending on this gene contribute to organismal function by testing whether flagellar synchronization is mechanical or hydrodynamic.","evidence":"Controlled external-flow hydrodynamics and high-speed imaging of vfl3 mutant Chlamydomonas flagella","pmids":["26684142"],"confidence":"Medium","gaps":["Uses mutant as a tool; does not define the protein's molecular function","Coupling structure not molecularly dissected"]},{"year":2017,"claim":"Connected the ortholog to a molecular recruitment event and basal body asymmetry, advancing from phenotype to a defined molecular dependency.","evidence":"RNAi depletion, immunofluorescence localization, and basal body/rootlet phenotyping in Paramecium tetraurelia","pmids":["28367320"],"confidence":"Medium","gaps":["Direct interaction with Centrin 3 not demonstrated","Mechanism of asymmetry specification unknown"]},{"year":2018,"claim":"Extended function into mitosis and into a defined human protein interaction, asking how CCDC61 contributes to spindle symmetry and microtubule stability.","evidence":"siRNA knockdown, microtubule tip-tracking, and co-immunoprecipitation in human cells","pmids":["30354798"],"confidence":"Medium","gaps":["Whether CCDC61 directly bridges Cep170-TBK1 vs indirectly is unresolved","Structural basis of spindle symmetry effect unknown"]},{"year":2019,"claim":"Defined the human protein's centriolar localization and a coherent set of structural roles, establishing it as a centrosome cohesion and positioning factor that binds microtubules and Cep170.","evidence":"Immunofluorescence, co-IP, microtubule co-sedimentation, and siRNA epistasis with the C-Nap1 linker in human cells","pmids":["31789463"],"confidence":"High","gaps":["Mechanism by which it enforces cohesion at the linker not detailed","Spatial relationship to subdistal appendage assembly unresolved"]},{"year":2020,"claim":"Provided the structural mechanism by revealing CCDC61 as a SAS6 paralog that polymerizes into filaments and directly binds microtubules via residues required for ciliary function.","evidence":"X-ray crystallography, in vitro microtubule-binding assay, and mutagenesis with Chlamydomonas ciliary complementation","pmids":["32375023"],"confidence":"High","gaps":["In vivo architecture of the filaments not visualized","How filament assembly couples to centriole/rootlet positioning unknown"]},{"year":2022,"claim":"Established the vertebrate multiciliated-cell role, showing CCDC61 is required for basal body docking, spacing, polarization, and cilia-driven fluid flow.","evidence":"Morpholino knockdown, immunofluorescence localization, and fluid-flow assay in Xenopus ciliated epidermis","pmids":["35067717"],"confidence":"Medium","gaps":["Molecular partners in vertebrate basal body/rootlet junction not identified","Relationship to apical cytoskeleton mechanistically undefined"]},{"year":2023,"claim":"Demonstrated the selectivity of CCDC61's centrosome binding by turning it into a one-step centrosome capture reagent.","evidence":"Affinity capture with a CCDC61-derived peptide, mass spectrometry, and electron microscopy verification of intact centrosomes","pmids":["37852252"],"confidence":"Medium","gaps":["Endogenous binding partner of the capture peptide not defined","Does not address native CCDC61 function"]},{"year":null,"claim":"How CCDC61 filament assembly is spatially regulated to coordinate subdistal appendage organization, centriole cohesion, and rootlet anchoring within a single mechanistic framework remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No in vivo structure of CCDC61 filaments at the centriole","Regulatory inputs governing its assembly/disassembly unknown","Direct vs indirect contribution to Cep170-TBK1 microtubule stability unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0]}],"complexes":[],"partners":["CEP170","TBK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y6R9","full_name":"Centrosomal protein CCDC61","aliases":["Coiled-coil domain-containing protein 61","VFL3 homolog"],"length_aa":512,"mass_kda":57.4,"function":"Microtubule-binding centrosomal protein required for centriole cohesion, independently of the centrosome-associated protein/CEP250 and rootletin/CROCC linker (PubMed:31789463). In interphase, required for anchoring microtubule at the mother centriole subdistal appendages and for centrosome positioning (PubMed:31789463). During mitosis, may be involved in spindle assembly and chromatin alignment by regulating the organization of spindle microtubules into a symmetrical structure (PubMed:30354798). Has been proposed to play a role in CEP170 recruitment to centrosomes (PubMed:30354798). However, this function could not be confirmed (PubMed:31789463). Plays a non-essential role in ciliogenesis (PubMed:31789463, PubMed:32375023)","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriolar satellite; Cytoplasm, cytoskeleton, cilium basal body","url":"https://www.uniprot.org/uniprotkb/Q9Y6R9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CCDC61","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CCDC61","total_profiled":1310},"omim":[{"mim_id":"620676","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 61; CCDC61","url":"https://www.omim.org/entry/620676"},{"mim_id":"617791","title":"LEUCINE-RICH REPEAT- AND COILED-COIL DOMAIN-CONTAINING CENTROSOMAL PROTEIN 1; LRRCC1","url":"https://www.omim.org/entry/617791"},{"mim_id":"613023","title":"CENTROSOMAL PROTEIN, 170-KD; CEP170","url":"https://www.omim.org/entry/613023"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CCDC61"},"hgnc":{"alias_symbol":["VFL3","hVFL3"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6R9","domains":[{"cath_id":"2.170.210.10","chopping":"7-101_112-144","consensus_level":"high","plddt":91.5352,"start":7,"end":144}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6R9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6R9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6R9-F1-predicted_aligned_error_v6.png","plddt_mean":70.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CCDC61","jax_strain_url":"https://www.jax.org/strain/search?query=CCDC61"},"sequence":{"accession":"Q9Y6R9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6R9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6R9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6R9"}},"corpus_meta":[{"pmid":"11267867","id":"PMC_11267867","title":"Kinetics and regulation of de novo centriole assembly. Implications for the mechanism of centriole duplication.","date":"2001","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/11267867","citation_count":112,"is_preprint":false},{"pmid":"2696598","id":"PMC_2696598","title":"Nucleus-basal body connector in Chlamydomonas: evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity.","date":"1989","source":"Cell motility and the cytoskeleton","url":"https://pubmed.ncbi.nlm.nih.gov/2696598","citation_count":59,"is_preprint":false},{"pmid":"26684142","id":"PMC_26684142","title":"Hydrodynamics Versus Intracellular Coupling in the Synchronization of Eukaryotic Flagella.","date":"2015","source":"Physical review letters","url":"https://pubmed.ncbi.nlm.nih.gov/26684142","citation_count":50,"is_preprint":false},{"pmid":"6699086","id":"PMC_6699086","title":"Flagellar waveform and rotational orientation in a Chlamydomonas mutant lacking normal striated fibers.","date":"1984","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/6699086","citation_count":32,"is_preprint":false},{"pmid":"28367320","id":"PMC_28367320","title":"Basal body positioning and anchoring in the multiciliated cell Paramecium tetraurelia: roles of OFD1 and VFL3.","date":"2017","source":"Cilia","url":"https://pubmed.ncbi.nlm.nih.gov/28367320","citation_count":24,"is_preprint":false},{"pmid":"30354798","id":"PMC_30354798","title":"Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry.","date":"2018","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/30354798","citation_count":21,"is_preprint":false},{"pmid":"31743665","id":"PMC_31743665","title":"Emergence of a Bilaterally Symmetric Pattern from Chiral Components in the Planarian Epidermis.","date":"2019","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/31743665","citation_count":20,"is_preprint":false},{"pmid":"37852252","id":"PMC_37852252","title":"Proteomic profiling of centrosomes across multiple mammalian cell and tissue types by an affinity capture method.","date":"2023","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/37852252","citation_count":18,"is_preprint":false},{"pmid":"23320108","id":"PMC_23320108","title":"Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23320108","citation_count":15,"is_preprint":false},{"pmid":"31789463","id":"PMC_31789463","title":"hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning.","date":"2019","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/31789463","citation_count":13,"is_preprint":false},{"pmid":"32375023","id":"PMC_32375023","title":"CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions.","date":"2020","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/32375023","citation_count":10,"is_preprint":false},{"pmid":"35067717","id":"PMC_35067717","title":"Lrrcc1 and Ccdc61 are conserved effectors of multiciliated cell function.","date":"2022","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/35067717","citation_count":9,"is_preprint":false},{"pmid":"32107856","id":"PMC_32107856","title":"Genetic basis of relapsing polychondritis revealed by family-based whole-exome sequencing.","date":"2020","source":"International journal of rheumatic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/32107856","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8184,"output_tokens":2311,"usd":0.029608,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9389,"output_tokens":2918,"usd":0.059948,"stage2_stop_reason":"end_turn"},"total_usd":0.089556,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"Ccdc61 localizes to centrosomes and is required for spindle assembly and symmetry in mitosis; its depletion causes loss of intrinsic symmetry of microtubule tracks within the spindle and impairs the binding between Cep170 and TANK-binding kinase 1 (TBK1), an interaction required for microtubule stability.\",\n      \"method\": \"siRNA knockdown, microtubule tip-tracking experiments, co-immunoprecipitation, fluorescence microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction data plus live microtubule dynamics in KD cells, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"30354798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"hVFL3/CCDC61 localizes to subdistal appendages (SAP) and proximal ends of the mother centriole, physically interacts with Cep170, is required for centrosome cohesion (depletion increases mother-daughter centriole distance and exacerbates centriole splitting when the rootletin/C-Nap1 linker is disrupted), is required for centrosome positioning in interphase cells, and directly binds microtubules.\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation (physical interaction with Cep170), siRNA depletion with phenotypic rescue, microtubule co-sedimentation assay\",\n      \"journal\": \"Biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, MT binding assay, KD epistasis with C-Nap1, positioning assay) in single lab, consistent with independent structural study\",\n      \"pmids\": [\"31789463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CCDC61 is a structural paralog of SAS6; crystal structures reveal it contains two homodimerization interfaces homologous to SAS6 but that drive formation of linear filaments rather than rings. CCDC61 binds microtubules, and residues involved in microtubule binding are required for ciliary function in Chlamydomonas.\",\n      \"method\": \"X-ray crystallography, in vitro microtubule-binding assay, mutagenesis of microtubule-binding residues, Chlamydomonas complementation/ciliary function assay\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro reconstitution of MT binding plus mutagenesis with functional readout in a single rigorous study\",\n      \"pmids\": [\"32375023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ccdc61 encodes a basal body component localizing proximally at the junction with striated rootlets in Xenopus multiciliated cells; morpholino knockdown causes defects in basal body docking, spacing, and polarization, impairs the apical cytoskeleton, and reduces ciliary beating and cilia-powered fluid flow.\",\n      \"method\": \"Morpholino knockdown in Xenopus embryonic ciliated epidermis, immunofluorescence localization, fluid-flow assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotypes at multiple organizational scales, single lab, vertebrate model\",\n      \"pmids\": [\"35067717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A synthetic peptide derived from the CCDC61 protein specifically captures intact centrosomes via affinity pull-down, enabling centrosome proteome isolation (CAPture-MS), demonstrating that CCDC61 provides a centrosome-specific molecular handle sufficient for one-step centrosome purification.\",\n      \"method\": \"Affinity capture using CCDC61-derived peptide coupled to beads, mass spectrometry, electron microscopy verification of intact centrosomes\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical demonstration of centrosome-specific binding activity of CCDC61 peptide, single study with multiple validation methods\",\n      \"pmids\": [\"37852252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In Paramecium tetraurelia, VFL3-A (ortholog of CCDC61/VFL3) localizes transiently near basal bodies at early duplication, at the junction between the striated rootlet and the basal body, is required for recruitment of Centrin 3, and is required for rotational asymmetry of the basal body that specifies assembly sites for appendages guiding basal body movement to the cell surface; depletion results in unanchored basal bodies with disorganized rootlet distribution.\",\n      \"method\": \"RNAi depletion in Paramecium, immunofluorescence localization, phenotypic analysis of basal body docking and rootlet organization\",\n      \"journal\": \"Cilia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined molecular (Centrin 3 recruitment) and structural phenotype, single lab, ciliate ortholog\",\n      \"pmids\": [\"28367320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Using the vfl3 mutant of Chlamydomonas (which lacks normal striated fibers connecting flagella), hydrodynamic synchronization experiments demonstrated that flagellar synchronization requires intracellular mechanical coupling through internal fibers rather than hydrodynamic forces; vfl3 cells with impaired mechanical connections between flagella cannot synchronize under physiological hydrodynamic forces.\",\n      \"method\": \"Controlled external flow hydrodynamics experiments, high-speed imaging of vfl3 mutant flagella\",\n      \"journal\": \"Physical review letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct mechanical perturbation experiment using vfl3 mutant as genetic tool, single study with clear functional readout\",\n      \"pmids\": [\"26684142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"In Chlamydomonas, a mutation in the VFL3 gene abolishes the templated centriole duplication pathway without eliminating de novo centriole assembly, establishing that VFL3 is specifically required for the templated (preexisting-centriole-dependent) pathway of centriole duplication.\",\n      \"method\": \"Genetic analysis of vfl3 mutant, quantification of de novo vs templated centriole assembly in centrioleless cells\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in defined mutant background with quantitative assembly assay, single lab, Chlamydomonas ortholog\",\n      \"pmids\": [\"11267867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"In Chlamydomonas vfl-3 mutant, which lacks normal striated fibers and microtubular rootlets, flagella beat vigorously but display variable rotational orientation of basal bodies, establishing that striated fibers and/or associated structures are required for establishing or maintaining correct rotational orientation of basal bodies rather than for flagellar beating per se.\",\n      \"method\": \"High-speed cinephotomicrography, structural polarity marker analysis in vfl-3 mutant\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined mutant with clear structural and functional phenotype, single lab, foundational Chlamydomonas ortholog study\",\n      \"pmids\": [\"6699086\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CCDC61/hVFL3 is a centrosomal protein — a structural paralog of SAS6 that forms linear filaments via two homodimerization interfaces — which localizes to subdistal appendages and proximal ends of the mother centriole, directly binds microtubules (with MT-binding residues required for ciliary function), physically interacts with Cep170 to support TBK1-mediated microtubule stability, is required for centrosome cohesion and positioning in interphase, and is required for spindle assembly and symmetry in mitosis; in unicellular eukaryotes its orthologs establish basal body rotational asymmetry and anchor striated rootlets/cytoskeletal fibers to enable proper ciliary orientation, templated centriole duplication, and flagellar synchronization through mechanical coupling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CCDC61 (hVFL3) is a centriolar/centrosomal structural protein that organizes the geometry and connectivity of basal bodies and centrioles across eukaryotes [#1, #2, #8]. Crystal structures establish it as a structural paralog of SAS6: it retains two homodimerization interfaces homologous to those of SAL6 but uses them to polymerize into linear filaments rather than rings, and it directly binds microtubules through residues required for ciliary function [#2]. In human cells it localizes to subdistal appendages and proximal ends of the mother centriole, where it directly binds microtubules, physically interacts with Cep170, and is required for centrosome cohesion and interphase centrosome positioning; loss of CCDC61 increases mother-daughter centriole distance and exacerbates splitting when the rootletin/C-Nap1 linker is disrupted [#1]. The Cep170 interaction supports the Cep170-TBK1 association required for microtubule stability, and CCDC61 depletion disrupts intrinsic spindle symmetry and mitotic spindle assembly [#0]. Across unicellular eukaryotes and Xenopus, its orthologs localize at the junction between basal bodies and striated rootlets and are required for basal body rotational asymmetry, docking, spacing and polarization, templated centriole duplication, and mechanical coupling that synchronizes flagellar beating [#3, #5, #6, #7, #8]. Its centrosome-specific binding is sufficiently selective that a CCDC61-derived peptide captures intact centrosomes for proteome isolation [#4].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1984,\n      \"claim\": \"Established the founding phenotype linking this gene to ciliary architecture by asking whether basal body orientation depends on associated fibers rather than on beating machinery.\",\n      \"evidence\": \"High-speed cinephotomicrography and polarity marker analysis of the Chlamydomonas vfl-3 mutant lacking striated fibers and microtubular rootlets\",\n      \"pmids\": [\"6699086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Gene product not molecularly identified\", \"Mechanism connecting fibers to rotational orientation unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined a specific requirement in centriole biogenesis by distinguishing templated from de novo assembly pathways.\",\n      \"evidence\": \"Genetic analysis of vfl3 mutant with quantification of de novo vs templated centriole assembly in centrioleless Chlamydomonas\",\n      \"pmids\": [\"11267867\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular role in templating unresolved\", \"No biochemical activity assigned\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed how the structures depending on this gene contribute to organismal function by testing whether flagellar synchronization is mechanical or hydrodynamic.\",\n      \"evidence\": \"Controlled external-flow hydrodynamics and high-speed imaging of vfl3 mutant Chlamydomonas flagella\",\n      \"pmids\": [\"26684142\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Uses mutant as a tool; does not define the protein's molecular function\", \"Coupling structure not molecularly dissected\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected the ortholog to a molecular recruitment event and basal body asymmetry, advancing from phenotype to a defined molecular dependency.\",\n      \"evidence\": \"RNAi depletion, immunofluorescence localization, and basal body/rootlet phenotyping in Paramecium tetraurelia\",\n      \"pmids\": [\"28367320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interaction with Centrin 3 not demonstrated\", \"Mechanism of asymmetry specification unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended function into mitosis and into a defined human protein interaction, asking how CCDC61 contributes to spindle symmetry and microtubule stability.\",\n      \"evidence\": \"siRNA knockdown, microtubule tip-tracking, and co-immunoprecipitation in human cells\",\n      \"pmids\": [\"30354798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CCDC61 directly bridges Cep170-TBK1 vs indirectly is unresolved\", \"Structural basis of spindle symmetry effect unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the human protein's centriolar localization and a coherent set of structural roles, establishing it as a centrosome cohesion and positioning factor that binds microtubules and Cep170.\",\n      \"evidence\": \"Immunofluorescence, co-IP, microtubule co-sedimentation, and siRNA epistasis with the C-Nap1 linker in human cells\",\n      \"pmids\": [\"31789463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which it enforces cohesion at the linker not detailed\", \"Spatial relationship to subdistal appendage assembly unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural mechanism by revealing CCDC61 as a SAS6 paralog that polymerizes into filaments and directly binds microtubules via residues required for ciliary function.\",\n      \"evidence\": \"X-ray crystallography, in vitro microtubule-binding assay, and mutagenesis with Chlamydomonas ciliary complementation\",\n      \"pmids\": [\"32375023\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo architecture of the filaments not visualized\", \"How filament assembly couples to centriole/rootlet positioning unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established the vertebrate multiciliated-cell role, showing CCDC61 is required for basal body docking, spacing, polarization, and cilia-driven fluid flow.\",\n      \"evidence\": \"Morpholino knockdown, immunofluorescence localization, and fluid-flow assay in Xenopus ciliated epidermis\",\n      \"pmids\": [\"35067717\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular partners in vertebrate basal body/rootlet junction not identified\", \"Relationship to apical cytoskeleton mechanistically undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated the selectivity of CCDC61's centrosome binding by turning it into a one-step centrosome capture reagent.\",\n      \"evidence\": \"Affinity capture with a CCDC61-derived peptide, mass spectrometry, and electron microscopy verification of intact centrosomes\",\n      \"pmids\": [\"37852252\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous binding partner of the capture peptide not defined\", \"Does not address native CCDC61 function\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CCDC61 filament assembly is spatially regulated to coordinate subdistal appendage organization, centriole cohesion, and rootlet anchoring within a single mechanistic framework remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo structure of CCDC61 filaments at the centriole\", \"Regulatory inputs governing its assembly/disassembly unknown\", \"Direct vs indirect contribution to Cep170-TBK1 microtubule stability unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CEP170\", \"TBK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}