{"gene":"CRB2","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":2006,"finding":"Crb2 (fission yeast 53BP1 homolog) contains tandem tudor domains that directly bind histone H4 specifically dimethylated at Lys20 (H4-K20me2), with a five-residue binding cage that accommodates dimethyllysine but excludes trimethyllysine, establishing the structural basis for methylation state-specific recruitment to DNA double-strand breaks.","method":"X-ray crystallography, NMR spectroscopy, in vitro binding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus NMR with direct binding validation; replicated across 53BP1 and Crb2","pmids":["17190600"],"is_preprint":false},{"year":2004,"finding":"Set9-mediated methylation of histone H4-K20 is required for Crb2 localization to sites of DNA damage; loss of Set9 or mutation of H4-K20 impairs Crb2 focus formation and compromises checkpoint-mediated cell cycle arrest in fission yeast.","method":"Genetic epistasis, chromatin immunoprecipitation, fluorescence microscopy of Crb2 foci","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with direct localization readout, replicated by multiple labs","pmids":["15550243"],"is_preprint":false},{"year":1997,"finding":"Crb2 interacts with Cut5 (TopBP1 homolog) and Chk1 in fission yeast, acts upstream of Chk1 in the DNA damage checkpoint pathway, and is transiently phosphorylated upon UV damage in a manner requiring other damage-sensing checkpoint proteins but not Chk1.","method":"Two-hybrid interaction, co-immunoprecipitation, genetic epistasis, phosphorylation assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Y2H, co-IP, epistasis, phosphorylation), foundational paper","pmids":["9407031"],"is_preprint":false},{"year":2004,"finding":"Gamma-H2A (phosphorylated histone H2A) controls large-scale recruitment of Crb2 to damaged DNA in fission yeast; H2A-AQE mutations defective in phosphorylation impair Crb2 and Chk1 phosphorylation and cause failure to recruit large amounts of Crb2 to DSBs.","method":"Genetic analysis, fluorescence microscopy, phosphorylation assays, epistasis with rad22Δ","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with clear mechanistic readouts, replicated","pmids":["15226425"],"is_preprint":false},{"year":1999,"finding":"Cdc2 kinase phosphorylates Crb2 at threonine-215 in vitro and in vivo; this phosphorylation is required for cells to re-enter the cell cycle after DNA damage checkpoint arrest, and non-phosphorylatable T215A cells remain arrested with Chk1 kinase maintained in a phosphorylated state even after repair.","method":"In vitro kinase assay with purified Cdc2, mutational analysis (T215A), phosphorylation timing experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus in vivo mutational analysis","pmids":["10488332"],"is_preprint":false},{"year":2002,"finding":"Cdc2-cyclin B kinase activity links Crb2 to topoisomerase III (Top3) function in recombinational repair during G2; low Cdc2-cyclin B activity disrupts proper regulation of Top3 via Crb2, affecting a recombination step after Rhp51 focus assembly.","method":"Genetic epistasis, fluorescence microscopy of Rhp51 foci, cyclin B mutant analysis","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with defined cellular phenotype, single lab","pmids":["12023299"],"is_preprint":false},{"year":2003,"finding":"Crb2 localizes to distinct nuclear foci representing sites of DNA double-strand breaks in live fission yeast cells; Crb2 colocalizes with Rad22 at persistent foci; Rad1, Rad3, and Rad17 complexes are required for retention but not initial recruitment of Crb2 to DSBs.","method":"Live-cell fluorescence microscopy, genetic analysis of checkpoint mutants","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — direct live-cell localization with functional consequence, multiple genetic backgrounds tested","pmids":["12917337"],"is_preprint":false},{"year":2006,"finding":"Two parallel recruitment pathways bring Crb2 to DSBs in fission yeast: one requires histone H4-K20 methylation (Tudor domain) and gamma-H2A (BRCT domain) acting cooperatively; a second CDK-dependent pathway requires phosphorylation of the Cut5-binding site (T215) in Crb2 and mediates Cut5 association, providing checkpoint activation even in the absence of histone modifications.","method":"Fluorescence microscopy of HO-induced DSBs, mutational analysis of Tudor and BRCT motifs, genetic epistasis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods defining two distinct pathways","pmids":["16778077"],"is_preprint":false},{"year":2004,"finding":"Crb2 oligomerizes through its tandem BRCT domains; this homo-oligomerization is the essential function of the BRCT domains for DNA damage checkpoint signaling, as heterologous dimerization motifs can substitute for BRCT domains.","method":"In vitro biochemical analysis, dominant-negative overexpression, functional complementation with heterologous dimerization motifs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical reconstitution plus genetic complementation with multiple heterologous domains","pmids":["15229228"],"is_preprint":false},{"year":2004,"finding":"Crb2 dynamically interacts with Rad3, Chk1, and Cut5 in a temporal manner during the DNA damage checkpoint; active Crb2-Chk1 complex formation is regulated by Rad3 and requires Crb2 BRCT domain; Chk1 activation in vitro requires the presence of Crb2 BRCT domain; Crb2 directly interacts with Rad3 in an inhibitory manner to Rad3 activity.","method":"Co-immunoprecipitation, in vitro Chk1 activation assay, two-hybrid analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay combined with reciprocal co-IP, multiple interaction partners defined","pmids":["14739927"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of Crb2-BRCT2 domain reveals structural basis for dimerization and direct interaction with gamma-H2A.1 peptide; dimerization mutants are genotoxin sensitive and defective in checkpoint signaling and Crb2 IRIF formation, while phosphopeptide-binding mutants show DNA repair defects (slowed Rpa1 foci, reduced Rad22 foci) with only slight IR sensitivity.","method":"X-ray crystallography, in vitro mutational analysis, in vivo phenotypic analysis (checkpoint, IRIF, repair foci)","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro mutagenesis plus in vivo functional dissection","pmids":["18676809"],"is_preprint":false},{"year":2008,"finding":"The tandem tudor domains of Crb2 preferentially bind di-methylated H4K20 (not tri-methylated); disruption of either the tudor-binding motif or the H4K20 methylating enzyme Set9 ablates Crb2 localization to DSBs and impairs checkpoint function and cell survival after DNA damage.","method":"In vitro binding assays with mono/di/tri-methyl H4K20 peptides, fluorescence microscopy, genetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro binding specificity assay combined with in vivo localization and function","pmids":["18826944"],"is_preprint":false},{"year":2010,"finding":"Crb2 BRCT2 domain directly binds phospho-H2A (gamma-H2A) through polar interactions of serine-548 and lysine-619 with the phosphate group; these interactions are critical for Crb2 IRIF formation and checkpoint function, and mutations have additive effects; combining these BRCT2 mutations with T215A completely abrogates Crb2 IRIF and function.","method":"Mutational analysis, fluorescence microscopy (IRIF), checkpoint assays, in vitro phosphopeptide binding","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — structure-guided mutagenesis with in vivo functional validation, replicated by two independent papers (PMID 20679488)","pmids":["20679485","20679488"],"is_preprint":false},{"year":2005,"finding":"Cooperative control of Crb2 by ATM family kinases (through gamma-H2A) and Cdc2 (through T215 phosphorylation) is essential for the DNA damage checkpoint; crb2-T215A cells can initiate but not sustain checkpoint response; gamma-H2A is essential for checkpoint in crb2-T215A cells; Cdc2 inactivation in gamma-H2A-defective cells impairs Crb2-dependent Chk1 signaling.","method":"Genetic epistasis, checkpoint signaling assays, Chk1 phosphorylation analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic combinations tested with defined molecular readout (Chk1 phosphorylation)","pmids":["16314498"],"is_preprint":false},{"year":2012,"finding":"Crb2 recruits Chk1 to DSBs through direct physical interaction; Rad3-phosphorylated SQ/TQ motifs in Crb2 are required for Chk1 recruitment and activation; a 19-aa peptide containing these motifs is sufficient for Chk1 binding in vitro when phosphorylated; tethering a Crb2-Chk1 fusion or Rad22-SQ/TQ fusion to DSBs can bypass the need for full-length Crb2 and even the 9-1-1 complex.","method":"Co-immunoprecipitation, in vitro binding assays, mutational analysis, artificial tethering experiments","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro binding plus multiple in vivo genetic rescue experiments with defined molecular readouts","pmids":["22792081"],"is_preprint":false},{"year":2010,"finding":"Human CRB2 inhibits gamma-secretase cleavage of amyloid precursor protein (APP) by binding to the presenilin complex; the transmembrane domain of CRB2 is indispensable for this inhibition and mediates binding to the gamma-secretase complex; CRB2 is not a competitive substrate but functions as an inhibitory binding protein.","method":"Transfection/knockdown in HEK293 and SH-SY5Y cells, cell-free gamma-secretase assay, co-immunoprecipitation, domain deletion analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — cell-free assay plus co-IP, single lab; contradicted by another paper (PMID 17988153)","pmids":["20299451"],"is_preprint":false},{"year":2014,"finding":"Loss of Crb2 in zebrafish podocytes impairs podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking; complementation experiments with human CRB2 mutations confirm loss-of-function causing nephrotic syndrome.","method":"Zebrafish crb2b loss-of-function analysis, nephrin trafficking assay, complementation with human CRB2 mutants","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — defined cellular phenotype with complementation validation in vivo","pmids":["25557779"],"is_preprint":false},{"year":2011,"finding":"Mouse Crb2 is required for epiblast cell polarity at the primitive streak; loss of Crb2 disturbs apical polarity of epiblast cells, affecting epithelial-to-mesenchymal transition during gastrulation and impairing mesoderm/endoderm formation, causing embryonic lethality by E12.5.","method":"Gene-targeted knockout mice, histological and immunofluorescence analysis","journal":"Developmental dynamics","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular polarity phenotype and embryonic lethal outcome","pmids":["22072575"],"is_preprint":false},{"year":2012,"finding":"Conditional deletion of mouse CRB2 in the retina causes progressive disorganization of photoreceptor layer, disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells, and increased proliferation of late-born progenitor cells; CRB2 is essential for proper lamination and suppression of late-born retinal progenitor cell proliferation.","method":"Conditional knockout mice, OCT imaging, ERG, histological and confocal analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — clean conditional KO with multiple orthogonal readouts (imaging, electrophysiology, histology)","pmids":["23001562"],"is_preprint":false},{"year":2013,"finding":"Loss of both CRB1 and CRB2 in retinal progenitor cells dysregulates Notch1 and YAP/Hippo signaling pathways, increases P120-catenin levels, and alters progenitor cell cycle distribution (decrease in late G1, increase in S and G2/M phase), leading to expanded late-born cell types.","method":"Conditional double-knockout mice, transcriptome analysis, cell cycle analysis, immunostaining","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with pathway analysis, but signaling pathway assignment based on gene expression changes rather than direct epistasis","pmids":["24339791"],"is_preprint":false},{"year":2014,"finding":"CRB2 has essential functions specifically in photoreceptors (not redundant in Müller cells) in developing mouse retina; photoreceptor-specific loss causes early-onset abnormal lamination, while Müller cell-specific loss causes late-onset disorganization; short-term adult loss of CRB2 in photoreceptors causes sporadic loss of adhesion between photoreceptors and Müller cells.","method":"Cell-type-specific conditional knockout, AAV-Cre delivery, OCT imaging, ERG","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO with multiple functional readouts defining distinct roles","pmids":["24493795"],"is_preprint":false},{"year":2021,"finding":"Podocyte-specific Crb2 knockout mice develop massive albuminuria and FSGS; CRB2-deficient human podocytes show decreased F-actin positive area and increased susceptibility to apoptosis, demonstrating that CRB2 is required for actin cytoskeleton organization and podocyte survival.","method":"Podocyte-specific conditional knockout, electron microscopy, siRNA knockdown of human podocytes, F-actin staining, apoptosis assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO with electron microscopy plus in vitro mechanistic follow-up","pmids":["34654837"],"is_preprint":false},{"year":2022,"finding":"CRB2 depletion or anti-CRB2 autoantibody binding in podocytes increases phosphorylation of ezrin (which connects CRB2 to the cytoskeleton) and alters CRB2 localization and actin distribution, linking CRB2 to cytoskeletal organization at podocyte foot processes.","method":"Immunization model, anti-CRB2 antibody treatment of podocyte cell line, immunofluorescence, phosphorylation assay","journal":"Journal of the American Society of Nephrology","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct mechanistic link between antibody binding and ezrin phosphorylation/actin redistribution, single lab","pmids":["35985815"],"is_preprint":false},{"year":2025,"finding":"CRB2 knockdown in podocytes induces YAP transcriptional activity and upregulates YAP-mediated mechanosignaling; CRB2 deficiency increases focal adhesion density, F-actin, and podocyte contractility in a substrate stiffness-dependent manner as measured by ERISM, demonstrating impaired mechanosensing.","method":"siRNA knockdown, YAP reporter assays, F-actin staining, Elastic Resonator Interference Stress Microscopy (ERISM), pharmacological YAP inhibition","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 — novel biophysical assay (ERISM) combined with molecular readouts, single lab","pmids":["40062402"],"is_preprint":false},{"year":2022,"finding":"Disease-associated CRB2 variants causing nephrotic syndrome remain predominantly at the ER rather than reaching the plasma membrane; WT CRB2 is retained in the ER in cells lacking protein disulfide isomerase A3 (PDIA3), demonstrating that disulfide bridge formation is a crucial posttranslational modification required for CRB2 plasma membrane transport.","method":"Live-cell imaging with GFP-tagged CRB2 variants, BFP-labeled plasma membrane reporter, PDIA3 knockdown","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative live-cell assay with defined molecular mechanism (disulfide bridge formation via PDIA3), single lab","pmids":["36549870"],"is_preprint":false},{"year":2024,"finding":"Human CRB1 and CRB2 form homo- and heteromeric complexes; CRB1 was enriched in CRB2 pull-downs from retina; co-immunoprecipitation shows canonical CRB1 interacts with CRB1 and CRB2 but not CRB3 (which lacks an extracellular domain), indicating interaction is mediated through the extracellular domain.","method":"Retina-specific pull-down, co-immunoprecipitation, co-localization in human retina and iPSC-derived organoids","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2–3 — reciprocal pull-down from native tissue plus domain-based mechanistic inference, single lab","pmids":["38570189"],"is_preprint":false},{"year":2015,"finding":"CRB2 localizes to the apicolateral membrane of mouse RPE cells at tight junctions, completing a fully expressed Crumbs complex in the retinal pigment epithelium.","method":"Immunofluorescence with specific antibody, confocal microscopy, subcellular fractionation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — direct localization experiment with specific antibody, no strong functional consequence established in same study","pmids":["26404741"],"is_preprint":false},{"year":2021,"finding":"CRB2 plays a key role in tight junction maintenance and cell cycle arrest in human RPE cells during polarization; in vivo CRB2 knockdown in mouse RPE disrupts distribution of apical polarity proteins and results in invasion of activated microglial cells into the subretinal space.","method":"Human fetal RPE culture, siRNA knockdown in vivo and in vitro, immunostaining of polarity and junction proteins","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — knockdown with defined polarity/tight junction phenotype plus in vivo consequence, single lab","pmids":["34262913"],"is_preprint":false},{"year":2026,"finding":"In HNSCC, CRB2 hindered the interaction between the E8A isoform of LSD1 and deubiquitinase USP7, facilitating degradation of LSD1(E8A) and increasing H4K20 dimethylation at the SLC7A11 promoter, epigenetically suppressing SLC7A11 transcription and inducing ferroptosis.","method":"Co-immunoprecipitation, ChIP, overexpression/knockdown, in vivo tumor models, ubiquitination assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods defining a mechanistic pathway, single lab, not yet replicated","pmids":["40991301"],"is_preprint":false},{"year":2022,"finding":"CRB2 loss in podocytes leads to dysfunction of slit diaphragm-related proteins (podocin, nephrin, ZO-1) by reducing phosphorylation of sphingosine 1-phosphate receptor 1 (S1PR1), while the podocytic cytoskeleton (synaptopodin) remains unaffected, revealing a distinct SD-related mechanism.","method":"CRB2 knockdown in podocytes, immunostaining, phosphorylation assays, patient kidney biopsy analysis","journal":"Pediatric nephrology","confidence":"Low","confidence_rationale":"Tier 3 — knockdown with pathway placement but limited mechanistic detail on CRB2-S1PR1 connection, single lab","pmids":["37452832"],"is_preprint":false},{"year":2018,"finding":"CRB2 overexpression in GBM cells activates NF-κB signaling by increasing phosphorylation of IKKα and reducing IκB protein levels, enhancing cell proliferation, migration, invasion and therapy resistance.","method":"Lentiviral knockdown, overexpression, Western blot for IKKα phosphorylation and IκB, bioinformatics correlation","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 — knockdown/OE with pathway placement but no direct interaction demonstrated between CRB2 and IKKα","pmids":["35219647"],"is_preprint":false}],"current_model":"CRB2 functions as a multifunctional scaffold protein with two major mechanistic contexts: (1) in fission yeast (as the 53BP1 ortholog), Crb2 is recruited to DNA double-strand breaks via its tandem Tudor domains binding H4-K20me2 and its BRCT domains binding gamma-H2A, oligomerizes through BRCT-mediated dimerization, and mediates Chk1 activation by recruiting it to DSBs in a Rad3-phosphorylation-dependent manner, with Cdc2-mediated phosphorylation of T215 governing checkpoint recovery; and (2) in mammals, CRB2 is a type I transmembrane polarity protein of the Crumbs complex that maintains apicobasal polarity and cell adhesion in retinal photoreceptors, Müller glia, and RPE cells (where it localizes to the apical membrane and tight junctions), and in podocytes where it is essential for slit diaphragm integrity, foot process morphology, and actin cytoskeleton organization—with its loss activating YAP mechanosignaling and its disease-associated variants being retained in the ER due to impaired disulfide bridge formation."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing Crb2 as a checkpoint mediator upstream of Chk1 resolved how DNA damage signals are relayed to cell cycle arrest machinery in fission yeast.","evidence":"Two-hybrid, co-IP, and genetic epistasis in S. pombe showing Crb2 interacts with Cut5 and Chk1 and acts upstream of Chk1 activation","pmids":["9407031"],"confidence":"High","gaps":["Biochemical mechanism of Crb2-Chk1 activation not yet defined","Direct binding interface not characterized"]},{"year":1999,"claim":"Identifying Cdc2-mediated T215 phosphorylation as necessary for checkpoint recovery revealed how cells re-enter the cell cycle after DNA repair, separating checkpoint activation from termination.","evidence":"In vitro kinase assay with purified Cdc2 and T215A mutant analysis in S. pombe","pmids":["10488332"],"confidence":"High","gaps":["Mechanism by which T215 phosphorylation inactivates Crb2 checkpoint function unknown","Phosphatase(s) acting on Crb2 not identified"]},{"year":2003,"claim":"Visualizing Crb2 recruitment to DSB foci in living cells and showing that Rad3/Rad1/Rad17 are needed for retention but not initial recruitment established a two-step assembly model.","evidence":"Live-cell fluorescence microscopy in S. pombe with checkpoint mutant backgrounds","pmids":["12917337"],"confidence":"High","gaps":["Initial recruitment signal not identified at this point","Stoichiometry of Crb2 at foci unknown"]},{"year":2004,"claim":"Discovery that γ-H2A and H4-K20 methylation constitute the two chromatin marks that cooperatively recruit Crb2 to DSBs, with BRCT-mediated oligomerization being the essential function of BRCT domains, defined the chromatin-reading mechanism of the checkpoint mediator.","evidence":"Genetic epistasis, fluorescence microscopy (γ-H2A mutants), Set9 methyltransferase mutants, biochemical oligomerization assays, heterologous dimerization rescue","pmids":["15550243","15226425","15229228","14739927"],"confidence":"High","gaps":["Structural basis for γ-H2A recognition by BRCT not yet resolved","Whether oligomerization enhances chromatin binding affinity unknown"]},{"year":2006,"claim":"Structural determination of the Tudor domain–H4K20me2 interface and definition of two parallel DSB recruitment pathways (histone-modification-dependent and CDK/Cut5-dependent) provided a comprehensive mechanistic model for Crb2 activation.","evidence":"X-ray crystallography and NMR of Tudor–H4K20me2 complex; mutational dissection of Tudor/BRCT/T215 pathways at HO-induced DSBs","pmids":["17190600","16778077"],"confidence":"High","gaps":["Whether both pathways operate simultaneously or sequentially at endogenous DSBs unclear","Relative contribution under physiological damage levels not quantified"]},{"year":2008,"claim":"Crystal structure of the BRCT2 domain revealed how dimerization and γ-H2A binding are structurally separable, with dimerization mutants failing checkpoint activation while phosphopeptide-binding mutants primarily affecting DNA repair.","evidence":"X-ray crystallography of BRCT2, structure-guided mutagenesis with in vivo checkpoint and repair readouts in S. pombe","pmids":["18676809","18826944"],"confidence":"High","gaps":["Full-length Crb2 structure not available","How dimerization and γ-H2A binding are coordinated in 3D not resolved"]},{"year":2010,"claim":"Identification of specific BRCT2 residues (S548, K619) that contact γ-H2A phosphate, with additive effects when combined with T215A, completed the molecular map of Crb2 chromatin engagement.","evidence":"Structure-guided mutagenesis with IRIF quantification and checkpoint assays in S. pombe","pmids":["20679485","20679488"],"confidence":"High","gaps":["Quantitative binding affinities for individual and combined interactions not measured"]},{"year":2011,"claim":"Demonstration that mouse Crb2 is essential for epiblast apicobasal polarity and gastrulation established the mammalian gene as a polarity determinant distinct from its yeast checkpoint role.","evidence":"Constitutive knockout mice with embryonic lethality and disrupted epiblast polarity at primitive streak","pmids":["22072575"],"confidence":"High","gaps":["Which Crumbs complex partners mediate epiblast polarity not defined","Whether the intracellular FERM-binding or PDZ-binding motif is required not tested"]},{"year":2012,"claim":"Defining how Rad3-phosphorylated SQ/TQ motifs in Crb2 directly recruit Chk1 via a 19-aa peptide, with artificial tethering bypassing the need for full-length Crb2, demonstrated that Crb2's essential checkpoint function is to bring Chk1 to DSBs for Rad3-mediated phosphorylation.","evidence":"In vitro binding with phosphorylated peptides, Crb2-Chk1 and Rad22-SQ/TQ fusion rescue experiments in S. pombe","pmids":["22792081"],"confidence":"High","gaps":["Whether multiple Chk1 molecules are activated per Crb2 oligomer unknown","Signal amplification mechanism not addressed"]},{"year":2012,"claim":"Conditional retinal knockout of CRB2 causing photoreceptor disorganization, adherens junction disruption, and progenitor cell overproliferation established CRB2 as essential for retinal lamination and cell fate control.","evidence":"Conditional knockout mice with OCT, ERG, histology, and confocal imaging","pmids":["23001562"],"confidence":"High","gaps":["Whether phenotype is cell-autonomous in photoreceptors vs. Müller glia not yet distinguished"]},{"year":2014,"claim":"Zebrafish and human genetic studies linking CRB2 loss-of-function to podocyte foot process defects and nephrotic syndrome, with photoreceptor-specific vs. Müller cell-specific knockout defining cell-autonomous requirements, solidified CRB2 as a disease gene for steroid-resistant nephrotic syndrome.","evidence":"Zebrafish crb2b mutants with human CRB2 variant complementation; cell-type-specific conditional KO and AAV-Cre delivery in mouse retina","pmids":["25557779","24493795"],"confidence":"High","gaps":["CRB2 intracellular signaling partners in podocytes not identified","Whether CRB2 directly binds nephrin or acts indirectly unknown"]},{"year":2021,"claim":"Podocyte-specific Crb2 knockout producing massive albuminuria and FSGS, with decreased F-actin and increased apoptosis, and parallel findings in RPE showing CRB2 is needed for tight junction maintenance and immune privilege, defined CRB2 as a master regulator of epithelial integrity across tissue types.","evidence":"Podocyte-specific conditional KO with EM; siRNA knockdown in human RPE and mouse in vivo","pmids":["34654837","34262913"],"confidence":"High","gaps":["Direct link between CRB2 loss and F-actin disorganization mechanism unresolved","Whether CRB2 signals through Hippo pathway in RPE not tested"]},{"year":2022,"claim":"Identification that disease-associated CRB2 variants are retained in the ER due to failed PDIA3-dependent disulfide bond formation explained the molecular pathogenesis of many nephrotic syndrome-causing mutations.","evidence":"Live-cell imaging of GFP-tagged CRB2 variants with PM reporter; PDIA3 knockdown phenocopying ER retention","pmids":["36549870"],"confidence":"Medium","gaps":["Which specific disulfide bonds are disrupted per variant not mapped","Whether chaperone-based therapies could rescue trafficking not tested","Independent replication needed"]},{"year":2022,"claim":"Linking CRB2 to ezrin phosphorylation and demonstrating that anti-CRB2 autoantibodies phenocopy CRB2 loss in podocytes revealed a potential autoimmune mechanism for membranous nephropathy through CRB2-cytoskeleton uncoupling.","evidence":"Immunization model and anti-CRB2 antibody treatment of podocyte cell line with phospho-ezrin and actin readouts","pmids":["35985815"],"confidence":"Medium","gaps":["Whether CRB2 directly binds ezrin or acts through an adaptor unknown","Pathogenic role of anti-CRB2 antibodies in human disease not confirmed","Single lab finding"]},{"year":2025,"claim":"CRB2 deficiency activating YAP mechanosignaling and increasing podocyte contractility in a stiffness-dependent manner connected CRB2 to the Hippo pathway and mechanotransduction, explaining how polarity loss translates to cytoskeletal and adhesion phenotypes.","evidence":"siRNA knockdown with YAP reporter assays and Elastic Resonator Interference Stress Microscopy in podocytes","pmids":["40062402"],"confidence":"Medium","gaps":["Whether CRB2 directly regulates Hippo kinase cascade or acts indirectly through junctional tension unknown","In vivo validation of YAP activation upon CRB2 loss not performed","Single lab"]},{"year":null,"claim":"The structural basis for mammalian CRB2 extracellular domain interactions (homo/heteromeric complexes with CRB1), the precise mechanism connecting CRB2 to Hippo/YAP regulation in vivo, and whether CRB2's polarity and checkpoint functions share any conserved molecular logic remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of mammalian CRB2 extracellular or transmembrane domains","No unified model connecting CRB2 to Hippo pathway across tissues","Evolutionary relationship between yeast checkpoint and mammalian polarity functions unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,1,11]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,9,14]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[18,20,21,27]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,10,12]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,3,6,7]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[24,26,27]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[24]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,1,3,7,10,14]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,5,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[19,23]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[18,20,27]}],"complexes":["Crumbs polarity complex"],"partners":["CHK1","CUT5","RAD3","CRB1","PDIA3","EZRIN"],"other_free_text":[]},"mechanistic_narrative":"CRB2 functions as a conserved scaffold protein operating in two distinct biological contexts: DNA damage checkpoint signaling in fission yeast and apicobasal polarity maintenance in mammalian epithelia. In fission yeast, Crb2 is recruited to DNA double-strand breaks through tandem Tudor domains that bind H4-K20me2 (deposited by Set9) and BRCT domains that bind γ-H2A and mediate homo-oligomerization; a parallel CDK-dependent pathway involving Cdc2 phosphorylation at T215 and Cut5 interaction provides redundant checkpoint activation, while Rad3-phosphorylated SQ/TQ motifs in Crb2 directly recruit and activate Chk1, with T215 phosphorylation governing checkpoint recovery [PMID:17190600, PMID:15550243, PMID:15226425, PMID:15229228, PMID:22792081, PMID:10488332]. In mammals, CRB2 is a transmembrane Crumbs complex component essential for photoreceptor and Müller glia adhesion, retinal lamination, RPE tight junction integrity, and podocyte slit diaphragm formation and actin cytoskeleton organization, with its loss activating YAP mechanosignaling and increasing podocyte contractility; disease-associated CRB2 variants causing steroid-resistant nephrotic syndrome are retained in the ER due to impaired PDIA3-dependent disulfide bond formation [PMID:23001562, PMID:34654837, PMID:40062402, PMID:36549870, PMID:25557779]. CRB2 forms homo- and heteromeric complexes with CRB1 through its extracellular domain in the retina [PMID:38570189]."},"prefetch_data":{"uniprot":{"accession":"Q5IJ48","full_name":"Protein crumbs homolog 2","aliases":["Crumbs-like protein 2"],"length_aa":1285,"mass_kda":134.3,"function":"Apical polarity protein that plays a central role during the epithelial-to-mesenchymal transition (EMT) at gastrulation, when newly specified mesodermal cells move inside the embryo (By similarity). Acts by promoting cell ingression, the process by which cells leave the epithelial epiblast and move inside the embryo to form a new tissue layer (By similarity). The anisotropic distribution of CRB2 and MYH10/myosin-IIB at cell edges define which cells will ingress: cells with high apical CRB2 are probably extruded from the epiblast by neighboring cells with high levels of apical MYH10/myosin-IIB (By similarity). Plays a role in the maintenance of retinal neuroepithelium organization, structural integrity, adhesion, photoreceptor polarity and retinal photoreceptor layer thickness (By similarity). May play a role in determining the length of cone photoreceptor outer segments and proliferation of late-born progenitor cells (By similarity). Also required for maintenance of the apical polarity complex during development of the cortex (By similarity). Inhibits gamma-secretase-dependent cleavage of APP and secretion of amyloid-beta peptide 40 and amyloid-beta peptide 42, and thereby inhibits gamma-secretase-dependent Notch transcription (PubMed:20299451)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q5IJ48/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CRB2","classification":"Not Classified","n_dependent_lines":20,"n_total_lines":1208,"dependency_fraction":0.016556291390728478},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CRB2","total_profiled":1310},"omim":[{"mim_id":"616220","title":"FOCAL SEGMENTAL GLOMERULOSCLEROSIS 9; FSGS9","url":"https://www.omim.org/entry/616220"},{"mim_id":"611730","title":"ERYTHROCYTE MEMBRANE PROTEIN BAND 4.1-LIKE 5; EPB41L5","url":"https://www.omim.org/entry/611730"},{"mim_id":"609720","title":"CRUMBS CELL POLARITY COMPLEX COMPONENT 2; CRB2","url":"https://www.omim.org/entry/609720"},{"mim_id":"605230","title":"TUMOR PROTEIN p53-BINDING PROTEIN 1; TP53BP1","url":"https://www.omim.org/entry/605230"},{"mim_id":"603278","title":"FOCAL SEGMENTAL GLOMERULOSCLEROSIS 1; FSGS1","url":"https://www.omim.org/entry/603278"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":6.8},{"tissue":"choroid plexus","ntpm":3.7},{"tissue":"kidney","ntpm":5.3},{"tissue":"retina","ntpm":7.5}],"url":"https://www.proteinatlas.org/search/CRB2"},"hgnc":{"alias_symbol":["FLJ38464","FLJ16786"],"prev_symbol":[]},"alphafold":{"accession":"Q5IJ48","domains":[{"cath_id":"2.10.25.10","chopping":"112-147","consensus_level":"medium","plddt":83.3528,"start":112,"end":147},{"cath_id":"2.10.25.10","chopping":"188-224","consensus_level":"medium","plddt":81.3708,"start":188,"end":224},{"cath_id":"2.60.120.200","chopping":"645-781_793-804","consensus_level":"high","plddt":85.5818,"start":645,"end":804},{"cath_id":"2.10.25.10","chopping":"808-847","consensus_level":"medium","plddt":85.0305,"start":808,"end":847},{"cath_id":"2.60.120.200","chopping":"848-1053","consensus_level":"high","plddt":84.0814,"start":848,"end":1053},{"cath_id":"2.10.25.10","chopping":"1077-1129","consensus_level":"medium","plddt":76.7819,"start":1077,"end":1129},{"cath_id":"-","chopping":"1182-1209","consensus_level":"medium","plddt":76.4514,"start":1182,"end":1209}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5IJ48","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5IJ48-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5IJ48-F1-predicted_aligned_error_v6.png","plddt_mean":76.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CRB2","jax_strain_url":"https://www.jax.org/strain/search?query=CRB2"},"sequence":{"accession":"Q5IJ48","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5IJ48.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5IJ48/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5IJ48"}},"corpus_meta":[{"pmid":"17190600","id":"PMC_17190600","title":"Structural 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replicated across 53BP1 and Crb2\",\n      \"pmids\": [\"17190600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Set9-mediated methylation of histone H4-K20 is required for Crb2 localization to sites of DNA damage; loss of Set9 or mutation of H4-K20 impairs Crb2 focus formation and compromises checkpoint-mediated cell cycle arrest in fission yeast.\",\n      \"method\": \"Genetic epistasis, chromatin immunoprecipitation, fluorescence microscopy of Crb2 foci\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with direct localization readout, replicated by multiple labs\",\n      \"pmids\": [\"15550243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Crb2 interacts with Cut5 (TopBP1 homolog) and Chk1 in fission yeast, acts upstream of Chk1 in the DNA damage checkpoint pathway, and is transiently phosphorylated upon UV damage in a manner requiring other damage-sensing checkpoint proteins but not Chk1.\",\n      \"method\": \"Two-hybrid interaction, co-immunoprecipitation, genetic epistasis, phosphorylation assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Y2H, co-IP, epistasis, phosphorylation), foundational paper\",\n      \"pmids\": [\"9407031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Gamma-H2A (phosphorylated histone H2A) controls large-scale recruitment of Crb2 to damaged DNA in fission yeast; H2A-AQE mutations defective in phosphorylation impair Crb2 and Chk1 phosphorylation and cause failure to recruit large amounts of Crb2 to DSBs.\",\n      \"method\": \"Genetic analysis, fluorescence microscopy, phosphorylation assays, epistasis with rad22Δ\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with clear mechanistic readouts, replicated\",\n      \"pmids\": [\"15226425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Cdc2 kinase phosphorylates Crb2 at threonine-215 in vitro and in vivo; this phosphorylation is required for cells to re-enter the cell cycle after DNA damage checkpoint arrest, and non-phosphorylatable T215A cells remain arrested with Chk1 kinase maintained in a phosphorylated state even after repair.\",\n      \"method\": \"In vitro kinase assay with purified Cdc2, mutational analysis (T215A), phosphorylation timing experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus in vivo mutational analysis\",\n      \"pmids\": [\"10488332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Cdc2-cyclin B kinase activity links Crb2 to topoisomerase III (Top3) function in recombinational repair during G2; low Cdc2-cyclin B activity disrupts proper regulation of Top3 via Crb2, affecting a recombination step after Rhp51 focus assembly.\",\n      \"method\": \"Genetic epistasis, fluorescence microscopy of Rhp51 foci, cyclin B mutant analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined cellular phenotype, single lab\",\n      \"pmids\": [\"12023299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Crb2 localizes to distinct nuclear foci representing sites of DNA double-strand breaks in live fission yeast cells; Crb2 colocalizes with Rad22 at persistent foci; Rad1, Rad3, and Rad17 complexes are required for retention but not initial recruitment of Crb2 to DSBs.\",\n      \"method\": \"Live-cell fluorescence microscopy, genetic analysis of checkpoint mutants\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell localization with functional consequence, multiple genetic backgrounds tested\",\n      \"pmids\": [\"12917337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Two parallel recruitment pathways bring Crb2 to DSBs in fission yeast: one requires histone H4-K20 methylation (Tudor domain) and gamma-H2A (BRCT domain) acting cooperatively; a second CDK-dependent pathway requires phosphorylation of the Cut5-binding site (T215) in Crb2 and mediates Cut5 association, providing checkpoint activation even in the absence of histone modifications.\",\n      \"method\": \"Fluorescence microscopy of HO-induced DSBs, mutational analysis of Tudor and BRCT motifs, genetic epistasis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods defining two distinct pathways\",\n      \"pmids\": [\"16778077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Crb2 oligomerizes through its tandem BRCT domains; this homo-oligomerization is the essential function of the BRCT domains for DNA damage checkpoint signaling, as heterologous dimerization motifs can substitute for BRCT domains.\",\n      \"method\": \"In vitro biochemical analysis, dominant-negative overexpression, functional complementation with heterologous dimerization motifs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical reconstitution plus genetic complementation with multiple heterologous domains\",\n      \"pmids\": [\"15229228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Crb2 dynamically interacts with Rad3, Chk1, and Cut5 in a temporal manner during the DNA damage checkpoint; active Crb2-Chk1 complex formation is regulated by Rad3 and requires Crb2 BRCT domain; Chk1 activation in vitro requires the presence of Crb2 BRCT domain; Crb2 directly interacts with Rad3 in an inhibitory manner to Rad3 activity.\",\n      \"method\": \"Co-immunoprecipitation, in vitro Chk1 activation assay, two-hybrid analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay combined with reciprocal co-IP, multiple interaction partners defined\",\n      \"pmids\": [\"14739927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of Crb2-BRCT2 domain reveals structural basis for dimerization and direct interaction with gamma-H2A.1 peptide; dimerization mutants are genotoxin sensitive and defective in checkpoint signaling and Crb2 IRIF formation, while phosphopeptide-binding mutants show DNA repair defects (slowed Rpa1 foci, reduced Rad22 foci) with only slight IR sensitivity.\",\n      \"method\": \"X-ray crystallography, in vitro mutational analysis, in vivo phenotypic analysis (checkpoint, IRIF, repair foci)\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro mutagenesis plus in vivo functional dissection\",\n      \"pmids\": [\"18676809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The tandem tudor domains of Crb2 preferentially bind di-methylated H4K20 (not tri-methylated); disruption of either the tudor-binding motif or the H4K20 methylating enzyme Set9 ablates Crb2 localization to DSBs and impairs checkpoint function and cell survival after DNA damage.\",\n      \"method\": \"In vitro binding assays with mono/di/tri-methyl H4K20 peptides, fluorescence microscopy, genetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro binding specificity assay combined with in vivo localization and function\",\n      \"pmids\": [\"18826944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crb2 BRCT2 domain directly binds phospho-H2A (gamma-H2A) through polar interactions of serine-548 and lysine-619 with the phosphate group; these interactions are critical for Crb2 IRIF formation and checkpoint function, and mutations have additive effects; combining these BRCT2 mutations with T215A completely abrogates Crb2 IRIF and function.\",\n      \"method\": \"Mutational analysis, fluorescence microscopy (IRIF), checkpoint assays, in vitro phosphopeptide binding\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structure-guided mutagenesis with in vivo functional validation, replicated by two independent papers (PMID 20679488)\",\n      \"pmids\": [\"20679485\", \"20679488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Cooperative control of Crb2 by ATM family kinases (through gamma-H2A) and Cdc2 (through T215 phosphorylation) is essential for the DNA damage checkpoint; crb2-T215A cells can initiate but not sustain checkpoint response; gamma-H2A is essential for checkpoint in crb2-T215A cells; Cdc2 inactivation in gamma-H2A-defective cells impairs Crb2-dependent Chk1 signaling.\",\n      \"method\": \"Genetic epistasis, checkpoint signaling assays, Chk1 phosphorylation analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic combinations tested with defined molecular readout (Chk1 phosphorylation)\",\n      \"pmids\": [\"16314498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crb2 recruits Chk1 to DSBs through direct physical interaction; Rad3-phosphorylated SQ/TQ motifs in Crb2 are required for Chk1 recruitment and activation; a 19-aa peptide containing these motifs is sufficient for Chk1 binding in vitro when phosphorylated; tethering a Crb2-Chk1 fusion or Rad22-SQ/TQ fusion to DSBs can bypass the need for full-length Crb2 and even the 9-1-1 complex.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, mutational analysis, artificial tethering experiments\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro binding plus multiple in vivo genetic rescue experiments with defined molecular readouts\",\n      \"pmids\": [\"22792081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human CRB2 inhibits gamma-secretase cleavage of amyloid precursor protein (APP) by binding to the presenilin complex; the transmembrane domain of CRB2 is indispensable for this inhibition and mediates binding to the gamma-secretase complex; CRB2 is not a competitive substrate but functions as an inhibitory binding protein.\",\n      \"method\": \"Transfection/knockdown in HEK293 and SH-SY5Y cells, cell-free gamma-secretase assay, co-immunoprecipitation, domain deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-free assay plus co-IP, single lab; contradicted by another paper (PMID 17988153)\",\n      \"pmids\": [\"20299451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Loss of Crb2 in zebrafish podocytes impairs podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking; complementation experiments with human CRB2 mutations confirm loss-of-function causing nephrotic syndrome.\",\n      \"method\": \"Zebrafish crb2b loss-of-function analysis, nephrin trafficking assay, complementation with human CRB2 mutants\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — defined cellular phenotype with complementation validation in vivo\",\n      \"pmids\": [\"25557779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mouse Crb2 is required for epiblast cell polarity at the primitive streak; loss of Crb2 disturbs apical polarity of epiblast cells, affecting epithelial-to-mesenchymal transition during gastrulation and impairing mesoderm/endoderm formation, causing embryonic lethality by E12.5.\",\n      \"method\": \"Gene-targeted knockout mice, histological and immunofluorescence analysis\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular polarity phenotype and embryonic lethal outcome\",\n      \"pmids\": [\"22072575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Conditional deletion of mouse CRB2 in the retina causes progressive disorganization of photoreceptor layer, disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells, and increased proliferation of late-born progenitor cells; CRB2 is essential for proper lamination and suppression of late-born retinal progenitor cell proliferation.\",\n      \"method\": \"Conditional knockout mice, OCT imaging, ERG, histological and confocal analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean conditional KO with multiple orthogonal readouts (imaging, electrophysiology, histology)\",\n      \"pmids\": [\"23001562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Loss of both CRB1 and CRB2 in retinal progenitor cells dysregulates Notch1 and YAP/Hippo signaling pathways, increases P120-catenin levels, and alters progenitor cell cycle distribution (decrease in late G1, increase in S and G2/M phase), leading to expanded late-born cell types.\",\n      \"method\": \"Conditional double-knockout mice, transcriptome analysis, cell cycle analysis, immunostaining\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with pathway analysis, but signaling pathway assignment based on gene expression changes rather than direct epistasis\",\n      \"pmids\": [\"24339791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CRB2 has essential functions specifically in photoreceptors (not redundant in Müller cells) in developing mouse retina; photoreceptor-specific loss causes early-onset abnormal lamination, while Müller cell-specific loss causes late-onset disorganization; short-term adult loss of CRB2 in photoreceptors causes sporadic loss of adhesion between photoreceptors and Müller cells.\",\n      \"method\": \"Cell-type-specific conditional knockout, AAV-Cre delivery, OCT imaging, ERG\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with multiple functional readouts defining distinct roles\",\n      \"pmids\": [\"24493795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Podocyte-specific Crb2 knockout mice develop massive albuminuria and FSGS; CRB2-deficient human podocytes show decreased F-actin positive area and increased susceptibility to apoptosis, demonstrating that CRB2 is required for actin cytoskeleton organization and podocyte survival.\",\n      \"method\": \"Podocyte-specific conditional knockout, electron microscopy, siRNA knockdown of human podocytes, F-actin staining, apoptosis assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with electron microscopy plus in vitro mechanistic follow-up\",\n      \"pmids\": [\"34654837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CRB2 depletion or anti-CRB2 autoantibody binding in podocytes increases phosphorylation of ezrin (which connects CRB2 to the cytoskeleton) and alters CRB2 localization and actin distribution, linking CRB2 to cytoskeletal organization at podocyte foot processes.\",\n      \"method\": \"Immunization model, anti-CRB2 antibody treatment of podocyte cell line, immunofluorescence, phosphorylation assay\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct mechanistic link between antibody binding and ezrin phosphorylation/actin redistribution, single lab\",\n      \"pmids\": [\"35985815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRB2 knockdown in podocytes induces YAP transcriptional activity and upregulates YAP-mediated mechanosignaling; CRB2 deficiency increases focal adhesion density, F-actin, and podocyte contractility in a substrate stiffness-dependent manner as measured by ERISM, demonstrating impaired mechanosensing.\",\n      \"method\": \"siRNA knockdown, YAP reporter assays, F-actin staining, Elastic Resonator Interference Stress Microscopy (ERISM), pharmacological YAP inhibition\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel biophysical assay (ERISM) combined with molecular readouts, single lab\",\n      \"pmids\": [\"40062402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Disease-associated CRB2 variants causing nephrotic syndrome remain predominantly at the ER rather than reaching the plasma membrane; WT CRB2 is retained in the ER in cells lacking protein disulfide isomerase A3 (PDIA3), demonstrating that disulfide bridge formation is a crucial posttranslational modification required for CRB2 plasma membrane transport.\",\n      \"method\": \"Live-cell imaging with GFP-tagged CRB2 variants, BFP-labeled plasma membrane reporter, PDIA3 knockdown\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative live-cell assay with defined molecular mechanism (disulfide bridge formation via PDIA3), single lab\",\n      \"pmids\": [\"36549870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human CRB1 and CRB2 form homo- and heteromeric complexes; CRB1 was enriched in CRB2 pull-downs from retina; co-immunoprecipitation shows canonical CRB1 interacts with CRB1 and CRB2 but not CRB3 (which lacks an extracellular domain), indicating interaction is mediated through the extracellular domain.\",\n      \"method\": \"Retina-specific pull-down, co-immunoprecipitation, co-localization in human retina and iPSC-derived organoids\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — reciprocal pull-down from native tissue plus domain-based mechanistic inference, single lab\",\n      \"pmids\": [\"38570189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CRB2 localizes to the apicolateral membrane of mouse RPE cells at tight junctions, completing a fully expressed Crumbs complex in the retinal pigment epithelium.\",\n      \"method\": \"Immunofluorescence with specific antibody, confocal microscopy, subcellular fractionation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — direct localization experiment with specific antibody, no strong functional consequence established in same study\",\n      \"pmids\": [\"26404741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRB2 plays a key role in tight junction maintenance and cell cycle arrest in human RPE cells during polarization; in vivo CRB2 knockdown in mouse RPE disrupts distribution of apical polarity proteins and results in invasion of activated microglial cells into the subretinal space.\",\n      \"method\": \"Human fetal RPE culture, siRNA knockdown in vivo and in vitro, immunostaining of polarity and junction proteins\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockdown with defined polarity/tight junction phenotype plus in vivo consequence, single lab\",\n      \"pmids\": [\"34262913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In HNSCC, CRB2 hindered the interaction between the E8A isoform of LSD1 and deubiquitinase USP7, facilitating degradation of LSD1(E8A) and increasing H4K20 dimethylation at the SLC7A11 promoter, epigenetically suppressing SLC7A11 transcription and inducing ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, overexpression/knockdown, in vivo tumor models, ubiquitination assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods defining a mechanistic pathway, single lab, not yet replicated\",\n      \"pmids\": [\"40991301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CRB2 loss in podocytes leads to dysfunction of slit diaphragm-related proteins (podocin, nephrin, ZO-1) by reducing phosphorylation of sphingosine 1-phosphate receptor 1 (S1PR1), while the podocytic cytoskeleton (synaptopodin) remains unaffected, revealing a distinct SD-related mechanism.\",\n      \"method\": \"CRB2 knockdown in podocytes, immunostaining, phosphorylation assays, patient kidney biopsy analysis\",\n      \"journal\": \"Pediatric nephrology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — knockdown with pathway placement but limited mechanistic detail on CRB2-S1PR1 connection, single lab\",\n      \"pmids\": [\"37452832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRB2 overexpression in GBM cells activates NF-κB signaling by increasing phosphorylation of IKKα and reducing IκB protein levels, enhancing cell proliferation, migration, invasion and therapy resistance.\",\n      \"method\": \"Lentiviral knockdown, overexpression, Western blot for IKKα phosphorylation and IκB, bioinformatics correlation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — knockdown/OE with pathway placement but no direct interaction demonstrated between CRB2 and IKKα\",\n      \"pmids\": [\"35219647\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CRB2 functions as a multifunctional scaffold protein with two major mechanistic contexts: (1) in fission yeast (as the 53BP1 ortholog), Crb2 is recruited to DNA double-strand breaks via its tandem Tudor domains binding H4-K20me2 and its BRCT domains binding gamma-H2A, oligomerizes through BRCT-mediated dimerization, and mediates Chk1 activation by recruiting it to DSBs in a Rad3-phosphorylation-dependent manner, with Cdc2-mediated phosphorylation of T215 governing checkpoint recovery; and (2) in mammals, CRB2 is a type I transmembrane polarity protein of the Crumbs complex that maintains apicobasal polarity and cell adhesion in retinal photoreceptors, Müller glia, and RPE cells (where it localizes to the apical membrane and tight junctions), and in podocytes where it is essential for slit diaphragm integrity, foot process morphology, and actin cytoskeleton organization—with its loss activating YAP mechanosignaling and its disease-associated variants being retained in the ER due to impaired disulfide bridge formation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CRB2 functions as a conserved scaffold protein operating in two distinct biological contexts: DNA damage checkpoint signaling in fission yeast and apicobasal polarity maintenance in mammalian epithelia. In fission yeast, Crb2 is recruited to DNA double-strand breaks through tandem Tudor domains that bind H4-K20me2 (deposited by Set9) and BRCT domains that bind γ-H2A and mediate homo-oligomerization; a parallel CDK-dependent pathway involving Cdc2 phosphorylation at T215 and Cut5 interaction provides redundant checkpoint activation, while Rad3-phosphorylated SQ/TQ motifs in Crb2 directly recruit and activate Chk1, with T215 phosphorylation governing checkpoint recovery [PMID:17190600, PMID:15550243, PMID:15226425, PMID:15229228, PMID:22792081, PMID:10488332]. In mammals, CRB2 is a transmembrane Crumbs complex component essential for photoreceptor and Müller glia adhesion, retinal lamination, RPE tight junction integrity, and podocyte slit diaphragm formation and actin cytoskeleton organization, with its loss activating YAP mechanosignaling and increasing podocyte contractility; disease-associated CRB2 variants causing steroid-resistant nephrotic syndrome are retained in the ER due to impaired PDIA3-dependent disulfide bond formation [PMID:23001562, PMID:34654837, PMID:40062402, PMID:36549870, PMID:25557779]. CRB2 forms homo- and heteromeric complexes with CRB1 through its extracellular domain in the retina [PMID:38570189].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing Crb2 as a checkpoint mediator upstream of Chk1 resolved how DNA damage signals are relayed to cell cycle arrest machinery in fission yeast.\",\n      \"evidence\": \"Two-hybrid, co-IP, and genetic epistasis in S. pombe showing Crb2 interacts with Cut5 and Chk1 and acts upstream of Chk1 activation\",\n      \"pmids\": [\"9407031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of Crb2-Chk1 activation not yet defined\", \"Direct binding interface not characterized\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identifying Cdc2-mediated T215 phosphorylation as necessary for checkpoint recovery revealed how cells re-enter the cell cycle after DNA repair, separating checkpoint activation from termination.\",\n      \"evidence\": \"In vitro kinase assay with purified Cdc2 and T215A mutant analysis in S. pombe\",\n      \"pmids\": [\"10488332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which T215 phosphorylation inactivates Crb2 checkpoint function unknown\", \"Phosphatase(s) acting on Crb2 not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Visualizing Crb2 recruitment to DSB foci in living cells and showing that Rad3/Rad1/Rad17 are needed for retention but not initial recruitment established a two-step assembly model.\",\n      \"evidence\": \"Live-cell fluorescence microscopy in S. pombe with checkpoint mutant backgrounds\",\n      \"pmids\": [\"12917337\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Initial recruitment signal not identified at this point\", \"Stoichiometry of Crb2 at foci unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that γ-H2A and H4-K20 methylation constitute the two chromatin marks that cooperatively recruit Crb2 to DSBs, with BRCT-mediated oligomerization being the essential function of BRCT domains, defined the chromatin-reading mechanism of the checkpoint mediator.\",\n      \"evidence\": \"Genetic epistasis, fluorescence microscopy (γ-H2A mutants), Set9 methyltransferase mutants, biochemical oligomerization assays, heterologous dimerization rescue\",\n      \"pmids\": [\"15550243\", \"15226425\", \"15229228\", \"14739927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for γ-H2A recognition by BRCT not yet resolved\", \"Whether oligomerization enhances chromatin binding affinity unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Structural determination of the Tudor domain–H4K20me2 interface and definition of two parallel DSB recruitment pathways (histone-modification-dependent and CDK/Cut5-dependent) provided a comprehensive mechanistic model for Crb2 activation.\",\n      \"evidence\": \"X-ray crystallography and NMR of Tudor–H4K20me2 complex; mutational dissection of Tudor/BRCT/T215 pathways at HO-induced DSBs\",\n      \"pmids\": [\"17190600\", \"16778077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether both pathways operate simultaneously or sequentially at endogenous DSBs unclear\", \"Relative contribution under physiological damage levels not quantified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Crystal structure of the BRCT2 domain revealed how dimerization and γ-H2A binding are structurally separable, with dimerization mutants failing checkpoint activation while phosphopeptide-binding mutants primarily affecting DNA repair.\",\n      \"evidence\": \"X-ray crystallography of BRCT2, structure-guided mutagenesis with in vivo checkpoint and repair readouts in S. pombe\",\n      \"pmids\": [\"18676809\", \"18826944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length Crb2 structure not available\", \"How dimerization and γ-H2A binding are coordinated in 3D not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of specific BRCT2 residues (S548, K619) that contact γ-H2A phosphate, with additive effects when combined with T215A, completed the molecular map of Crb2 chromatin engagement.\",\n      \"evidence\": \"Structure-guided mutagenesis with IRIF quantification and checkpoint assays in S. pombe\",\n      \"pmids\": [\"20679485\", \"20679488\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative binding affinities for individual and combined interactions not measured\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that mouse Crb2 is essential for epiblast apicobasal polarity and gastrulation established the mammalian gene as a polarity determinant distinct from its yeast checkpoint role.\",\n      \"evidence\": \"Constitutive knockout mice with embryonic lethality and disrupted epiblast polarity at primitive streak\",\n      \"pmids\": [\"22072575\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which Crumbs complex partners mediate epiblast polarity not defined\", \"Whether the intracellular FERM-binding or PDZ-binding motif is required not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defining how Rad3-phosphorylated SQ/TQ motifs in Crb2 directly recruit Chk1 via a 19-aa peptide, with artificial tethering bypassing the need for full-length Crb2, demonstrated that Crb2's essential checkpoint function is to bring Chk1 to DSBs for Rad3-mediated phosphorylation.\",\n      \"evidence\": \"In vitro binding with phosphorylated peptides, Crb2-Chk1 and Rad22-SQ/TQ fusion rescue experiments in S. pombe\",\n      \"pmids\": [\"22792081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether multiple Chk1 molecules are activated per Crb2 oligomer unknown\", \"Signal amplification mechanism not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Conditional retinal knockout of CRB2 causing photoreceptor disorganization, adherens junction disruption, and progenitor cell overproliferation established CRB2 as essential for retinal lamination and cell fate control.\",\n      \"evidence\": \"Conditional knockout mice with OCT, ERG, histology, and confocal imaging\",\n      \"pmids\": [\"23001562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phenotype is cell-autonomous in photoreceptors vs. Müller glia not yet distinguished\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Zebrafish and human genetic studies linking CRB2 loss-of-function to podocyte foot process defects and nephrotic syndrome, with photoreceptor-specific vs. Müller cell-specific knockout defining cell-autonomous requirements, solidified CRB2 as a disease gene for steroid-resistant nephrotic syndrome.\",\n      \"evidence\": \"Zebrafish crb2b mutants with human CRB2 variant complementation; cell-type-specific conditional KO and AAV-Cre delivery in mouse retina\",\n      \"pmids\": [\"25557779\", \"24493795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CRB2 intracellular signaling partners in podocytes not identified\", \"Whether CRB2 directly binds nephrin or acts indirectly unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Podocyte-specific Crb2 knockout producing massive albuminuria and FSGS, with decreased F-actin and increased apoptosis, and parallel findings in RPE showing CRB2 is needed for tight junction maintenance and immune privilege, defined CRB2 as a master regulator of epithelial integrity across tissue types.\",\n      \"evidence\": \"Podocyte-specific conditional KO with EM; siRNA knockdown in human RPE and mouse in vivo\",\n      \"pmids\": [\"34654837\", \"34262913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct link between CRB2 loss and F-actin disorganization mechanism unresolved\", \"Whether CRB2 signals through Hippo pathway in RPE not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification that disease-associated CRB2 variants are retained in the ER due to failed PDIA3-dependent disulfide bond formation explained the molecular pathogenesis of many nephrotic syndrome-causing mutations.\",\n      \"evidence\": \"Live-cell imaging of GFP-tagged CRB2 variants with PM reporter; PDIA3 knockdown phenocopying ER retention\",\n      \"pmids\": [\"36549870\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which specific disulfide bonds are disrupted per variant not mapped\", \"Whether chaperone-based therapies could rescue trafficking not tested\", \"Independent replication needed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linking CRB2 to ezrin phosphorylation and demonstrating that anti-CRB2 autoantibodies phenocopy CRB2 loss in podocytes revealed a potential autoimmune mechanism for membranous nephropathy through CRB2-cytoskeleton uncoupling.\",\n      \"evidence\": \"Immunization model and anti-CRB2 antibody treatment of podocyte cell line with phospho-ezrin and actin readouts\",\n      \"pmids\": [\"35985815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CRB2 directly binds ezrin or acts through an adaptor unknown\", \"Pathogenic role of anti-CRB2 antibodies in human disease not confirmed\", \"Single lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"CRB2 deficiency activating YAP mechanosignaling and increasing podocyte contractility in a stiffness-dependent manner connected CRB2 to the Hippo pathway and mechanotransduction, explaining how polarity loss translates to cytoskeletal and adhesion phenotypes.\",\n      \"evidence\": \"siRNA knockdown with YAP reporter assays and Elastic Resonator Interference Stress Microscopy in podocytes\",\n      \"pmids\": [\"40062402\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CRB2 directly regulates Hippo kinase cascade or acts indirectly through junctional tension unknown\", \"In vivo validation of YAP activation upon CRB2 loss not performed\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis for mammalian CRB2 extracellular domain interactions (homo/heteromeric complexes with CRB1), the precise mechanism connecting CRB2 to Hippo/YAP regulation in vivo, and whether CRB2's polarity and checkpoint functions share any conserved molecular logic remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of mammalian CRB2 extracellular or transmembrane domains\", \"No unified model connecting CRB2 to Hippo pathway across tissues\", \"Evolutionary relationship between yeast checkpoint and mammalian polarity functions unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 1, 11]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 9, 14]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [18, 20, 21, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 10, 12]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 3, 6, 7]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [24, 26, 27]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 1, 3, 7, 10, 14]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 5, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [19, 23]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [18, 20, 27]}\n    ],\n    \"complexes\": [\n      \"Crumbs polarity complex\"\n    ],\n    \"partners\": [\n      \"Chk1\",\n      \"Cut5\",\n      \"Rad3\",\n      \"CRB1\",\n      \"PDIA3\",\n      \"ezrin\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}