{"gene":"CEP104","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2013,"finding":"CEP104 (Chlamydomonas FAP256) localizes to the tips of both central pair and outer doublet microtubules in cilia/flagella, and remains at the tip during flagellar assembly and disassembly. Loss-of-function (null mutant in Chlamydomonas, RNAi in vertebrate cells) causes ciliogenesis defects and structural deformities at ciliary tips.","method":"Quantitative comparative proteomics (tip-enrichment assay), immunolocalization, null mutant analysis in Chlamydomonas, RNAi knockdown in vertebrate cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (proteomics, localization, loss-of-function in two organisms) with defined cellular phenotype","pmids":["23970417"],"is_preprint":false},{"year":2016,"finding":"CEP104 contains a tubulin-binding TOG (tumor overexpressed gene) domain and a novel C2HC zinc finger array. The kinase NEK1 binds the zinc finger array and competes with the distal centriole-capping protein CP110 for binding to CEP104.","method":"X-ray crystallography (structural determination of TOG domain), biochemical binding assays, competitive binding experiments","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with biochemical binding assays in a single rigorous study","pmids":["28017521"],"is_preprint":false},{"year":2016,"finding":"The TOG domain of CEP104 directly binds tubulin. CEP104 also interacts with CP110, CEP97, and EB (end-binding) proteins. Crystal structure of the TOG domain was solved.","method":"X-ray crystallography, biophysical characterization (sequence analysis, binding assays)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure solved and tubulin interaction biophysically characterized, replicated across two independent structural studies (PMID 28017521 and 27402853)","pmids":["27402853"],"is_preprint":false},{"year":2019,"finding":"CEP104 interacts with the Joubert syndrome-associated ciliary tip protein CSPP1 at microtubules to regulate axoneme length. An intra-ciliary CEP104-CSPP1 complex is required for Smoothened ciliary translocation in response to Hedgehog pathway stimulation. CEP104 is not required for ciliary recruitment of CSPP1.","method":"Co-immunoprecipitation, zebrafish cep104 silencing (in vivo phenotypic analysis of cilia length/laterality), hTERT-RPE1 cell knockdown with Hedgehog pathway readout (Smoothened localization)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction demonstrated, in vivo and in vitro orthogonal analyses, defined pathway placement for Hedgehog signaling","pmids":["31412255"],"is_preprint":false},{"year":2020,"finding":"The TOG domain of CEP104 possesses microtubule-polymerizing activity that is essential for cilium elongation. The N-terminal jelly-roll (JR) fold partially contributes to cilium elongation. The zinc-finger region mediates CP110 binding and the SXIP motif mediates EB1 binding, but neither interaction is required for cilium elongation activity. CEP104 functions after CP110 removal from the mother centriole during ciliogenesis. CEP104 is required for ciliary entry of Smoothened and export of GPR161 upon Hedgehog activation, with the TOG domain playing a critical role.","method":"Knockdown/rescue experiments with domain deletion/point mutants, in vitro microtubule polymerization assay, immunofluorescence of CP110 removal, Smoothened and GPR161 ciliary localization assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic activity assay combined with domain-specific mutagenesis and multiple cellular readouts in a single systematic study","pmids":["32820051"],"is_preprint":false},{"year":2023,"finding":"By cryo-electron tomography and subtomogram averaging of Tetrahymena cilia lacking CEP104/FAP256, CEP104 was found to be part of a central pair cap complex at ciliary tips. Loss of CEP104/FAP256 destabilizes ciliary tip microtubule organization and affects the central pair cap complex composition.","method":"Cryo-electron tomography, subtomogram averaging, proteomics of CEP104/FAP256-null cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-ET structural analysis combined with proteomics in loss-of-function cells, single lab but multiple orthogonal methods","pmids":["37756660"],"is_preprint":false},{"year":2023,"finding":"In Drosophila, Cep104 is a component of the distal tip complex (DTC) of centrioles. Cep104 promotes centriole elongation through its microtubule-binding TOG domain. Cep104 null flies show defects in spermatid alignment and individualization during spermiogenesis, and Cep104 cooperates with Cep97 in this process. Cep97 is the central scaffolding unit required to recruit DTC components (including Cep104) to the distal tip.","method":"Proximity-labeling screen in Drosophila cells, null mutant analysis, domain function analysis (TOG domain), DTC interactome mapping","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — proximity labeling interactome, null mutant with defined phenotype, domain-function analysis, and scaffolding hierarchy established in single study","pmids":["37729913"],"is_preprint":false},{"year":2025,"finding":"Chlamydomonas MAK kinase (CrMAK) directly phosphorylates FAP256/CEP104 to regulate axonemal microtubule assembly. CrMAK acts downstream of the LF1/LF2/LF3 ciliary length-regulatory complex. Loss of CrMAK or its kinase activity causes aciliation. Dephosphorylation of FAP256/CEP104 impairs ciliary assembly.","method":"In vitro kinase assay (phosphorylation of FAP256/CEP104 by CrMAK), genetic epistasis (CrMAK loss-of-function relative to LF complex), loss-of-function phenotypic analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay demonstrating phosphorylation, combined with genetic epistasis and loss-of-function analysis","pmids":["41231942"],"is_preprint":false},{"year":2025,"finding":"In Xenopus, CEP104 localizes to the ends of cytoplasmic microtubules (not only ciliary tip) and influences their stability. Downregulation of CEP104 leads to cytoplasmic microtubule instability and defects in multiciliated cell intercalation. CEP104 is required for neural tube closure through regulation of apical constriction, a process that cannot be explained solely by its ciliary or Hedgehog signaling roles.","method":"Live imaging and localization studies in Xenopus embryos, CEP104 knockdown with phenotypic readouts (neural tube closure, apical constriction, microtubule stability, multiciliated cell intercalation)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct localization experiment with functional consequence in a model organism, single preprint lab, multiple phenotypic readouts but not yet peer reviewed","pmids":["bio_10.1101_2025.06.12.659327"],"is_preprint":true},{"year":2015,"finding":"CEP104 loss-of-function mutations (splice-site, nonsense, frameshift) cause Joubert syndrome. Knockdown of CEP104 in RPE1 cells results in severe ciliogenesis defects, and CEP104 acts early during cilia formation by regulating conversion of the mother centriole into the cilia basal body.","method":"Exome/targeted sequencing for variant identification, RNAi knockdown in RPE1 cells with ciliogenesis readout","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with defined cellular phenotype, consistent with prior Chlamydomonas/vertebrate data but pathway placement based on single-lab RPE1 experiments","pmids":["26477546"],"is_preprint":false}],"current_model":"CEP104 is a conserved centrosomal and ciliary tip protein that promotes ciliogenesis and axoneme elongation via its TOG domain, which directly binds tubulin and has microtubule-polymerizing activity; it localizes to centriole distal tips and the ciliary tip, interacts with CP110/CEP97 (via its zinc-finger array), EB proteins (via SXIP motif), NEK1, and the Joubert syndrome protein CSPP1, with the CEP104-CSPP1 intra-ciliary complex controlling axoneme length and Hedgehog signaling competence (Smoothened entry / GPR161 export); its activity is regulated by MAK kinase phosphorylation; and in addition to its ciliary roles, it stabilizes cytoplasmic microtubules to support processes such as neural tube closure."},"narrative":{"mechanistic_narrative":"CEP104 is a conserved centriolar and ciliary-tip protein that drives ciliogenesis and axoneme elongation through direct, microtubule-polymerizing activity of its TOG domain [PMID:23970417, PMID:27402853, PMID:32820051]. The TOG domain directly binds tubulin and is essential for cilium elongation, while CEP104 functions downstream of CP110 removal from the mother centriole during ciliary assembly [PMID:32820051, PMID:26477546]. Structurally, CEP104 carries a C2HC zinc-finger array that mediates binding to the centriole-capping CP110/CEP97 module and is competed for by the kinase NEK1, and an SXIP motif that recruits EB proteins—though neither interaction is required for elongation activity [PMID:28017521, PMID:27402853, PMID:32820051]. At the ciliary tip CEP104 forms part of a central-pair cap complex whose loss destabilizes tip microtubule organization [PMID:23970417, PMID:37756660], and it partners with the Joubert syndrome protein CSPP1 in an intra-ciliary complex that controls axoneme length and Hedgehog signaling competence, governing Smoothened ciliary entry and GPR161 export upon pathway activation [PMID:31412255, PMID:32820051]. Its activity is regulated by MAK kinase, which directly phosphorylates CEP104 downstream of the LF1/LF2/LF3 length-control complex to permit axonemal assembly [PMID:41231942]. Loss-of-function mutations in CEP104 cause Joubert syndrome, consistent with its requirement for converting the mother centriole into a ciliary basal body [PMID:26477546]. Beyond cilia, CEP104 localizes to cytoplasmic microtubule ends and stabilizes them to support neural tube closure and apical constriction [PMID:bio_10.1101_2025.06.12.659327].","teleology":[{"year":2013,"claim":"Established CEP104 as a bona fide ciliary-tip protein required for normal ciliogenesis, answering where it acts and that its loss disrupts ciliary structure.","evidence":"Tip-enrichment proteomics, immunolocalization, and loss-of-function in Chlamydomonas and vertebrate cells","pmids":["23970417"],"confidence":"High","gaps":["Molecular activity at the tip undefined","No structural basis for microtubule association"]},{"year":2015,"claim":"Linked CEP104 to human disease and placed it early in ciliogenesis, showing it regulates conversion of the mother centriole into the basal body.","evidence":"Exome sequencing of Joubert syndrome families plus RNAi in RPE1 cells with ciliogenesis readout","pmids":["26477546"],"confidence":"Medium","gaps":["Mechanism of basal-body conversion not resolved","Pathway placement based on single-lab RPE1 experiments"]},{"year":2016,"claim":"Defined the molecular architecture of CEP104, identifying a tubulin-binding TOG domain, a C2HC zinc-finger array, and partner-binding interfaces for CP110/CEP97, EB proteins, and NEK1.","evidence":"X-ray crystallography of the TOG domain with biochemical and competitive binding assays (two independent structural studies)","pmids":["28017521","27402853"],"confidence":"High","gaps":["Functional consequence of NEK1/CP110 competition not tested in cells","Whether TOG-tubulin binding confers polymerase activity not yet shown"]},{"year":2019,"claim":"Connected CEP104 to Hedgehog signaling by identifying an intra-ciliary CEP104-CSPP1 complex controlling axoneme length and Smoothened translocation.","evidence":"Co-immunoprecipitation, zebrafish silencing, and RPE1 knockdown with Smoothened localization readout","pmids":["31412255"],"confidence":"High","gaps":["How the complex gates Smoothened entry mechanistically unclear","CSPP1 recruitment shown to be CEP104-independent, leaving recruitment hierarchy open"]},{"year":2020,"claim":"Assigned microtubule-polymerizing activity to the TOG domain as the essential determinant of cilium elongation and dissected which interactions are dispensable.","evidence":"In vitro microtubule polymerization assay with domain-deletion/point-mutant rescue and Smoothened/GPR161 ciliary localization assays","pmids":["32820051"],"confidence":"High","gaps":["Role of zinc-finger/SXIP interactions in vivo beyond elongation unresolved","How phosphoregulation modulates polymerase activity not addressed here"]},{"year":2023,"claim":"Placed CEP104 structurally within the central-pair cap complex at ciliary tips and within the centriolar distal-tip complex, establishing a CEP97-anchored recruitment hierarchy.","evidence":"Cryo-electron tomography and proteomics in Tetrahymena nulls; proximity-labeling interactome and null-mutant analysis in Drosophila","pmids":["37756660","37729913"],"confidence":"High","gaps":["Stoichiometry of CEP104 within the cap complex undefined","Conservation of DTC hierarchy in vertebrate cilia not directly tested"]},{"year":2025,"claim":"Identified MAK kinase as a direct upstream regulator phosphorylating CEP104 to control axonemal assembly, integrating it into a length-control signaling axis.","evidence":"In vitro kinase assay, genetic epistasis relative to the LF1/LF2/LF3 complex, and loss-of-function phenotyping in Chlamydomonas","pmids":["41231942"],"confidence":"High","gaps":["Phosphosites on CEP104 and their functional consequence not mapped","Whether MAK regulation operates in vertebrates unknown"]},{"year":2025,"claim":"Extended CEP104 function beyond cilia, showing it stabilizes cytoplasmic microtubules to drive neural tube closure via apical constriction.","evidence":"Live imaging and knockdown phenotyping in Xenopus embryos (preprint)","pmids":["bio_10.1101_2025.06.12.659327"],"confidence":"Medium","gaps":["Not yet peer reviewed","Molecular basis for cytoplasmic microtubule stabilization distinct from ciliary role unresolved"]},{"year":null,"claim":"How CEP104's TOG-driven polymerase activity, partner competition (NEK1/CP110), and phosphoregulation are coordinated to switch between ciliary and cytoplasmic microtubule functions remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model of spatial/temporal regulation across compartments","Mechanism linking phosphorylation to polymerase output not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[8]}],"complexes":["central pair cap complex","centriole distal tip complex (DTC)","CEP104-CSPP1 intra-ciliary complex"],"partners":["CSPP1","CP110","CEP97","NEK1","EB1","MAK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60308","full_name":"Centrosomal protein of 104 kDa","aliases":[],"length_aa":925,"mass_kda":104.4,"function":"Required for ciliogenesis and for structural integrity at the ciliary tip","subcellular_location":"Cell projection, cilium; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, spindle pole","url":"https://www.uniprot.org/uniprotkb/O60308/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CEP104","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CEP104","total_profiled":1310},"omim":[{"mim_id":"619988","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 77; MRT77","url":"https://www.omim.org/entry/619988"},{"mim_id":"616781","title":"JOUBERT SYNDROME 25; JBTS25","url":"https://www.omim.org/entry/616781"},{"mim_id":"616690","title":"CENTROSOMAL PROTEIN, 104-KD; CEP104","url":"https://www.omim.org/entry/616690"},{"mim_id":"615948","title":"OROFACIODIGITAL SYNDROME XIV; OFD14","url":"https://www.omim.org/entry/615948"},{"mim_id":"615944","title":"C2 CALCIUM-DEPENDENT DOMAIN-CONTAINING PROTEIN 3; C2CD3","url":"https://www.omim.org/entry/615944"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CEP104"},"hgnc":{"alias_symbol":["GlyBP","RP1-286D6.4","CFAP256","ROC22","JBTS25"],"prev_symbol":["KIAA0562"]},"alphafold":{"accession":"O60308","domains":[{"cath_id":"2.60.120.260","chopping":"2-83_90-158","consensus_level":"medium","plddt":89.6087,"start":2,"end":158},{"cath_id":"-","chopping":"206-298","consensus_level":"medium","plddt":85.928,"start":206,"end":298},{"cath_id":"-","chopping":"767-879","consensus_level":"high","plddt":85.8603,"start":767,"end":879}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60308","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60308-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60308-F1-predicted_aligned_error_v6.png","plddt_mean":76.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CEP104","jax_strain_url":"https://www.jax.org/strain/search?query=CEP104"},"sequence":{"accession":"O60308","fasta_url":"https://rest.uniprot.org/uniprotkb/O60308.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60308/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60308"}},"corpus_meta":[{"pmid":"23970417","id":"PMC_23970417","title":"Centrosomal protein CEP104 (Chlamydomonas FAP256) moves to the ciliary tip during ciliary assembly.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23970417","citation_count":63,"is_preprint":false},{"pmid":"26477546","id":"PMC_26477546","title":"Joubert Syndrome in French Canadians and Identification of Mutations in CEP104.","date":"2015","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26477546","citation_count":62,"is_preprint":false},{"pmid":"31412255","id":"PMC_31412255","title":"A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling.","date":"2019","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/31412255","citation_count":41,"is_preprint":false},{"pmid":"28017521","id":"PMC_28017521","title":"The Ciliopathy-Associated Cep104 Protein Interacts with Tubulin and Nek1 Kinase.","date":"2016","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/28017521","citation_count":32,"is_preprint":false},{"pmid":"27402853","id":"PMC_27402853","title":"Biophysical and Structural Characterization of the Centriolar Protein Cep104 Interaction Network.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27402853","citation_count":31,"is_preprint":false},{"pmid":"37756660","id":"PMC_37756660","title":"CEP104/FAP256 and associated cap complex maintain stability of the ciliary tip.","date":"2023","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/37756660","citation_count":18,"is_preprint":false},{"pmid":"32820051","id":"PMC_32820051","title":"Roles of TOG and jelly-roll domains of centrosomal protein CEP104 in its functions in cilium elongation and Hedgehog signaling.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32820051","citation_count":13,"is_preprint":false},{"pmid":"31625690","id":"PMC_31625690","title":"Whole exome sequencing reveals novel CEP104 mutations in a Chinese patient with Joubert syndrome.","date":"2019","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31625690","citation_count":10,"is_preprint":false},{"pmid":"34196201","id":"PMC_34196201","title":"CEP104 and CEP290; Genes with Ciliary Functions Cause Intellectual Disability in Multiple Families.","date":"2021","source":"Archives of Iranian medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34196201","citation_count":6,"is_preprint":false},{"pmid":"37729913","id":"PMC_37729913","title":"Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis.","date":"2023","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/37729913","citation_count":5,"is_preprint":false},{"pmid":"35359234","id":"PMC_35359234","title":"CEP104 gene may involve in the pathogenesis of a new developmental disorder other than joubert syndrome.","date":"2022","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/35359234","citation_count":4,"is_preprint":false},{"pmid":"41231942","id":"PMC_41231942","title":"Chlamydomonas protein kinase MAK phosphorylates FAP256/CEP104 and regulates axonemal microtubule assembly.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41231942","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.12.659327","title":"Ciliary and non-ciliary functions of CEP104 in Xenopus","date":"2025-06-15","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.12.659327","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7969,"output_tokens":2741,"usd":0.032511,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9962,"output_tokens":2979,"usd":0.062143,"stage2_stop_reason":"end_turn"},"total_usd":0.094654,"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\": 2013,\n      \"finding\": \"CEP104 (Chlamydomonas FAP256) localizes to the tips of both central pair and outer doublet microtubules in cilia/flagella, and remains at the tip during flagellar assembly and disassembly. Loss-of-function (null mutant in Chlamydomonas, RNAi in vertebrate cells) causes ciliogenesis defects and structural deformities at ciliary tips.\",\n      \"method\": \"Quantitative comparative proteomics (tip-enrichment assay), immunolocalization, null mutant analysis in Chlamydomonas, RNAi knockdown in vertebrate cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (proteomics, localization, loss-of-function in two organisms) with defined cellular phenotype\",\n      \"pmids\": [\"23970417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CEP104 contains a tubulin-binding TOG (tumor overexpressed gene) domain and a novel C2HC zinc finger array. The kinase NEK1 binds the zinc finger array and competes with the distal centriole-capping protein CP110 for binding to CEP104.\",\n      \"method\": \"X-ray crystallography (structural determination of TOG domain), biochemical binding assays, competitive binding experiments\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with biochemical binding assays in a single rigorous study\",\n      \"pmids\": [\"28017521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The TOG domain of CEP104 directly binds tubulin. CEP104 also interacts with CP110, CEP97, and EB (end-binding) proteins. Crystal structure of the TOG domain was solved.\",\n      \"method\": \"X-ray crystallography, biophysical characterization (sequence analysis, binding assays)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure solved and tubulin interaction biophysically characterized, replicated across two independent structural studies (PMID 28017521 and 27402853)\",\n      \"pmids\": [\"27402853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CEP104 interacts with the Joubert syndrome-associated ciliary tip protein CSPP1 at microtubules to regulate axoneme length. An intra-ciliary CEP104-CSPP1 complex is required for Smoothened ciliary translocation in response to Hedgehog pathway stimulation. CEP104 is not required for ciliary recruitment of CSPP1.\",\n      \"method\": \"Co-immunoprecipitation, zebrafish cep104 silencing (in vivo phenotypic analysis of cilia length/laterality), hTERT-RPE1 cell knockdown with Hedgehog pathway readout (Smoothened localization)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction demonstrated, in vivo and in vitro orthogonal analyses, defined pathway placement for Hedgehog signaling\",\n      \"pmids\": [\"31412255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The TOG domain of CEP104 possesses microtubule-polymerizing activity that is essential for cilium elongation. The N-terminal jelly-roll (JR) fold partially contributes to cilium elongation. The zinc-finger region mediates CP110 binding and the SXIP motif mediates EB1 binding, but neither interaction is required for cilium elongation activity. CEP104 functions after CP110 removal from the mother centriole during ciliogenesis. CEP104 is required for ciliary entry of Smoothened and export of GPR161 upon Hedgehog activation, with the TOG domain playing a critical role.\",\n      \"method\": \"Knockdown/rescue experiments with domain deletion/point mutants, in vitro microtubule polymerization assay, immunofluorescence of CP110 removal, Smoothened and GPR161 ciliary localization assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic activity assay combined with domain-specific mutagenesis and multiple cellular readouts in a single systematic study\",\n      \"pmids\": [\"32820051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"By cryo-electron tomography and subtomogram averaging of Tetrahymena cilia lacking CEP104/FAP256, CEP104 was found to be part of a central pair cap complex at ciliary tips. Loss of CEP104/FAP256 destabilizes ciliary tip microtubule organization and affects the central pair cap complex composition.\",\n      \"method\": \"Cryo-electron tomography, subtomogram averaging, proteomics of CEP104/FAP256-null cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-ET structural analysis combined with proteomics in loss-of-function cells, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"37756660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Drosophila, Cep104 is a component of the distal tip complex (DTC) of centrioles. Cep104 promotes centriole elongation through its microtubule-binding TOG domain. Cep104 null flies show defects in spermatid alignment and individualization during spermiogenesis, and Cep104 cooperates with Cep97 in this process. Cep97 is the central scaffolding unit required to recruit DTC components (including Cep104) to the distal tip.\",\n      \"method\": \"Proximity-labeling screen in Drosophila cells, null mutant analysis, domain function analysis (TOG domain), DTC interactome mapping\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proximity labeling interactome, null mutant with defined phenotype, domain-function analysis, and scaffolding hierarchy established in single study\",\n      \"pmids\": [\"37729913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Chlamydomonas MAK kinase (CrMAK) directly phosphorylates FAP256/CEP104 to regulate axonemal microtubule assembly. CrMAK acts downstream of the LF1/LF2/LF3 ciliary length-regulatory complex. Loss of CrMAK or its kinase activity causes aciliation. Dephosphorylation of FAP256/CEP104 impairs ciliary assembly.\",\n      \"method\": \"In vitro kinase assay (phosphorylation of FAP256/CEP104 by CrMAK), genetic epistasis (CrMAK loss-of-function relative to LF complex), loss-of-function phenotypic analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay demonstrating phosphorylation, combined with genetic epistasis and loss-of-function analysis\",\n      \"pmids\": [\"41231942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Xenopus, CEP104 localizes to the ends of cytoplasmic microtubules (not only ciliary tip) and influences their stability. Downregulation of CEP104 leads to cytoplasmic microtubule instability and defects in multiciliated cell intercalation. CEP104 is required for neural tube closure through regulation of apical constriction, a process that cannot be explained solely by its ciliary or Hedgehog signaling roles.\",\n      \"method\": \"Live imaging and localization studies in Xenopus embryos, CEP104 knockdown with phenotypic readouts (neural tube closure, apical constriction, microtubule stability, multiciliated cell intercalation)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct localization experiment with functional consequence in a model organism, single preprint lab, multiple phenotypic readouts but not yet peer reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.12.659327\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CEP104 loss-of-function mutations (splice-site, nonsense, frameshift) cause Joubert syndrome. Knockdown of CEP104 in RPE1 cells results in severe ciliogenesis defects, and CEP104 acts early during cilia formation by regulating conversion of the mother centriole into the cilia basal body.\",\n      \"method\": \"Exome/targeted sequencing for variant identification, RNAi knockdown in RPE1 cells with ciliogenesis readout\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with defined cellular phenotype, consistent with prior Chlamydomonas/vertebrate data but pathway placement based on single-lab RPE1 experiments\",\n      \"pmids\": [\"26477546\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CEP104 is a conserved centrosomal and ciliary tip protein that promotes ciliogenesis and axoneme elongation via its TOG domain, which directly binds tubulin and has microtubule-polymerizing activity; it localizes to centriole distal tips and the ciliary tip, interacts with CP110/CEP97 (via its zinc-finger array), EB proteins (via SXIP motif), NEK1, and the Joubert syndrome protein CSPP1, with the CEP104-CSPP1 intra-ciliary complex controlling axoneme length and Hedgehog signaling competence (Smoothened entry / GPR161 export); its activity is regulated by MAK kinase phosphorylation; and in addition to its ciliary roles, it stabilizes cytoplasmic microtubules to support processes such as neural tube closure.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CEP104 is a conserved centriolar and ciliary-tip protein that drives ciliogenesis and axoneme elongation through direct, microtubule-polymerizing activity of its TOG domain [#0, #2, #4]. The TOG domain directly binds tubulin and is essential for cilium elongation, while CEP104 functions downstream of CP110 removal from the mother centriole during ciliary assembly [#4, #9]. Structurally, CEP104 carries a C2HC zinc-finger array that mediates binding to the centriole-capping CP110/CEP97 module and is competed for by the kinase NEK1, and an SXIP motif that recruits EB proteins—though neither interaction is required for elongation activity [#1, #2, #4]. At the ciliary tip CEP104 forms part of a central-pair cap complex whose loss destabilizes tip microtubule organization [#0, #5], and it partners with the Joubert syndrome protein CSPP1 in an intra-ciliary complex that controls axoneme length and Hedgehog signaling competence, governing Smoothened ciliary entry and GPR161 export upon pathway activation [#3, #4]. Its activity is regulated by MAK kinase, which directly phosphorylates CEP104 downstream of the LF1/LF2/LF3 length-control complex to permit axonemal assembly [#7]. Loss-of-function mutations in CEP104 cause Joubert syndrome, consistent with its requirement for converting the mother centriole into a ciliary basal body [#9]. Beyond cilia, CEP104 localizes to cytoplasmic microtubule ends and stabilizes them to support neural tube closure and apical constriction [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established CEP104 as a bona fide ciliary-tip protein required for normal ciliogenesis, answering where it acts and that its loss disrupts ciliary structure.\",\n      \"evidence\": \"Tip-enrichment proteomics, immunolocalization, and loss-of-function in Chlamydomonas and vertebrate cells\",\n      \"pmids\": [\"23970417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular activity at the tip undefined\", \"No structural basis for microtubule association\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked CEP104 to human disease and placed it early in ciliogenesis, showing it regulates conversion of the mother centriole into the basal body.\",\n      \"evidence\": \"Exome sequencing of Joubert syndrome families plus RNAi in RPE1 cells with ciliogenesis readout\",\n      \"pmids\": [\"26477546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of basal-body conversion not resolved\", \"Pathway placement based on single-lab RPE1 experiments\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the molecular architecture of CEP104, identifying a tubulin-binding TOG domain, a C2HC zinc-finger array, and partner-binding interfaces for CP110/CEP97, EB proteins, and NEK1.\",\n      \"evidence\": \"X-ray crystallography of the TOG domain with biochemical and competitive binding assays (two independent structural studies)\",\n      \"pmids\": [\"28017521\", \"27402853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of NEK1/CP110 competition not tested in cells\", \"Whether TOG-tubulin binding confers polymerase activity not yet shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected CEP104 to Hedgehog signaling by identifying an intra-ciliary CEP104-CSPP1 complex controlling axoneme length and Smoothened translocation.\",\n      \"evidence\": \"Co-immunoprecipitation, zebrafish silencing, and RPE1 knockdown with Smoothened localization readout\",\n      \"pmids\": [\"31412255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the complex gates Smoothened entry mechanistically unclear\", \"CSPP1 recruitment shown to be CEP104-independent, leaving recruitment hierarchy open\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Assigned microtubule-polymerizing activity to the TOG domain as the essential determinant of cilium elongation and dissected which interactions are dispensable.\",\n      \"evidence\": \"In vitro microtubule polymerization assay with domain-deletion/point-mutant rescue and Smoothened/GPR161 ciliary localization assays\",\n      \"pmids\": [\"32820051\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of zinc-finger/SXIP interactions in vivo beyond elongation unresolved\", \"How phosphoregulation modulates polymerase activity not addressed here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed CEP104 structurally within the central-pair cap complex at ciliary tips and within the centriolar distal-tip complex, establishing a CEP97-anchored recruitment hierarchy.\",\n      \"evidence\": \"Cryo-electron tomography and proteomics in Tetrahymena nulls; proximity-labeling interactome and null-mutant analysis in Drosophila\",\n      \"pmids\": [\"37756660\", \"37729913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of CEP104 within the cap complex undefined\", \"Conservation of DTC hierarchy in vertebrate cilia not directly tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified MAK kinase as a direct upstream regulator phosphorylating CEP104 to control axonemal assembly, integrating it into a length-control signaling axis.\",\n      \"evidence\": \"In vitro kinase assay, genetic epistasis relative to the LF1/LF2/LF3 complex, and loss-of-function phenotyping in Chlamydomonas\",\n      \"pmids\": [\"41231942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosites on CEP104 and their functional consequence not mapped\", \"Whether MAK regulation operates in vertebrates unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended CEP104 function beyond cilia, showing it stabilizes cytoplasmic microtubules to drive neural tube closure via apical constriction.\",\n      \"evidence\": \"Live imaging and knockdown phenotyping in Xenopus embryos (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.12.659327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not yet peer reviewed\", \"Molecular basis for cytoplasmic microtubule stabilization distinct from ciliary role unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CEP104's TOG-driven polymerase activity, partner competition (NEK1/CP110), and phosphoregulation are coordinated to switch between ciliary and cytoplasmic microtubule functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model of spatial/temporal regulation across compartments\", \"Mechanism linking phosphorylation to polymerase output not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"central pair cap complex\", \"centriole distal tip complex (DTC)\", \"CEP104-CSPP1 intra-ciliary complex\"],\n    \"partners\": [\"CSPP1\", \"CP110\", \"CEP97\", \"NEK1\", \"EB1\", \"MAK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}