{"gene":"CRB3","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":2004,"finding":"CRB3 directly binds Par6 via its C-terminal ERLI motif (PDZ-binding domain), and this interaction regulates tight junction morphogenesis in epithelial cells. Expression of CRB3 or a chimera containing its transmembrane and cytoplasmic domains slowed development of functional tight junctions in MDCK cells, dependent on the ERLI motif.","method":"Direct binding assay, chimeric protein expression, loss-of-function in MDCK cells with TJ permeability readout","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — direct binding established with mutagenesis (ERLI deletion), functional rescue, replicated across constructs","pmids":["14718572"],"is_preprint":false},{"year":2013,"finding":"The WD40 protein Morg1 directly binds Par6 and interacts with CRB3 to facilitate apical targeting of the Par6-aPKC complex. Cdc42 reinforces Par6-aPKC binding to CRB3. Morg1 depletion disrupts tight junction development and cyst formation, rescued by forced apical targeting of aPKC.","method":"Co-IP, pulldown, siRNA knockdown in MDCK cells, 3D cyst assay, epistasis rescue by aPKC apical targeting","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, genetic epistasis rescue, multiple orthogonal methods in single study","pmids":["23439680"],"is_preprint":false},{"year":2016,"finding":"CRB3 in Sertoli cells interacts with the actin branching protein Arp3 and barbed-end capping/bundling protein Eps8. CRB3 knockdown reorganizes actin microfilaments (truncation and excessive branching), destabilizing F-actin-based adhesion complexes at the blood-testis barrier and disrupting spermatid transport and polarity.","method":"Co-IP (Arp3, Eps8), RNAi knockdown in Sertoli cells with TJ-permeability barrier assay, in vivo RNAi via jetPEI with spermatid transport readout","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2/3 — Co-IP for binding partners, KD with defined actin and functional phenotype, single lab","pmids":["27358069"],"is_preprint":false},{"year":2016,"finding":"CRB3, localized at the tips of the peripheral myelin sheath, activates the Hippo pathway to limit YAP transcriptional activity, thereby restraining myelin elongation. YAP promotes myelin gene transcription and excessive myelin growth when CRB3-Hippo signaling is reduced.","method":"Loss-of-function in vivo (Dy2j/2j mice with reduced internode lengths), YAP nuclear localization rescue, Hippo pathway activity assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo model with defined molecular pathway, YAP nuclear correction rescue, single lab","pmids":["27435623"],"is_preprint":false},{"year":2016,"finding":"MUC1-C associates with ZEB1 on the CRB3 promoter to repress CRB3 transcription, thereby suppressing the CRB3-driven Hippo pathway (LATS1/2 phosphorylation), activating YAP, and promoting YAP/β-catenin-mediated MYC induction.","method":"ChIP (MUC1-C/ZEB1 on CRB3 promoter), siRNA knockdown, phospho-LATS1 assay, co-IP (MUC1-C/YAP), luciferase reporter","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP + Co-IP + phosphorylation assay, multiple methods, single lab","pmids":["27658423"],"is_preprint":false},{"year":2017,"finding":"CRB3 acts as an upstream activator of the Hippo pathway in mammary epithelial cells by recruiting Kibra and/or FRMD6, leading to LATS activation, YAP inactivation, and contact inhibition. CRB3 overexpression decreases proliferation, promotes apoptosis, and enhances tight and adherens junction formation.","method":"Co-IP (CRB3 with Kibra, FRMD6), CRB3 overexpression/knockdown with proliferation, apoptosis, and junction assays; in vivo tumor growth assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2/3 — Co-IP for upstream Hippo recruitment, KD/OE with pathway and cellular readouts, single lab","pmids":["28079891"],"is_preprint":false},{"year":2017,"finding":"CRB3 downregulation in breast epithelial MCF10A cells induces epithelial-mesenchymal transition and activates TAZ and β-catenin, conferring cancer stem cell properties. TAZ and β-catenin activation downstream of CRB3 loss is sufficient to drive the stem cell phenotype.","method":"CRB3 knockdown, TAZ/β-catenin expression and activity assays, mammosphere formation, gain-of-function rescue experiments","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2/3 — KD with pathway activation readout and rescue, single lab, multiple methods","pmids":["28436991"],"is_preprint":false},{"year":2018,"finding":"CRB3 promotes proliferation via its FERM-binding domain (FBD), not its PDZ-binding domain, by inducing secretion of amphiregulin (AREG) through increased endocytic trafficking. The FERM-domain protein EPB41L4B is an essential mediator of CRB3-driven proliferation and endosomal changes.","method":"CRB3 domain mutants (FBD vs. PDZ deletion), 3D acinar culture, AREG secretion assay, siRNA knockdown of EPB41L4B, endosome quantification","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — domain dissection with functional rescue, binding partner identification, multiple orthogonal readouts, single lab","pmids":["30440051"],"is_preprint":false},{"year":2022,"finding":"METTL3 promotes m6A methylation on CRB3 mRNA, leading to YTHDF2-dependent mRNA degradation and reduced CRB3 protein. YTHDF2 directly binds CRB3 mRNA (verified by RIP assay). Increased CRB3 activates the Hippo pathway and reduces nuclear YAP; CRB3 knockdown reverses the Hippo activation caused by METTL3 depletion.","method":"m6A epitranscriptomic microarray, MeRIP-qPCR, RNA stability assay, luciferase reporter, RIP (YTHDF2-CRB3 mRNA), siRNA knockdown epistasis","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 — direct m6A mapping, RIP confirmation, epistasis knockdown, multiple methods, single lab","pmids":["35012593"],"is_preprint":false},{"year":2023,"finding":"CRB3 localizes to the basal body and interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, enabling intact γ-tubulin ring complex (γTuRC) assembly. CRB3 depletion causes defective ciliogenesis, loss of Hh pathway responsiveness, and altered Wnt signaling. Crb3 knockout mice show severe primary cilium defects in mammary ductal lumen and renal tubule.","method":"Co-IP (CRB3-Rab11, GCP6-Rab11-CEP290), Crb3 knockout mice (mammary- and whole-body), immunofluorescence for basal body localization, Hh/Wnt signaling assays, γTuRC assembly assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1/2 — Co-IP of complex, in vivo KO with defined ciliary phenotype, mechanistic reconstitution of γTuRC assembly, multiple orthogonal methods","pmids":["37737843"],"is_preprint":false},{"year":2023,"finding":"In Xenopus MCCs, Crb3.L localizes to cytoplasmic vesicles near ascending centrioles/basal bodies, partially colocalizing with Rab11a. Crb3.L depletion causes abnormal centriole/basal body migration, reduced apical surface, disorganized actin meshwork, and defective ciliogenesis. Rab11a depletion phenocopies Crb3.L loss, suggesting Crb3.L controls centriole migration via Rab11a-dependent apical trafficking. Crb3.L anchors phospho-activated ERM at the growing apical domain to enable actin-dependent apical membrane expansion.","method":"Morpholino knockdown of Crb3.L and Rab11a in Xenopus MCCs, live imaging/colocalization, pERM co-localization, actin organization assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — morpholino KD with phenotypic rescue, colocalization, epistasis via Rab11a phenocopy, Xenopus ortholog","pmids":["37840525"],"is_preprint":false},{"year":2025,"finding":"CRB3A interacts with the actin cytoskeletal linker Merlin (NF2) via its FERM-binding domain (FBD). CRB3 loss leads to increased RhoA activity, hypercontractile perijunctional actomyosin, and impaired apical junctional complex (AJC) assembly. NF2 knockdown phenocopies CRB3 loss. ROCK-II or myosin II inhibition restores junctional architecture in CRB3-null cells.","method":"Conditional Crb3 knockout colonoids (Crb3ERΔIEC), Co-IP (CRB3A-NF2 via FBD), RhoA activity assay, NF2 siRNA phenocopy, ROCK-II/myosin II inhibitor rescue, barrier function assay, vinculin tension readout","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 1/2 — Co-IP with domain mapping, conditional KO in primary cells, pharmacological rescue, multiple orthogonal methods, independent preprint/peer-reviewed pair","pmids":["41122968"],"is_preprint":false}],"current_model":"CRB3 is an apical transmembrane polarity protein that regulates epithelial architecture through multiple mechanisms: its PDZ-binding domain (ERLI) directly recruits Par6-aPKC to the apical membrane to organize tight junctions; its FERM-binding domain interacts with Merlin (NF2) and EPB41L4B to control perijunctional actomyosin contractility, actin organization, and endocytic trafficking; CRB3 navigates Rab11-positive vesicles to deliver GCP6 to CEP290 for γTuRC assembly during ciliogenesis; and CRB3 acts upstream of the Hippo pathway (via Kibra/FRMD6 recruitment and LATS activation) to suppress YAP/TAZ, thereby regulating contact inhibition, tissue polarity, and tumor suppression."},"narrative":{"teleology":[{"year":2004,"claim":"Establishing CRB3 as a polarity protein that directly recruits the Par6-aPKC complex to regulate tight junction formation answered how Crumbs-family proteins connect to the conserved Par complex in mammalian epithelia.","evidence":"Direct binding assay and ERLI-motif mutagenesis in MDCK cells with tight junction permeability readout","pmids":["14718572"],"confidence":"High","gaps":["Upstream signals initiating CRB3 apical delivery were unknown","Whether CRB3-Par6 interaction is sufficient or requires co-factors was unresolved"]},{"year":2013,"claim":"Identification of Morg1 as a bridging adaptor between CRB3 and Par6-aPKC, reinforced by Cdc42, resolved how CRB3 efficiently recruits the polarity complex to the apical membrane.","evidence":"Co-IP, pulldown, siRNA in MDCK cells, 3D cyst assay with epistasis rescue by forced apical aPKC targeting","pmids":["23439680"],"confidence":"High","gaps":["Whether Morg1 is required in all epithelial subtypes was untested","Structural basis of the tripartite CRB3-Morg1-Par6 complex unknown"]},{"year":2016,"claim":"Three parallel studies revealed CRB3 functions beyond tight junctions: regulating actin organization at the blood–testis barrier, activating the Hippo pathway to limit YAP-driven myelin elongation, and being transcriptionally repressed by MUC1-C/ZEB1 to modulate Hippo-YAP-MYC signaling in cancer.","evidence":"Co-IP of CRB3 with Arp3/Eps8 and Sertoli cell RNAi (actin); in vivo Dy2j/2j mouse model with YAP nuclear rescue (myelin); ChIP of MUC1-C/ZEB1 on CRB3 promoter with phospho-LATS and luciferase reporter (cancer)","pmids":["27358069","27435623","27658423"],"confidence":"Medium","gaps":["Direct interaction between CRB3 and Hippo core kinases was not demonstrated","Whether CRB3-actin and CRB3-Hippo branches operate independently or converge was unclear","Myelin phenotype studied in a single disease model"]},{"year":2017,"claim":"Demonstrating that CRB3 recruits Kibra and FRMD6 to activate LATS and suppress YAP/TAZ established CRB3 as an upstream scaffold for the Hippo pathway that enforces contact inhibition and opposes EMT-driven stemness.","evidence":"Co-IP of CRB3 with Kibra/FRMD6, overexpression/knockdown in mammary epithelial cells with proliferation, apoptosis, junction, and tumor growth assays; CRB3 KD inducing TAZ/β-catenin and mammosphere formation in MCF10A cells","pmids":["28079891","28436991"],"confidence":"Medium","gaps":["Whether CRB3-Kibra interaction is direct or scaffolded was unresolved","Relative contributions of TAZ versus β-catenin downstream of CRB3 loss not dissected"]},{"year":2018,"claim":"Domain dissection showed that the FERM-binding domain—not the PDZ-binding domain—drives CRB3-dependent proliferation through EPB41L4B-mediated endocytic trafficking and amphiregulin secretion, revealing a second functional axis of the CRB3 cytoplasmic tail.","evidence":"CRB3 FBD versus PDZ deletion mutants in 3D acinar culture with AREG secretion, EPB41L4B siRNA knockdown, and endosome quantification","pmids":["30440051"],"confidence":"Medium","gaps":["How EPB41L4B couples CRB3-FBD to endosomal machinery was not defined","Whether AREG secretion is the sole proliferative effector downstream of the FBD was untested"]},{"year":2022,"claim":"Showing that METTL3-mediated m6A methylation of CRB3 mRNA triggers YTHDF2-dependent degradation revealed an epitranscriptomic layer controlling CRB3 protein levels and, consequently, Hippo pathway activity.","evidence":"MeRIP-qPCR, RIP of YTHDF2 on CRB3 mRNA, RNA stability assay, epistasis knockdown of CRB3 reversing METTL3-depletion effects on YAP","pmids":["35012593"],"confidence":"Medium","gaps":["Specific m6A sites on CRB3 mRNA were not mapped at nucleotide resolution","Whether this regulatory axis operates in normal epithelia or only in cancer cells was unclear"]},{"year":2023,"claim":"Discovery that CRB3 localizes to the basal body and navigates Rab11-positive vesicles carrying GCP6 to CEP290 for γTuRC assembly established CRB3 as a direct regulator of ciliogenesis, linking it to Hedgehog and Wnt signaling.","evidence":"Co-IP of CRB3-Rab11 and GCP6-Rab11-CEP290, Crb3 knockout mice with ciliary defects in mammary and renal epithelia, γTuRC assembly assay, Hh/Wnt signaling readouts","pmids":["37737843"],"confidence":"High","gaps":["Whether CRB3 directly contacts GCP6 or acts solely through Rab11 was not resolved","Structural basis of CRB3 recognition by Rab11 unknown"]},{"year":2023,"claim":"Parallel work in Xenopus multiciliated cells confirmed CRB3 colocalizes with Rab11a on vesicles near ascending basal bodies and anchors phospho-ERM to enable actin-dependent apical membrane expansion, generalizing the CRB3-Rab11 ciliogenesis mechanism across vertebrates.","evidence":"Morpholino knockdown of Crb3.L and Rab11a in Xenopus MCCs with live imaging, pERM colocalization, and Rab11a phenocopy","pmids":["37840525"],"confidence":"Medium","gaps":["Rescue experiments with human CRB3 in Xenopus MCCs were not performed","Whether ERM anchoring is a direct CRB3 interaction or indirect via scaffold proteins was not established"]},{"year":2025,"claim":"Identifying Merlin/NF2 as a direct FERM-binding domain partner of CRB3 that restrains RhoA-ROCK-myosin II contractility at junctions unified the polarity and actomyosin branches and explained how CRB3 loss causes junctional collapse via hypercontractility.","evidence":"Conditional Crb3 knockout colonoids, Co-IP of CRB3A-NF2 via FBD, NF2 siRNA phenocopy, ROCK-II/myosin II inhibitor rescue of junctional architecture","pmids":["41122968"],"confidence":"High","gaps":["Whether NF2 links CRB3-FBD to the Hippo kinase cascade directly or through separate effectors was not resolved","Contribution of EPB41L4B versus NF2 at the FBD under different cellular contexts not compared"]},{"year":null,"claim":"How the PDZ-binding (Par6-aPKC) and FERM-binding (NF2/EPB41L4B) arms of CRB3 are coordinately regulated in space and time, and whether the junctional and ciliary pools of CRB3 share trafficking machinery, remain open mechanistic questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of the CRB3 cytoplasmic tail in complex with its partners","Relative stoichiometry and competition between NF2, EPB41L4B, Kibra, and FRMD6 at the FBD is undefined","Whether CRB3 isoforms (CRB3A vs CRB3B) partition between junctional and ciliary functions has not been rigorously tested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5,9,11]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,2,11]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[9,10]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[9,10]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[9,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,5,6,8]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,1,11]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[9,10]}],"complexes":["CRB3-Par6-aPKC apical polarity complex","CRB3-Rab11-GCP6 ciliogenesis trafficking complex"],"partners":["PARD6B","PRKCZ","NF2","EPB41L4B","RAB11A","KIBRA","FRMD6","MORG1"],"other_free_text":[]},"mechanistic_narrative":"CRB3 is an apical transmembrane polarity determinant that organizes epithelial architecture, tight junctions, and primary cilia through distinct cytoplasmic domain interactions and upstream regulation of the Hippo pathway. Its C-terminal PDZ-binding motif (ERLI) recruits Par6-aPKC to the apical membrane to govern tight junction morphogenesis, facilitated by the adaptor Morg1 and Cdc42 [PMID:14718572, PMID:23439680], while its FERM-binding domain engages Merlin/NF2 and EPB41L4B to control perijunctional actomyosin contractility, endocytic trafficking, and junctional complex assembly [PMID:41122968, PMID:30440051]. CRB3 recruits Kibra/FRMD6 to activate LATS and suppress YAP/TAZ, enforcing contact inhibition and restraining proliferation; loss of CRB3 activates TAZ/β-catenin signaling, promotes epithelial–mesenchymal transition, and confers stem-cell properties [PMID:28079891, PMID:28436991]. CRB3 also localizes to the basal body, where it navigates Rab11-positive vesicles carrying GCP6 to CEP290 for γ-tubulin ring complex assembly, and its depletion causes severe ciliogenesis defects, loss of Hedgehog responsiveness, and disrupted centriole migration in vivo [PMID:37737843, PMID:37840525]."},"prefetch_data":{"uniprot":{"accession":"Q9BUF7","full_name":"Protein crumbs homolog 3","aliases":[],"length_aa":120,"mass_kda":12.9,"function":"Involved in the establishment of cell polarity in mammalian epithelial cells (PubMed:12771187, PubMed:14718572, PubMed:23439680). Regulates the morphogenesis of tight junctions (PubMed:12771187, PubMed:14718572). Involved in promoting phosphorylation and cytoplasmic retention of transcriptional coactivators YAP1 and WWTR1/TAZ which leads to suppression of TGFB1-dependent transcription of target genes such as CCN2/CTGF, SERPINE1/PAI1, SNAI1/SNAIL1 and SMAD7 (By similarity)","subcellular_location":"Apical cell membrane; Cell junction, tight junction","url":"https://www.uniprot.org/uniprotkb/Q9BUF7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CRB3","classification":"Not Classified","n_dependent_lines":17,"n_total_lines":1208,"dependency_fraction":0.014072847682119206},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CRB3","total_profiled":1310},"omim":[{"mim_id":"616850","title":"WD REPEAT-CONTAINING PROTEIN 83; WDR83","url":"https://www.omim.org/entry/616850"},{"mim_id":"611730","title":"ERYTHROCYTE MEMBRANE PROTEIN BAND 4.1-LIKE 5; EPB41L5","url":"https://www.omim.org/entry/611730"},{"mim_id":"610973","title":"MEMBRANE PROTEIN, PALMITOYLATED 7; MPP7","url":"https://www.omim.org/entry/610973"},{"mim_id":"609737","title":"CRUMBS CELL POLARITY COMPLEX COMPONENT 3; CRB3","url":"https://www.omim.org/entry/609737"},{"mim_id":"607484","title":"PAR6 FAMILY CELL POLARITY REGULATOR ALPHA; PARD6A","url":"https://www.omim.org/entry/607484"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cell Junctions","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in 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disease.","date":"2024","source":"Immunity, inflammation and disease","url":"https://pubmed.ncbi.nlm.nih.gov/38353316","citation_count":2,"is_preprint":false},{"pmid":"39868176","id":"PMC_39868176","title":"Crb3 and NF2: A dynamic duo that controls assembly of the apical junctions and barrier function via Rho/ROCK signaling.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39868176","citation_count":0,"is_preprint":false},{"pmid":"37840525","id":"PMC_37840525","title":"Crb3 is required to organize the apical domain of multiciliated cells.","date":"2023","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/37840525","citation_count":0,"is_preprint":false},{"pmid":"41122968","id":"PMC_41122968","title":"CRB3 and NF2 orchestrate cytoskeletal dynamics to control epithelial barrier assembly.","date":"2025","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/41122968","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12396,"output_tokens":3322,"usd":0.043509},"stage2":{"model":"claude-opus-4-6","input_tokens":6704,"output_tokens":3111,"usd":0.166942},"total_usd":0.210451,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"CRB3 directly binds Par6 via its C-terminal ERLI motif (PDZ-binding domain), and this interaction regulates tight junction morphogenesis in epithelial cells. Expression of CRB3 or a chimera containing its transmembrane and cytoplasmic domains slowed development of functional tight junctions in MDCK cells, dependent on the ERLI motif.\",\n      \"method\": \"Direct binding assay, chimeric protein expression, loss-of-function in MDCK cells with TJ permeability readout\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding established with mutagenesis (ERLI deletion), functional rescue, replicated across constructs\",\n      \"pmids\": [\"14718572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The WD40 protein Morg1 directly binds Par6 and interacts with CRB3 to facilitate apical targeting of the Par6-aPKC complex. Cdc42 reinforces Par6-aPKC binding to CRB3. Morg1 depletion disrupts tight junction development and cyst formation, rescued by forced apical targeting of aPKC.\",\n      \"method\": \"Co-IP, pulldown, siRNA knockdown in MDCK cells, 3D cyst assay, epistasis rescue by aPKC apical targeting\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, genetic epistasis rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"23439680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CRB3 in Sertoli cells interacts with the actin branching protein Arp3 and barbed-end capping/bundling protein Eps8. CRB3 knockdown reorganizes actin microfilaments (truncation and excessive branching), destabilizing F-actin-based adhesion complexes at the blood-testis barrier and disrupting spermatid transport and polarity.\",\n      \"method\": \"Co-IP (Arp3, Eps8), RNAi knockdown in Sertoli cells with TJ-permeability barrier assay, in vivo RNAi via jetPEI with spermatid transport readout\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — Co-IP for binding partners, KD with defined actin and functional phenotype, single lab\",\n      \"pmids\": [\"27358069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CRB3, localized at the tips of the peripheral myelin sheath, activates the Hippo pathway to limit YAP transcriptional activity, thereby restraining myelin elongation. YAP promotes myelin gene transcription and excessive myelin growth when CRB3-Hippo signaling is reduced.\",\n      \"method\": \"Loss-of-function in vivo (Dy2j/2j mice with reduced internode lengths), YAP nuclear localization rescue, Hippo pathway activity assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo model with defined molecular pathway, YAP nuclear correction rescue, single lab\",\n      \"pmids\": [\"27435623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MUC1-C associates with ZEB1 on the CRB3 promoter to repress CRB3 transcription, thereby suppressing the CRB3-driven Hippo pathway (LATS1/2 phosphorylation), activating YAP, and promoting YAP/β-catenin-mediated MYC induction.\",\n      \"method\": \"ChIP (MUC1-C/ZEB1 on CRB3 promoter), siRNA knockdown, phospho-LATS1 assay, co-IP (MUC1-C/YAP), luciferase reporter\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP + Co-IP + phosphorylation assay, multiple methods, single lab\",\n      \"pmids\": [\"27658423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CRB3 acts as an upstream activator of the Hippo pathway in mammary epithelial cells by recruiting Kibra and/or FRMD6, leading to LATS activation, YAP inactivation, and contact inhibition. CRB3 overexpression decreases proliferation, promotes apoptosis, and enhances tight and adherens junction formation.\",\n      \"method\": \"Co-IP (CRB3 with Kibra, FRMD6), CRB3 overexpression/knockdown with proliferation, apoptosis, and junction assays; in vivo tumor growth assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — Co-IP for upstream Hippo recruitment, KD/OE with pathway and cellular readouts, single lab\",\n      \"pmids\": [\"28079891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CRB3 downregulation in breast epithelial MCF10A cells induces epithelial-mesenchymal transition and activates TAZ and β-catenin, conferring cancer stem cell properties. TAZ and β-catenin activation downstream of CRB3 loss is sufficient to drive the stem cell phenotype.\",\n      \"method\": \"CRB3 knockdown, TAZ/β-catenin expression and activity assays, mammosphere formation, gain-of-function rescue experiments\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — KD with pathway activation readout and rescue, single lab, multiple methods\",\n      \"pmids\": [\"28436991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRB3 promotes proliferation via its FERM-binding domain (FBD), not its PDZ-binding domain, by inducing secretion of amphiregulin (AREG) through increased endocytic trafficking. The FERM-domain protein EPB41L4B is an essential mediator of CRB3-driven proliferation and endosomal changes.\",\n      \"method\": \"CRB3 domain mutants (FBD vs. PDZ deletion), 3D acinar culture, AREG secretion assay, siRNA knockdown of EPB41L4B, endosome quantification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain dissection with functional rescue, binding partner identification, multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"30440051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"METTL3 promotes m6A methylation on CRB3 mRNA, leading to YTHDF2-dependent mRNA degradation and reduced CRB3 protein. YTHDF2 directly binds CRB3 mRNA (verified by RIP assay). Increased CRB3 activates the Hippo pathway and reduces nuclear YAP; CRB3 knockdown reverses the Hippo activation caused by METTL3 depletion.\",\n      \"method\": \"m6A epitranscriptomic microarray, MeRIP-qPCR, RNA stability assay, luciferase reporter, RIP (YTHDF2-CRB3 mRNA), siRNA knockdown epistasis\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct m6A mapping, RIP confirmation, epistasis knockdown, multiple methods, single lab\",\n      \"pmids\": [\"35012593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CRB3 localizes to the basal body and interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, enabling intact γ-tubulin ring complex (γTuRC) assembly. CRB3 depletion causes defective ciliogenesis, loss of Hh pathway responsiveness, and altered Wnt signaling. Crb3 knockout mice show severe primary cilium defects in mammary ductal lumen and renal tubule.\",\n      \"method\": \"Co-IP (CRB3-Rab11, GCP6-Rab11-CEP290), Crb3 knockout mice (mammary- and whole-body), immunofluorescence for basal body localization, Hh/Wnt signaling assays, γTuRC assembly assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — Co-IP of complex, in vivo KO with defined ciliary phenotype, mechanistic reconstitution of γTuRC assembly, multiple orthogonal methods\",\n      \"pmids\": [\"37737843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Xenopus MCCs, Crb3.L localizes to cytoplasmic vesicles near ascending centrioles/basal bodies, partially colocalizing with Rab11a. Crb3.L depletion causes abnormal centriole/basal body migration, reduced apical surface, disorganized actin meshwork, and defective ciliogenesis. Rab11a depletion phenocopies Crb3.L loss, suggesting Crb3.L controls centriole migration via Rab11a-dependent apical trafficking. Crb3.L anchors phospho-activated ERM at the growing apical domain to enable actin-dependent apical membrane expansion.\",\n      \"method\": \"Morpholino knockdown of Crb3.L and Rab11a in Xenopus MCCs, live imaging/colocalization, pERM co-localization, actin organization assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — morpholino KD with phenotypic rescue, colocalization, epistasis via Rab11a phenocopy, Xenopus ortholog\",\n      \"pmids\": [\"37840525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRB3A interacts with the actin cytoskeletal linker Merlin (NF2) via its FERM-binding domain (FBD). CRB3 loss leads to increased RhoA activity, hypercontractile perijunctional actomyosin, and impaired apical junctional complex (AJC) assembly. NF2 knockdown phenocopies CRB3 loss. ROCK-II or myosin II inhibition restores junctional architecture in CRB3-null cells.\",\n      \"method\": \"Conditional Crb3 knockout colonoids (Crb3ERΔIEC), Co-IP (CRB3A-NF2 via FBD), RhoA activity assay, NF2 siRNA phenocopy, ROCK-II/myosin II inhibitor rescue, barrier function assay, vinculin tension readout\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — Co-IP with domain mapping, conditional KO in primary cells, pharmacological rescue, multiple orthogonal methods, independent preprint/peer-reviewed pair\",\n      \"pmids\": [\"41122968\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CRB3 is an apical transmembrane polarity protein that regulates epithelial architecture through multiple mechanisms: its PDZ-binding domain (ERLI) directly recruits Par6-aPKC to the apical membrane to organize tight junctions; its FERM-binding domain interacts with Merlin (NF2) and EPB41L4B to control perijunctional actomyosin contractility, actin organization, and endocytic trafficking; CRB3 navigates Rab11-positive vesicles to deliver GCP6 to CEP290 for γTuRC assembly during ciliogenesis; and CRB3 acts upstream of the Hippo pathway (via Kibra/FRMD6 recruitment and LATS activation) to suppress YAP/TAZ, thereby regulating contact inhibition, tissue polarity, and tumor suppression.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CRB3 is an apical transmembrane polarity determinant that organizes epithelial architecture, tight junctions, and primary cilia through distinct cytoplasmic domain interactions and upstream regulation of the Hippo pathway. Its C-terminal PDZ-binding motif (ERLI) recruits Par6-aPKC to the apical membrane to govern tight junction morphogenesis, facilitated by the adaptor Morg1 and Cdc42 [PMID:14718572, PMID:23439680], while its FERM-binding domain engages Merlin/NF2 and EPB41L4B to control perijunctional actomyosin contractility, endocytic trafficking, and junctional complex assembly [PMID:41122968, PMID:30440051]. CRB3 recruits Kibra/FRMD6 to activate LATS and suppress YAP/TAZ, enforcing contact inhibition and restraining proliferation; loss of CRB3 activates TAZ/β-catenin signaling, promotes epithelial–mesenchymal transition, and confers stem-cell properties [PMID:28079891, PMID:28436991]. CRB3 also localizes to the basal body, where it navigates Rab11-positive vesicles carrying GCP6 to CEP290 for γ-tubulin ring complex assembly, and its depletion causes severe ciliogenesis defects, loss of Hedgehog responsiveness, and disrupted centriole migration in vivo [PMID:37737843, PMID:37840525].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing CRB3 as a polarity protein that directly recruits the Par6-aPKC complex to regulate tight junction formation answered how Crumbs-family proteins connect to the conserved Par complex in mammalian epithelia.\",\n      \"evidence\": \"Direct binding assay and ERLI-motif mutagenesis in MDCK cells with tight junction permeability readout\",\n      \"pmids\": [\"14718572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals initiating CRB3 apical delivery were unknown\", \"Whether CRB3-Par6 interaction is sufficient or requires co-factors was unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of Morg1 as a bridging adaptor between CRB3 and Par6-aPKC, reinforced by Cdc42, resolved how CRB3 efficiently recruits the polarity complex to the apical membrane.\",\n      \"evidence\": \"Co-IP, pulldown, siRNA in MDCK cells, 3D cyst assay with epistasis rescue by forced apical aPKC targeting\",\n      \"pmids\": [\"23439680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Morg1 is required in all epithelial subtypes was untested\", \"Structural basis of the tripartite CRB3-Morg1-Par6 complex unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Three parallel studies revealed CRB3 functions beyond tight junctions: regulating actin organization at the blood–testis barrier, activating the Hippo pathway to limit YAP-driven myelin elongation, and being transcriptionally repressed by MUC1-C/ZEB1 to modulate Hippo-YAP-MYC signaling in cancer.\",\n      \"evidence\": \"Co-IP of CRB3 with Arp3/Eps8 and Sertoli cell RNAi (actin); in vivo Dy2j/2j mouse model with YAP nuclear rescue (myelin); ChIP of MUC1-C/ZEB1 on CRB3 promoter with phospho-LATS and luciferase reporter (cancer)\",\n      \"pmids\": [\"27358069\", \"27435623\", \"27658423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interaction between CRB3 and Hippo core kinases was not demonstrated\", \"Whether CRB3-actin and CRB3-Hippo branches operate independently or converge was unclear\", \"Myelin phenotype studied in a single disease model\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating that CRB3 recruits Kibra and FRMD6 to activate LATS and suppress YAP/TAZ established CRB3 as an upstream scaffold for the Hippo pathway that enforces contact inhibition and opposes EMT-driven stemness.\",\n      \"evidence\": \"Co-IP of CRB3 with Kibra/FRMD6, overexpression/knockdown in mammary epithelial cells with proliferation, apoptosis, junction, and tumor growth assays; CRB3 KD inducing TAZ/β-catenin and mammosphere formation in MCF10A cells\",\n      \"pmids\": [\"28079891\", \"28436991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CRB3-Kibra interaction is direct or scaffolded was unresolved\", \"Relative contributions of TAZ versus β-catenin downstream of CRB3 loss not dissected\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Domain dissection showed that the FERM-binding domain—not the PDZ-binding domain—drives CRB3-dependent proliferation through EPB41L4B-mediated endocytic trafficking and amphiregulin secretion, revealing a second functional axis of the CRB3 cytoplasmic tail.\",\n      \"evidence\": \"CRB3 FBD versus PDZ deletion mutants in 3D acinar culture with AREG secretion, EPB41L4B siRNA knockdown, and endosome quantification\",\n      \"pmids\": [\"30440051\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How EPB41L4B couples CRB3-FBD to endosomal machinery was not defined\", \"Whether AREG secretion is the sole proliferative effector downstream of the FBD was untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that METTL3-mediated m6A methylation of CRB3 mRNA triggers YTHDF2-dependent degradation revealed an epitranscriptomic layer controlling CRB3 protein levels and, consequently, Hippo pathway activity.\",\n      \"evidence\": \"MeRIP-qPCR, RIP of YTHDF2 on CRB3 mRNA, RNA stability assay, epistasis knockdown of CRB3 reversing METTL3-depletion effects on YAP\",\n      \"pmids\": [\"35012593\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific m6A sites on CRB3 mRNA were not mapped at nucleotide resolution\", \"Whether this regulatory axis operates in normal epithelia or only in cancer cells was unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that CRB3 localizes to the basal body and navigates Rab11-positive vesicles carrying GCP6 to CEP290 for γTuRC assembly established CRB3 as a direct regulator of ciliogenesis, linking it to Hedgehog and Wnt signaling.\",\n      \"evidence\": \"Co-IP of CRB3-Rab11 and GCP6-Rab11-CEP290, Crb3 knockout mice with ciliary defects in mammary and renal epithelia, γTuRC assembly assay, Hh/Wnt signaling readouts\",\n      \"pmids\": [\"37737843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CRB3 directly contacts GCP6 or acts solely through Rab11 was not resolved\", \"Structural basis of CRB3 recognition by Rab11 unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Parallel work in Xenopus multiciliated cells confirmed CRB3 colocalizes with Rab11a on vesicles near ascending basal bodies and anchors phospho-ERM to enable actin-dependent apical membrane expansion, generalizing the CRB3-Rab11 ciliogenesis mechanism across vertebrates.\",\n      \"evidence\": \"Morpholino knockdown of Crb3.L and Rab11a in Xenopus MCCs with live imaging, pERM colocalization, and Rab11a phenocopy\",\n      \"pmids\": [\"37840525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Rescue experiments with human CRB3 in Xenopus MCCs were not performed\", \"Whether ERM anchoring is a direct CRB3 interaction or indirect via scaffold proteins was not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying Merlin/NF2 as a direct FERM-binding domain partner of CRB3 that restrains RhoA-ROCK-myosin II contractility at junctions unified the polarity and actomyosin branches and explained how CRB3 loss causes junctional collapse via hypercontractility.\",\n      \"evidence\": \"Conditional Crb3 knockout colonoids, Co-IP of CRB3A-NF2 via FBD, NF2 siRNA phenocopy, ROCK-II/myosin II inhibitor rescue of junctional architecture\",\n      \"pmids\": [\"41122968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NF2 links CRB3-FBD to the Hippo kinase cascade directly or through separate effectors was not resolved\", \"Contribution of EPB41L4B versus NF2 at the FBD under different cellular contexts not compared\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the PDZ-binding (Par6-aPKC) and FERM-binding (NF2/EPB41L4B) arms of CRB3 are coordinately regulated in space and time, and whether the junctional and ciliary pools of CRB3 share trafficking machinery, remain open mechanistic questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of the CRB3 cytoplasmic tail in complex with its partners\", \"Relative stoichiometry and competition between NF2, EPB41L4B, Kibra, and FRMD6 at the FBD is undefined\", \"Whether CRB3 isoforms (CRB3A vs CRB3B) partition between junctional and ciliary functions has not been rigorously tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5, 9, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 2, 11]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [9, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 5, 6, 8]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 1, 11]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [9, 10]}\n    ],\n    \"complexes\": [\n      \"CRB3-Par6-aPKC apical polarity complex\",\n      \"CRB3-Rab11-GCP6 ciliogenesis trafficking complex\"\n    ],\n    \"partners\": [\n      \"PARD6B\",\n      \"PRKCZ\",\n      \"NF2\",\n      \"EPB41L4B\",\n      \"RAB11A\",\n      \"KIBRA\",\n      \"FRMD6\",\n      \"MORG1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}