{"gene":"CGNL1","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2004,"finding":"JACOP (CGNL1) is a novel cytoplasmic plaque protein that localizes to the apical junctional complex in epithelial and endothelial cells. It contains a coiled-coil domain with sequence similarity to cingulin. Overexpression studies showed it is recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts, suggesting a role in anchoring the apical junctional complex (especially tight junctions) to actin-based cytoskeletons.","method":"Monoclonal antibody cloning, immunofluorescence, immunoelectron microscopy, overexpression studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional consequence (overexpression), single lab, multiple imaging methods","pmids":["15292197"],"is_preprint":false},{"year":2012,"finding":"JACOP/paracingulin (CGNL1) forms a complex with SH3BP1 (a Cdc42/Rac GAP) and CD2AP (a scaffolding protein) at sites of active membrane remodeling during junction formation. Both JACOP and CD2AP were required for normal Cdc42 signaling and junction formation. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity and stalled membrane remodeling.","method":"siRNA screen, co-immunoprecipitation, depletion/rescue experiments, immunofluorescence, functional junction assembly assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional siRNA screen, epistasis with rescue, multiple orthogonal methods in single study","pmids":["22891260"],"is_preprint":false},{"year":2014,"finding":"CGNL1 (paracingulin) depletion inhibits Rac1 activation during junction assembly. MgcRacGAP colocalizes with CGN and CGNL1 at tight junctions, forms a complex with them, and interacts directly in vitro with both. Depletion of either CGN or CGNL1 results in decreased junctional localization of MgcRacGAP. Unexpectedly, double knockdown of both CGN and CGNL1 restores normal Rac1 activation because MgcRacGAP expression is also decreased, and exogenous MgcRacGAP rescues barrier and Rac1 activation phenotypes.","method":"siRNA knockdown, in vitro direct interaction assay, co-immunoprecipitation, immunofluorescence, Rac1 activation assays, transepithelial resistance measurements","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus in vitro direct interaction, functional knockdown with rescue, multiple orthogonal methods","pmids":["24807907"],"is_preprint":false},{"year":2015,"finding":"In endothelial cells, ZO-1 is required for junctional recruitment of JACOP (CGNL1), which in turn recruits p114RhoGEF. Downregulation of JAM-A, JACOP, or p114RhoGEF phenocopied ZO-1 effects on mechanotransducers (redistribution of active myosin II, loss of junctional vinculin and PAK2), establishing a ZO-1→JACOP→p114RhoGEF signaling axis at endothelial junctions.","method":"siRNA depletion, immunofluorescence, FRET-based tension sensors, rescue experiments","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional depletion with phenotypic rescue, epistasis, single lab","pmids":["25753039"],"is_preprint":false},{"year":2017,"finding":"Cgnl1 is enriched in endothelial cells during vascular growth and is required for multicellular tubule formation. Cgnl1 promotes Ve-cadherin association with the actin cytoskeleton to stabilize adherens junctions, and regulates focal adhesion assembly by promoting vinculin and paxillin recruitment and focal adhesion kinase signaling. In vivo, Cgnl1 is required for postnatal retinal neovascular growth and stability.","method":"Loss-of-function assays (3D co-culture), immunofluorescence, in vivo postnatal retinal vascular model, biochemical co-association assays","journal":"Cardiovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular and in vivo phenotypes, single lab, multiple readouts","pmids":["29016873"],"is_preprint":false},{"year":2022,"finding":"CGNL1 (paracingulin) binds to the C-terminal ZU5 domain of ZO-1, and this interaction is required for CGNL1 recruitment to tight junctions and to phase-separated ZO-1 condensates. Knockout of CGNL1 (unlike CGN KO) does not significantly decrease ZO-1 accumulation at tight junctions.","method":"GST pulldown, immunofluorescence microscopy, FRAP, structured illumination microscopy, KO cell lines","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — GST pulldown (in vitro direct interaction), KO cells, structured illumination microscopy, FRAP, multiple orthogonal methods in single rigorous study","pmids":["35259394"],"is_preprint":false},{"year":2023,"finding":"CGNL1 (paracingulin) interacts directly with nonmuscle myosins NM2A and NM2B through its C-terminal coiled-coil sequences. CGNL1 knockout results in myosin-dependent fragmentation of adherens junction complexes. CGNL1 expression promotes junctional accumulation of both NM2A and NM2B, indicating CGNL1 mechanically couples the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.","method":"In vitro direct binding assays, KO and rescue experiments with WT and mutant proteins, immunofluorescence, atomic force microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro binding with domain mutagenesis, KO rescue with mutants, multiple orthogonal methods","pmids":["37204781"],"is_preprint":false},{"year":2023,"finding":"CGNL1 (paracingulin) recruits CAMSAP3 (a microtubule minus-end-binding protein) to tight junctions through a direct interaction mediated by their respective coiled-coil regions. KO of CGNL1 causes loss of junctional CAMSAP3, disorganized cytoplasmic microtubules, irregular nuclei alignment in intestinal epithelial cells, altered cyst morphogenesis, and disrupted planar apical microtubules. The ZO-1-associated pool of CGNL1 tethers CAMSAP3-capped microtubules to junctions.","method":"KO mouse model, KO cultured cells, GST pulldown, ultrastructure expansion microscopy, immunofluorescence","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — GST pulldown showing direct interaction, KO in vivo and in vitro with multiple phenotypic readouts, expansion microscopy","pmids":["37013686"],"is_preprint":false},{"year":2023,"finding":"KO of CGN or CGNL1 or both in MDCK cells causes modest but significant increase in transepithelial resistance and decreased junctional accumulation of claudin-2, which is rescued by CGN or CGNL1 overexpression but not by ZO-1 overexpression. This indicates CGN and CGNL1 regulate claudin-2 junctional localization but are dispensable for overall epithelial barrier function.","method":"CRISPR KO, transepithelial resistance measurements, dextran permeability assays, immunofluorescence, Western blot, mRNA quantification, rescue overexpression","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with multiple functional readouts and rescue, single lab","pmids":["37566083"],"is_preprint":false},{"year":2024,"finding":"Recombinant purified CGNL1 (paracingulin) decorates microtubules and co-pellets with microtubules in vitro. This interaction is mediated by a central region of the CGNL1 head domain (residues 250–420), and deletion of a basic amino-acid stretch (residues 365–377) within this region abolishes both co-pelleting with and decoration of microtubules.","method":"Negative staining electron microscopy, microtubule co-pelleting assay, recombinant protein fragments, deletion mutagenesis","journal":"microPublication biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with domain mutagenesis, single lab, single study","pmids":["39469042"],"is_preprint":false},{"year":2025,"finding":"CGNL1 KO mice do not develop hypertension under a unilateral nephrectomy + angiotensin II (N+A) protocol that induces hypertension in WT mice. CGNL1 is expressed in kidney tubules and endothelium. CGNL1 KO blunts N+A-induced changes in tubular ion transporters (NHE3 and NCC expression/activation) and in angiotensin II-dependent changes in AMPK, ERK, and myosin light chain levels/activation, without affecting vascular contractility. This indicates CGNL1 couples angiotensin II signaling to sodium transport in kidney tubules.","method":"CGNL1 KO mouse model, blood pressure measurement, immunolocalization, transcriptomics, immunoblot, myography","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined in vivo phenotype, multiple molecular readouts, single lab","pmids":["40204428"],"is_preprint":false},{"year":2026,"finding":"CRISPR KO of CGNL1 in porcine intestinal epithelial cells (IPEC-J2) markedly alleviates AFB1-induced cytotoxicity and oxidative stress, and attenuates AFB1-triggered aberrant expression of immune response genes. This identifies CGNL1 as a modulator of epithelial susceptibility to AFB1-induced oxidative stress.","method":"CRISPR/Cas9 KO, cell viability assays, ROS measurement, RNA-seq transcriptomics","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, KO with phenotype but limited mechanistic pathway placement for CGNL1 specifically","pmids":["42123512"],"is_preprint":false}],"current_model":"CGNL1 (paracingulin/JACOP) is a cytoplasmic tight junction scaffold protein that is recruited to junctions via binding of its head domain to the ZU5 domain of ZO-1; at junctions it directly binds nonmuscle myosins NM2A/NM2B through its C-terminal coiled-coil to mechanically couple actomyosin to the plasma membrane, recruits CAMSAP3 to tether microtubule minus-ends to junctions (via coiled-coil interaction), directly interacts with microtubules through a basic stretch in its head domain, recruits p114RhoGEF to regulate RhoA/actomyosin organization, forms a complex with SH3BP1 and CD2AP to spatially restrict Cdc42 activity during junction assembly, recruits MgcRacGAP to down-regulate Rac1 at mature junctions, promotes Ve-cadherin–actin linkage and focal adhesion signaling in endothelial cells, regulates claudin-2 junctional accumulation, and in the kidney couples angiotensin II signaling to tubular ion transporter (NHE3/NCC) activity to influence blood pressure."},"narrative":{"mechanistic_narrative":"CGNL1 (paracingulin/JACOP) is a cytoplasmic plaque protein of the apical junctional complex that scaffolds the actomyosin and microtubule cytoskeletons to epithelial and endothelial cell-cell junctions [PMID:15292197]. It is recruited to tight junctions and to phase-separated ZO-1 condensates through a direct interaction between CGNL1 and the C-terminal ZU5 domain of ZO-1 [PMID:35259394]. From this junctional position CGNL1 mechanically couples the cytoskeleton to the membrane: it binds nonmuscle myosins NM2A and NM2B directly through its C-terminal coiled-coil to promote their junctional accumulation and prevent myosin-dependent fragmentation of adherens junctions [PMID:37204781], tethers CAMSAP3-capped microtubule minus-ends to junctions via a coiled-coil interaction to organize cytoplasmic and planar apical microtubules [PMID:37013686], and directly decorates microtubules through a basic stretch in its head domain (residues 365–377) [PMID:39469042]. CGNL1 also functions as a spatial organizer of small-GTPase signaling at forming and mature junctions, forming a complex with the Cdc42/Rac GAP SH3BP1 and CD2AP to spatially restrict Cdc42 activity during junction assembly [PMID:22891260], recruiting MgcRacGAP to control Rac1 activation [PMID:24807907], and acting in a ZO-1→CGNL1→p114RhoGEF axis that organizes junctional myosin II tension in endothelial cells [PMID:25753039]. In endothelium it stabilizes VE-cadherin–actin linkage and focal adhesion signaling and is required for vascular tubule formation and postnatal retinal neovascular growth [PMID:29016873], and it governs junctional accumulation of claudin-2 [PMID:37566083]. In vivo, CGNL1 couples angiotensin II signaling to tubular sodium transporter (NHE3/NCC) activity in the kidney, with loss of CGNL1 protecting mice from angiotensin II–induced hypertension [PMID:40204428].","teleology":[{"year":2004,"claim":"Established that CGNL1 exists as a distinct cingulin-related junctional plaque protein, defining its baseline localization and candidate cytoskeletal-anchoring role.","evidence":"Monoclonal antibody cloning, immunofluorescence, immunoelectron microscopy and overexpression in epithelial/endothelial cells and fibroblasts","pmids":["15292197"],"confidence":"Medium","gaps":["No direct binding partners identified","Anchoring function inferred from overexpression, not loss-of-function","No molecular mechanism of recruitment"]},{"year":2012,"claim":"Resolved how CGNL1 contributes to junction assembly by placing it in a complex that spatially confines Cdc42 activity during membrane remodeling.","evidence":"siRNA screen, reciprocal co-immunoprecipitation, depletion/rescue and junction assembly assays in epithelial cells","pmids":["22891260"],"confidence":"High","gaps":["Direct vs indirect binding within the CGNL1–SH3BP1–CD2AP complex not fully delineated","How the complex senses sites of active remodeling unknown"]},{"year":2014,"claim":"Extended CGNL1's GTPase-regulatory role to Rac1 by showing it recruits MgcRacGAP, while revealing a compensatory expression relationship with cingulin.","evidence":"siRNA knockdown, in vitro direct interaction, co-IP, Rac1 activation and TER assays","pmids":["24807907"],"confidence":"High","gaps":["Mechanism coupling MgcRacGAP recruitment to Rac1 dynamics not detailed","Basis of co-regulation of MgcRacGAP expression unresolved"]},{"year":2015,"claim":"Defined a directional endothelial signaling axis (ZO-1→CGNL1→p114RhoGEF) controlling junctional myosin II tension and mechanotransduction.","evidence":"siRNA depletion, FRET tension sensors, immunofluorescence and rescue in endothelial cells","pmids":["25753039"],"confidence":"Medium","gaps":["Direct CGNL1–p114RhoGEF binding not biochemically established here","Single lab","RhoA activation step inferred from phenotype"]},{"year":2017,"claim":"Demonstrated an in vivo angiogenic requirement for CGNL1, linking it to VE-cadherin–actin coupling and focal adhesion signaling.","evidence":"3D co-culture loss-of-function, immunofluorescence, biochemical co-association and postnatal retinal vascular model","pmids":["29016873"],"confidence":"Medium","gaps":["Direct molecular partners at adherens junctions/focal adhesions not mapped","Mechanism linking junctional and focal-adhesion roles unclear"]},{"year":2022,"claim":"Identified the molecular basis of CGNL1 junctional recruitment via direct binding to the ZO-1 ZU5 domain and to ZO-1 phase-separated condensates.","evidence":"GST pulldown, FRAP, structured illumination microscopy in KO cell lines","pmids":["35259394"],"confidence":"High","gaps":["Functional consequence of condensate partitioning not fully resolved","Whether other recruitment routes exist not addressed"]},{"year":2023,"claim":"Established CGNL1 as a direct actomyosin coupler at junctions by mapping NM2A/NM2B binding to its C-terminal coiled-coil and showing myosin-dependent junction fragmentation upon loss.","evidence":"In vitro direct binding with domain mutagenesis, KO/rescue, immunofluorescence and atomic force microscopy","pmids":["37204781"],"confidence":"High","gaps":["Force regulation dynamics not quantified","Interplay with the RhoGEF/GAP modules not integrated"]},{"year":2023,"claim":"Defined CGNL1 as a microtubule-tethering scaffold by mapping a direct coiled-coil interaction with CAMSAP3 that anchors minus-ends and organizes apical microtubule architecture.","evidence":"KO mouse and cell models, GST pulldown, ultrastructure expansion microscopy","pmids":["37013686"],"confidence":"High","gaps":["How microtubule and actomyosin functions are coordinated unknown","Tissue-specific consequences beyond intestine not explored"]},{"year":2023,"claim":"Showed CGNL1 (with cingulin) governs claudin-2 junctional accumulation but is dispensable for overall barrier integrity, refining its role in junction composition.","evidence":"CRISPR KO, TER and dextran permeability, immunofluorescence and rescue in MDCK cells","pmids":["37566083"],"confidence":"Medium","gaps":["Mechanism linking CGNL1 to claudin-2 localization unknown","Redundancy with cingulin not fully separated"]},{"year":2024,"claim":"Demonstrated a direct, autonomous microtubule-binding activity of CGNL1 independent of CAMSAP3, localized to a basic stretch in the head domain.","evidence":"Negative-stain EM, microtubule co-pelleting with recombinant fragments and deletion mutagenesis","pmids":["39469042"],"confidence":"Medium","gaps":["Cellular significance of direct microtubule binding vs CAMSAP3 tethering not resolved","Single in vitro study"]},{"year":2025,"claim":"Connected CGNL1 to organ-level physiology by showing it couples angiotensin II signaling to renal tubular sodium transport and blood pressure control.","evidence":"CGNL1 KO mouse, blood pressure measurement, immunolocalization, transcriptomics, immunoblot and myography","pmids":["40204428"],"confidence":"Medium","gaps":["Direct molecular link between CGNL1 and NHE3/NCC regulation unknown","Whether junctional scaffolding functions underlie the renal phenotype unclear"]},{"year":2026,"claim":"Implicated CGNL1 as a modulator of epithelial susceptibility to aflatoxin-induced oxidative stress and immune gene responses.","evidence":"CRISPR KO in porcine IPEC-J2 cells, viability, ROS and RNA-seq","pmids":["42123512"],"confidence":"Low","gaps":["Limited mechanistic placement of CGNL1 in oxidative-stress pathways","Single lab, non-human cell line","No direct molecular target identified"]},{"year":null,"claim":"How CGNL1 integrates its parallel actomyosin coupling, microtubule tethering, and small-GTPase regulatory modules into a unified, spatially coordinated program of junction mechanoregulation remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of full-length CGNL1 or its multivalent assemblies","Hierarchy and crosstalk among ZO-1, myosin, CAMSAP3 and GTPase modules not established","Direct molecular link to renal ion-transport phenotype unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,3,5,6,7]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[6,7,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[1,3,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2,3,10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4]}],"complexes":["CGNL1–SH3BP1–CD2AP complex","tight junction cytoplasmic plaque"],"partners":["TJP1","MYH9","MYH10","CAMSAP3","SH3BP1","CD2AP","RACGAP1","ARHGEF18"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q0VF96","full_name":"Cingulin-like protein 1","aliases":["Junction-associated coiled-coil protein","Paracingulin"],"length_aa":1302,"mass_kda":149.1,"function":"May be involved in anchoring the apical junctional complex, especially tight junctions, to actin-based cytoskeletons","subcellular_location":"Cell junction, tight junction","url":"https://www.uniprot.org/uniprotkb/Q0VF96/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CGNL1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CGNL1","total_profiled":1310},"omim":[{"mim_id":"617368","title":"SH3 DOMAIN-BINDING PROTEIN 1; SH3BP1","url":"https://www.omim.org/entry/617368"},{"mim_id":"614578","title":"PROGESTIN AND ADIPOQ RECEPTOR FAMILY, MEMBER 4; PAQR4","url":"https://www.omim.org/entry/614578"},{"mim_id":"607856","title":"CINGULIN-LIKE 1; CGNL1","url":"https://www.omim.org/entry/607856"},{"mim_id":"605112","title":"TROPOMODULIN 3; TMOD3","url":"https://www.omim.org/entry/605112"},{"mim_id":"139300","title":"AROMATASE EXCESS SYNDROME; AEXS","url":"https://www.omim.org/entry/139300"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cell Junctions","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"kidney","ntpm":63.8}],"url":"https://www.proteinatlas.org/search/CGNL1"},"hgnc":{"alias_symbol":["FLJ14957","JACOP","KIAA1749","PCING"],"prev_symbol":[]},"alphafold":{"accession":"Q0VF96","domains":[{"cath_id":"-","chopping":"895-1199","consensus_level":"medium","plddt":88.6939,"start":895,"end":1199},{"cath_id":"1.20.5","chopping":"1202-1251","consensus_level":"medium","plddt":83.0034,"start":1202,"end":1251}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q0VF96","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q0VF96-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q0VF96-F1-predicted_aligned_error_v6.png","plddt_mean":63.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CGNL1","jax_strain_url":"https://www.jax.org/strain/search?query=CGNL1"},"sequence":{"accession":"Q0VF96","fasta_url":"https://rest.uniprot.org/uniprotkb/Q0VF96.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q0VF96/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q0VF96"}},"corpus_meta":[{"pmid":"17210054","id":"PMC_17210054","title":"A 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It contains a coiled-coil domain with sequence similarity to cingulin. Overexpression studies showed it is recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts, suggesting a role in anchoring the apical junctional complex (especially tight junctions) to actin-based cytoskeletons.\",\n      \"method\": \"Monoclonal antibody cloning, immunofluorescence, immunoelectron microscopy, overexpression studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional consequence (overexpression), single lab, multiple imaging methods\",\n      \"pmids\": [\"15292197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"JACOP/paracingulin (CGNL1) forms a complex with SH3BP1 (a Cdc42/Rac GAP) and CD2AP (a scaffolding protein) at sites of active membrane remodeling during junction formation. Both JACOP and CD2AP were required for normal Cdc42 signaling and junction formation. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity and stalled membrane remodeling.\",\n      \"method\": \"siRNA screen, co-immunoprecipitation, depletion/rescue experiments, immunofluorescence, functional junction assembly assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional siRNA screen, epistasis with rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"22891260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CGNL1 (paracingulin) depletion inhibits Rac1 activation during junction assembly. MgcRacGAP colocalizes with CGN and CGNL1 at tight junctions, forms a complex with them, and interacts directly in vitro with both. Depletion of either CGN or CGNL1 results in decreased junctional localization of MgcRacGAP. Unexpectedly, double knockdown of both CGN and CGNL1 restores normal Rac1 activation because MgcRacGAP expression is also decreased, and exogenous MgcRacGAP rescues barrier and Rac1 activation phenotypes.\",\n      \"method\": \"siRNA knockdown, in vitro direct interaction assay, co-immunoprecipitation, immunofluorescence, Rac1 activation assays, transepithelial resistance measurements\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus in vitro direct interaction, functional knockdown with rescue, multiple orthogonal methods\",\n      \"pmids\": [\"24807907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In endothelial cells, ZO-1 is required for junctional recruitment of JACOP (CGNL1), which in turn recruits p114RhoGEF. Downregulation of JAM-A, JACOP, or p114RhoGEF phenocopied ZO-1 effects on mechanotransducers (redistribution of active myosin II, loss of junctional vinculin and PAK2), establishing a ZO-1→JACOP→p114RhoGEF signaling axis at endothelial junctions.\",\n      \"method\": \"siRNA depletion, immunofluorescence, FRET-based tension sensors, rescue experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional depletion with phenotypic rescue, epistasis, single lab\",\n      \"pmids\": [\"25753039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cgnl1 is enriched in endothelial cells during vascular growth and is required for multicellular tubule formation. Cgnl1 promotes Ve-cadherin association with the actin cytoskeleton to stabilize adherens junctions, and regulates focal adhesion assembly by promoting vinculin and paxillin recruitment and focal adhesion kinase signaling. In vivo, Cgnl1 is required for postnatal retinal neovascular growth and stability.\",\n      \"method\": \"Loss-of-function assays (3D co-culture), immunofluorescence, in vivo postnatal retinal vascular model, biochemical co-association assays\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular and in vivo phenotypes, single lab, multiple readouts\",\n      \"pmids\": [\"29016873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CGNL1 (paracingulin) binds to the C-terminal ZU5 domain of ZO-1, and this interaction is required for CGNL1 recruitment to tight junctions and to phase-separated ZO-1 condensates. Knockout of CGNL1 (unlike CGN KO) does not significantly decrease ZO-1 accumulation at tight junctions.\",\n      \"method\": \"GST pulldown, immunofluorescence microscopy, FRAP, structured illumination microscopy, KO cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — GST pulldown (in vitro direct interaction), KO cells, structured illumination microscopy, FRAP, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"35259394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CGNL1 (paracingulin) interacts directly with nonmuscle myosins NM2A and NM2B through its C-terminal coiled-coil sequences. CGNL1 knockout results in myosin-dependent fragmentation of adherens junction complexes. CGNL1 expression promotes junctional accumulation of both NM2A and NM2B, indicating CGNL1 mechanically couples the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.\",\n      \"method\": \"In vitro direct binding assays, KO and rescue experiments with WT and mutant proteins, immunofluorescence, atomic force microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro binding with domain mutagenesis, KO rescue with mutants, multiple orthogonal methods\",\n      \"pmids\": [\"37204781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CGNL1 (paracingulin) recruits CAMSAP3 (a microtubule minus-end-binding protein) to tight junctions through a direct interaction mediated by their respective coiled-coil regions. KO of CGNL1 causes loss of junctional CAMSAP3, disorganized cytoplasmic microtubules, irregular nuclei alignment in intestinal epithelial cells, altered cyst morphogenesis, and disrupted planar apical microtubules. The ZO-1-associated pool of CGNL1 tethers CAMSAP3-capped microtubules to junctions.\",\n      \"method\": \"KO mouse model, KO cultured cells, GST pulldown, ultrastructure expansion microscopy, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — GST pulldown showing direct interaction, KO in vivo and in vitro with multiple phenotypic readouts, expansion microscopy\",\n      \"pmids\": [\"37013686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KO of CGN or CGNL1 or both in MDCK cells causes modest but significant increase in transepithelial resistance and decreased junctional accumulation of claudin-2, which is rescued by CGN or CGNL1 overexpression but not by ZO-1 overexpression. This indicates CGN and CGNL1 regulate claudin-2 junctional localization but are dispensable for overall epithelial barrier function.\",\n      \"method\": \"CRISPR KO, transepithelial resistance measurements, dextran permeability assays, immunofluorescence, Western blot, mRNA quantification, rescue overexpression\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with multiple functional readouts and rescue, single lab\",\n      \"pmids\": [\"37566083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Recombinant purified CGNL1 (paracingulin) decorates microtubules and co-pellets with microtubules in vitro. This interaction is mediated by a central region of the CGNL1 head domain (residues 250–420), and deletion of a basic amino-acid stretch (residues 365–377) within this region abolishes both co-pelleting with and decoration of microtubules.\",\n      \"method\": \"Negative staining electron microscopy, microtubule co-pelleting assay, recombinant protein fragments, deletion mutagenesis\",\n      \"journal\": \"microPublication biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with domain mutagenesis, single lab, single study\",\n      \"pmids\": [\"39469042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CGNL1 KO mice do not develop hypertension under a unilateral nephrectomy + angiotensin II (N+A) protocol that induces hypertension in WT mice. CGNL1 is expressed in kidney tubules and endothelium. CGNL1 KO blunts N+A-induced changes in tubular ion transporters (NHE3 and NCC expression/activation) and in angiotensin II-dependent changes in AMPK, ERK, and myosin light chain levels/activation, without affecting vascular contractility. This indicates CGNL1 couples angiotensin II signaling to sodium transport in kidney tubules.\",\n      \"method\": \"CGNL1 KO mouse model, blood pressure measurement, immunolocalization, transcriptomics, immunoblot, myography\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined in vivo phenotype, multiple molecular readouts, single lab\",\n      \"pmids\": [\"40204428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CRISPR KO of CGNL1 in porcine intestinal epithelial cells (IPEC-J2) markedly alleviates AFB1-induced cytotoxicity and oxidative stress, and attenuates AFB1-triggered aberrant expression of immune response genes. This identifies CGNL1 as a modulator of epithelial susceptibility to AFB1-induced oxidative stress.\",\n      \"method\": \"CRISPR/Cas9 KO, cell viability assays, ROS measurement, RNA-seq transcriptomics\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, KO with phenotype but limited mechanistic pathway placement for CGNL1 specifically\",\n      \"pmids\": [\"42123512\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CGNL1 (paracingulin/JACOP) is a cytoplasmic tight junction scaffold protein that is recruited to junctions via binding of its head domain to the ZU5 domain of ZO-1; at junctions it directly binds nonmuscle myosins NM2A/NM2B through its C-terminal coiled-coil to mechanically couple actomyosin to the plasma membrane, recruits CAMSAP3 to tether microtubule minus-ends to junctions (via coiled-coil interaction), directly interacts with microtubules through a basic stretch in its head domain, recruits p114RhoGEF to regulate RhoA/actomyosin organization, forms a complex with SH3BP1 and CD2AP to spatially restrict Cdc42 activity during junction assembly, recruits MgcRacGAP to down-regulate Rac1 at mature junctions, promotes Ve-cadherin–actin linkage and focal adhesion signaling in endothelial cells, regulates claudin-2 junctional accumulation, and in the kidney couples angiotensin II signaling to tubular ion transporter (NHE3/NCC) activity to influence blood pressure.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CGNL1 (paracingulin/JACOP) is a cytoplasmic plaque protein of the apical junctional complex that scaffolds the actomyosin and microtubule cytoskeletons to epithelial and endothelial cell-cell junctions [#0]. It is recruited to tight junctions and to phase-separated ZO-1 condensates through a direct interaction between CGNL1 and the C-terminal ZU5 domain of ZO-1 [#5]. From this junctional position CGNL1 mechanically couples the cytoskeleton to the membrane: it binds nonmuscle myosins NM2A and NM2B directly through its C-terminal coiled-coil to promote their junctional accumulation and prevent myosin-dependent fragmentation of adherens junctions [#6], tethers CAMSAP3-capped microtubule minus-ends to junctions via a coiled-coil interaction to organize cytoplasmic and planar apical microtubules [#7], and directly decorates microtubules through a basic stretch in its head domain (residues 365\\u2013377) [#9]. CGNL1 also functions as a spatial organizer of small-GTPase signaling at forming and mature junctions, forming a complex with the Cdc42/Rac GAP SH3BP1 and CD2AP to spatially restrict Cdc42 activity during junction assembly [#1], recruiting MgcRacGAP to control Rac1 activation [#2], and acting in a ZO-1\\u2192CGNL1\\u2192p114RhoGEF axis that organizes junctional myosin II tension in endothelial cells [#3]. In endothelium it stabilizes VE-cadherin\\u2013actin linkage and focal adhesion signaling and is required for vascular tubule formation and postnatal retinal neovascular growth [#4], and it governs junctional accumulation of claudin-2 [#8]. In vivo, CGNL1 couples angiotensin II signaling to tubular sodium transporter (NHE3/NCC) activity in the kidney, with loss of CGNL1 protecting mice from angiotensin II\\u2013induced hypertension [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that CGNL1 exists as a distinct cingulin-related junctional plaque protein, defining its baseline localization and candidate cytoskeletal-anchoring role.\",\n      \"evidence\": \"Monoclonal antibody cloning, immunofluorescence, immunoelectron microscopy and overexpression in epithelial/endothelial cells and fibroblasts\",\n      \"pmids\": [\"15292197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct binding partners identified\", \"Anchoring function inferred from overexpression, not loss-of-function\", \"No molecular mechanism of recruitment\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved how CGNL1 contributes to junction assembly by placing it in a complex that spatially confines Cdc42 activity during membrane remodeling.\",\n      \"evidence\": \"siRNA screen, reciprocal co-immunoprecipitation, depletion/rescue and junction assembly assays in epithelial cells\",\n      \"pmids\": [\"22891260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect binding within the CGNL1\\u2013SH3BP1\\u2013CD2AP complex not fully delineated\", \"How the complex senses sites of active remodeling unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended CGNL1's GTPase-regulatory role to Rac1 by showing it recruits MgcRacGAP, while revealing a compensatory expression relationship with cingulin.\",\n      \"evidence\": \"siRNA knockdown, in vitro direct interaction, co-IP, Rac1 activation and TER assays\",\n      \"pmids\": [\"24807907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling MgcRacGAP recruitment to Rac1 dynamics not detailed\", \"Basis of co-regulation of MgcRacGAP expression unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a directional endothelial signaling axis (ZO-1\\u2192CGNL1\\u2192p114RhoGEF) controlling junctional myosin II tension and mechanotransduction.\",\n      \"evidence\": \"siRNA depletion, FRET tension sensors, immunofluorescence and rescue in endothelial cells\",\n      \"pmids\": [\"25753039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CGNL1\\u2013p114RhoGEF binding not biochemically established here\", \"Single lab\", \"RhoA activation step inferred from phenotype\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated an in vivo angiogenic requirement for CGNL1, linking it to VE-cadherin\\u2013actin coupling and focal adhesion signaling.\",\n      \"evidence\": \"3D co-culture loss-of-function, immunofluorescence, biochemical co-association and postnatal retinal vascular model\",\n      \"pmids\": [\"29016873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular partners at adherens junctions/focal adhesions not mapped\", \"Mechanism linking junctional and focal-adhesion roles unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the molecular basis of CGNL1 junctional recruitment via direct binding to the ZO-1 ZU5 domain and to ZO-1 phase-separated condensates.\",\n      \"evidence\": \"GST pulldown, FRAP, structured illumination microscopy in KO cell lines\",\n      \"pmids\": [\"35259394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of condensate partitioning not fully resolved\", \"Whether other recruitment routes exist not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established CGNL1 as a direct actomyosin coupler at junctions by mapping NM2A/NM2B binding to its C-terminal coiled-coil and showing myosin-dependent junction fragmentation upon loss.\",\n      \"evidence\": \"In vitro direct binding with domain mutagenesis, KO/rescue, immunofluorescence and atomic force microscopy\",\n      \"pmids\": [\"37204781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Force regulation dynamics not quantified\", \"Interplay with the RhoGEF/GAP modules not integrated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined CGNL1 as a microtubule-tethering scaffold by mapping a direct coiled-coil interaction with CAMSAP3 that anchors minus-ends and organizes apical microtubule architecture.\",\n      \"evidence\": \"KO mouse and cell models, GST pulldown, ultrastructure expansion microscopy\",\n      \"pmids\": [\"37013686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How microtubule and actomyosin functions are coordinated unknown\", \"Tissue-specific consequences beyond intestine not explored\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed CGNL1 (with cingulin) governs claudin-2 junctional accumulation but is dispensable for overall barrier integrity, refining its role in junction composition.\",\n      \"evidence\": \"CRISPR KO, TER and dextran permeability, immunofluorescence and rescue in MDCK cells\",\n      \"pmids\": [\"37566083\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking CGNL1 to claudin-2 localization unknown\", \"Redundancy with cingulin not fully separated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated a direct, autonomous microtubule-binding activity of CGNL1 independent of CAMSAP3, localized to a basic stretch in the head domain.\",\n      \"evidence\": \"Negative-stain EM, microtubule co-pelleting with recombinant fragments and deletion mutagenesis\",\n      \"pmids\": [\"39469042\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular significance of direct microtubule binding vs CAMSAP3 tethering not resolved\", \"Single in vitro study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected CGNL1 to organ-level physiology by showing it couples angiotensin II signaling to renal tubular sodium transport and blood pressure control.\",\n      \"evidence\": \"CGNL1 KO mouse, blood pressure measurement, immunolocalization, transcriptomics, immunoblot and myography\",\n      \"pmids\": [\"40204428\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between CGNL1 and NHE3/NCC regulation unknown\", \"Whether junctional scaffolding functions underlie the renal phenotype unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Implicated CGNL1 as a modulator of epithelial susceptibility to aflatoxin-induced oxidative stress and immune gene responses.\",\n      \"evidence\": \"CRISPR KO in porcine IPEC-J2 cells, viability, ROS and RNA-seq\",\n      \"pmids\": [\"42123512\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Limited mechanistic placement of CGNL1 in oxidative-stress pathways\", \"Single lab, non-human cell line\", \"No direct molecular target identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CGNL1 integrates its parallel actomyosin coupling, microtubule tethering, and small-GTPase regulatory modules into a unified, spatially coordinated program of junction mechanoregulation remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of full-length CGNL1 or its multivalent assemblies\", \"Hierarchy and crosstalk among ZO-1, myosin, CAMSAP3 and GTPase modules not established\", \"Direct molecular link to renal ion-transport phenotype unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 3, 5, 6, 7]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [6, 7, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005923\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [1, 3, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 3, 10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\n      \"CGNL1\\u2013SH3BP1\\u2013CD2AP complex\",\n      \"tight junction cytoplasmic plaque\"\n    ],\n    \"partners\": [\n      \"TJP1\",\n      \"MYH9\",\n      \"MYH10\",\n      \"CAMSAP3\",\n      \"SH3BP1\",\n      \"CD2AP\",\n      \"RACGAP1\",\n      \"ARHGEF18\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}