{"gene":"MAPRE3","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2000,"finding":"EB3 is a novel member of the EB1 family that associates with the cytoplasmic microtubule network and binds directly to APCL (APC2), a CNS-specific homologue of APC, as demonstrated by yeast two-hybrid screening and confocal microscopy showing co-localization in perinucleus and microtubule network.","method":"Yeast two-hybrid, confocal microscopy, sequence analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid and co-localization, single lab, two methods but no in vitro reconstitution","pmids":["10644998"],"is_preprint":false},{"year":2003,"finding":"EB3-GFP tracks exclusively to the growing plus-ends of microtubules in live neurons, marking microtubule growth events; microtubules grow slower in neurons than in glia/COS-1 cells, and ~65% of EB3-GFP movements in proximal dendrites are directed distally vs. ~35% toward the cell body, reflecting the mixed microtubule polarity of dendrites.","method":"Live-cell fluorescence microscopy with EB3-GFP in cultured neurons","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct live-cell imaging with quantitative analysis, replicated across multiple cell types and compartments, foundational study","pmids":["12684451"],"is_preprint":false},{"year":2005,"finding":"EB1 and EB3 directly bind CLIP-170 and CLIP-115 through their C-terminal tyrosine residues, and control CLIP dissociation kinetics from microtubule plus-ends; RNAi depletion of EB1 and EB3 accelerates CLIP dissociation from tips, which is rescued by EB1 but not EB2 expression.","method":"RNA interference, direct binding assay, live-cell imaging of CLIP dynamics","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays plus RNAi rescue experiments, multiple orthogonal methods in single study","pmids":["16148041"],"is_preprint":false},{"year":2007,"finding":"EB3 is specifically upregulated upon myogenic differentiation; knockdown of EB3 (but not EB1) prevents myoblast elongation and fusion, impairs microtubule capture at the cell cortex, and disrupts microtubule dynamics; two specific amino acids in the calponin-like domain of EB3 are required for myoblast fusion.","method":"RNAi knockdown, live-cell imaging, EB1/EB3 chimera expression, C2C12 myoblast differentiation assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined phenotype, domain-mapping with chimeras, multiple orthogonal approaches in single study","pmids":["17658256"],"is_preprint":false},{"year":2007,"finding":"EB3 interacts with the p53-target DDA3 protein; the interaction requires intact microtubules, maps to aa 118-329 of DDA3 and both N- and C-termini of EB3; DDA3 and EB3 cooperate for microtubule binding in vitro; ectopic expression of DDA3 and EB3 enhances beta-catenin-dependent transactivation and cyclin D1 production.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, in vitro microtubule-binding assay, immunofluorescence, reporter assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in single lab, direct binding confirmed in vitro","pmids":["17310996"],"is_preprint":false},{"year":2008,"finding":"EB3 directly binds drebrin (an F-actin-associated protein); in growth cones this interaction occurs specifically when drebrin is on F-actin in the proximal region of filopodia and EB3 is at microtubule tips invading filopodia; disruption of this interaction impairs growth cone formation and neurite extension.","method":"Direct binding assay (in vitro), co-localization, dominant-negative disruption, neurite outgrowth assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding established in vitro, functional loss-of-interaction phenotype, multiple methods","pmids":["18806788"],"is_preprint":false},{"year":2009,"finding":"EB3 stability is regulated during mitosis by Aurora-A and Aurora-B kinases that phosphorylate EB3 at Ser-176, disrupting the EB3-SIAH-1 complex; SIAH-1 ubiquitin ligase mediates EB3 polyubiquitination and proteasomal degradation during G1; phosphorylation-induced stabilization of EB3 during mitosis facilitates cell cycle progression at prometaphase.","method":"In vitro kinase assay, co-immunoprecipitation, RNAi knockdown, proteasome inhibition, cell cycle analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase phosphorylation, co-IP of complex disruption, RNAi knockdown, multiple methods in single study","pmids":["19696028"],"is_preprint":false},{"year":2010,"finding":"In vitro reconstitution demonstrated that EB3 restores robust microtubule growth in the presence of MCAK (which alone blocks assembly) and targets MCAK to growing microtubule ends by increasing its association rate; the EB3-dependent targeting requires direct EB3-MCAK interaction and enhances MCAK's capacity to induce catastrophes without affecting growth/shortening velocities.","method":"In vitro reconstitution of microtubule dynamics, TIRF microscopy, quantitative analysis of dynamics","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified components, quantitative dynamics measurements, multiple conditions tested","pmids":["20850319"],"is_preprint":false},{"year":2011,"finding":"EB3 (and EB1) directly interact with the AIS scaffold protein ankyrin G (ankG); EB3 is concentrated and stabilized at the axon initial segment (AIS) in mature neurons; knockdown of ankG leads to cell-wide upregulation of EB3 comets; EB3/EB1 participate in AIS maintenance.","method":"Direct interaction assay, live-cell imaging, immunofluorescence, ankG knockdown, fractionation/localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct interaction identified, subcellular localization with functional consequence via knockdown, multiple orthogonal methods","pmids":["21551097"],"is_preprint":false},{"year":2011,"finding":"EB3 (and EB1) are required for assembly of primary cilia; cells lacking EB1 or EB3 have defective MT minus-end anchoring at centrosome/basal body and short cilia stumps; GST pull-down and mass spectrometry showed EB1/EB3 interact with proteins implicated in MT minus-end anchoring and vesicular trafficking to cilia base; EB3 localizes to the tip of motile cilia and affects centriole-associated rootlet filament formation.","method":"Protein depletion (siRNA), dominant-negative expression, electron microscopy, GST pull-down, mass spectrometry, immunoprecipitation, live imaging","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined structural phenotype, protein interactions confirmed by multiple methods, direct localization","pmids":["21768326"],"is_preprint":false},{"year":2011,"finding":"The SH3 domain of PSD-95 interacts with a proline-rich region within EB3; overexpression of PSD-95 decreases the lifetime of EB3 comets in dendrites, leading to less organized microtubules at dendritic branch points and decreased dendritic branching.","method":"Co-immunoprecipitation, live-cell imaging (EB3 comet analysis), overexpression in neurons","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus live imaging of comet dynamics, single lab, two orthogonal methods","pmids":["21248129"],"is_preprint":false},{"year":2012,"finding":"VE-cadherin outside-in signaling activates Src and PLCγ2, causing Ca2+ release from ER stores, activating calcineurin (CaN); CaN downregulation leads to phosphorylation of EB3 at Ser-162, destabilizing the EB3 dimer, suppressing microtubule growth, and enabling adherens junction assembly; phospho-defective S162A EB3 mutant induces MT growth in confluent monolayers and disassembles AJs.","method":"Phospho-specific mutagenesis, calcium imaging, pharmacological inhibition, co-immunoprecipitation, endothelial permeability assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — site-specific phospho-mutagenesis with defined functional consequence, pharmacological pathway dissection, multiple orthogonal methods in single study","pmids":["23159740"],"is_preprint":false},{"year":2012,"finding":"Drebrin E and EB3 form a complex with myosin IIB and βII-spectrin at the apical domain of columnar epithelial cells; depletion of drebrin E disrupts apical accumulation of EB3 and impairs cell elongation; EB3 depletion produces a similar elongation defect; the complex connects F-actin and microtubule networks apically during epithelial morphogenesis.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence, morphometric analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP of complex, loss-of-function phenotype, single lab, multiple components tested","pmids":["22275434"],"is_preprint":false},{"year":2013,"finding":"Aurora B phosphorylates EB3 at Ser-176 at the midbody to control cortical microtubule growth; EB3 stabilizes focal adhesions and coordinates daughter cell spreading during mitotic exit, promotes midbody microtubule stability, and is required for efficient cytokinesis; EB1 and EB3 play temporally distinct roles in cell division, with EB1 involved in spindle orientation before anaphase.","method":"Phospho-specific mutagenesis, live-cell imaging, RNAi knockdown, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — site-specific phospho-mutagenesis with functional readouts, live-cell imaging, loss-of-function, multiple orthogonal methods","pmids":["23712260"],"is_preprint":false},{"year":2014,"finding":"BPAG1a/b C-terminal isoform-specific tails bind both EB1 and EB3 and are sufficient to bundle microtubules; knockdown of BPAG1a/b in C2.7 myoblasts impairs directness of cell migration and disrupts Golgi structure.","method":"GST pull-down, co-immunoprecipitation, microtubule bundling assay, siRNA knockdown, migration assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct binding demonstrated, loss-of-function phenotype, single lab","pmids":["25244344"],"is_preprint":false},{"year":2015,"finding":"EB3 binds to IP3 receptors (IP3R3) through an S/TxIP EB-binding motif; in endothelial cells, EB3 depletion or mutation of the TxIP motif of IP3R3 prevents α-thrombin-induced IP3R3 clustering, Ca2+ increase, myosin light chain phosphorylation, and vascular permeability increase; selective EB3 gene deletion in mouse endothelial cells abrogates α-thrombin-induced endothelial permeability.","method":"Co-immunoprecipitation, mutagenesis of binding motif, siRNA knockdown, conditional knockout mouse, Ca2+ imaging, permeability assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding via motif mutagenesis, in vitro and in vivo loss-of-function, multiple orthogonal methods, in vivo validation","pmids":["26119739"],"is_preprint":false},{"year":2017,"finding":"EB3 (and EB1) form a complex with myomegalin that acts as a membrane-microtubule tether at Golgi membranes; CRISPR/Cas9 knockout of EB2/EB3 and C-terminal half of EB1 reduces CAMSAP2-decorated microtubule minus end lengths, detaches microtubules from Golgi membranes, compacts the Golgi complex, and disrupts cell migration, polarity, and focal adhesion distribution.","method":"CRISPR/Cas9 knockout, co-immunoprecipitation, electron microscopy, live-cell imaging, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR knockout with defined structural phenotypes, interaction validated by co-IP, multiple orthogonal methods, rigorous controls","pmids":["28814570"],"is_preprint":false},{"year":2017,"finding":"STIM2 forms a Ca2+-dependent complex with EB3 via a Ser-x-Ile-Pro (SxIP) amino acid motif; disruption of STIM2-EB3 interaction results in loss of mushroom dendritic spines; EB3 overexpression rescues mushroom spine loss in a PS1-M146V knock-in Alzheimer's disease model, while STIM2 overexpression fails to rescue spines after EB3 knockdown.","method":"Co-immunoprecipitation, mutagenesis of SxIP motif, siRNA knockdown, overexpression, spine morphometry in hippocampal neurons","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP with motif identification, epistasis via rescue experiments, single lab","pmids":["29247211"],"is_preprint":false},{"year":2018,"finding":"IP3K-A (ITPKA) binds to EB3, and this interaction is regulated by PKA-dependent phosphorylation of IP3K-A at Ser119; the IP3K-A/EB3 complex dissociates and reassociates rapidly during chemically-induced LTP conditions.","method":"Co-immunoprecipitation, phospho-specific mutagenesis, chemical LTP induction","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP with phospho-mutant, single lab, single method per finding","pmids":["30466786"],"is_preprint":false},{"year":2019,"finding":"EB3 comet intensity profiles in mouse fibroblasts frequently exhibit secondary EB3-binding peaks (EB3-islands) on 56% of growing microtubule comets, which are stationary and originate from EB3 comets moving with growing MT tips; modeling suggests additional protein factors beyond GTP hydrolysis contribute to EB3 residence time on MTs.","method":"Live-cell fluorescence microscopy (EB3-RFP), quantitative image analysis, computational modeling","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct live-cell imaging with quantitative analysis and modeling, single lab","pmids":["31141458"],"is_preprint":false},{"year":2021,"finding":"Loss of EB3 (but not EB1) inhibits neuritogenesis in embryonic cortical neurons; EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia, enabling continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin; pharmacological blockade of microtubule dynamics is associated with selective loss of EB3 (not EB1) from plus-ends.","method":"Gene editing (CRISPR), pharmacological inhibition, overexpression, live-cell imaging, morphometric analysis","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function by gene editing plus live imaging of plus-end dynamics, multiple orthogonal approaches, EB1 vs EB3 specificity established","pmids":["34478582"],"is_preprint":false},{"year":2023,"finding":"Fidgetin (microtubule-severing enzyme) trims tyrosinated (labile) microtubules by interacting with EB3; fidgetin depletion increases EB3 at neurite ends, elongates the labile microtubule domain, and increases axon length and branching; EB3 knockdown does not change fidgetin expression but fidgetin deletion markedly increases EB3 levels.","method":"RNA interference, overexpression, rat SCI model, immunofluorescence, EB3 localization analysis","journal":"Neural regeneration research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function for both proteins with epistatic analysis, in vivo model, single lab","pmids":["37449637"],"is_preprint":false},{"year":2023,"finding":"A 14-amino acid peptide (CIPRI) targeting the EB3-IP3R3 interface disrupts EB3-IP3R3 interaction in vitro and in mouse lungs; disruption mitigates Ca2+ release from ER stores, prevents VE-cadherin junction disassembly, and reduces inflammation-induced lung injury and microvascular leakage in mice.","method":"Peptide competition assay, co-immunoprecipitation, Ca2+ imaging, in vivo endotoxemia/sepsis mouse model, vascular permeability assay","journal":"American journal of respiratory cell and molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct interface disruption with peptide, both in vitro and in vivo validation, multiple functional readouts","pmids":["37290041"],"is_preprint":false},{"year":2024,"finding":"EZH2-mediated H3K27 trimethylation epigenetically represses MAPRE3 (EB3) expression at its promoter in ovarian cancer; EZH2 knockdown decreases H3K27me3 enrichment at the MAPRE3 promoter and increases EB3 expression; MAPRE3 overexpression induces cell cycle arrest and apoptosis in ovarian cancer cells.","method":"ChIP assay (H3K27me3 at MAPRE3 promoter), EZH2 knockdown, overexpression, xenograft tumor model","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP showing epigenetic repression with functional consequences, single lab, in vitro and in vivo validation","pmids":["38199479"],"is_preprint":false},{"year":2024,"finding":"EB3 undergoes liquid-liquid phase separation (LLPS) with significantly higher propensity than EB1 despite 67% sequence identity; this difference involves contributions from multiple protein regions with histidine residues in the N-terminal domain playing a key role; EB3 condensates have lower dynamics (less fluid) than EB1 and higher capacity to recruit tubulin and nucleate polymerization; EB3/CLIP-170 droplets show higher tubulin polymerization capacity than EB1/CLIP-170.","method":"In vitro LLPS assay, fluorescence recovery after photobleaching (FRAP), mutagenesis, in vitro tubulin polymerization assay, cell-based condensate imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of LLPS, mutagenesis, functional tubulin polymerization assay, multiple orthogonal methods in single study","pmids":["41135671"],"is_preprint":false},{"year":2024,"finding":"In vitro reconstitution showed that EB3-dependent comet size at microtubule ends is independent of GTP-cap size; a phenomenological model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate fully describes stalled and freely growing microtubule lifetime distributions; modulation of microtubule growth velocity in cells does not consistently alter EB3 comet length.","method":"In vitro reconstitution with microfabricated barriers, GFP-EB3 live imaging, mathematical modeling","journal":"Biophysical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with modeling, single lab, novel mechanistic insight about cap independence","pmids":["39604262"],"is_preprint":false}],"current_model":"MAPRE3/EB3 is a microtubule plus-end tracking protein (+TIP) that autonomously recognizes the GTP/GDP-Pi cap at growing microtubule ends, undergoes liquid-liquid phase separation to nucleate tubulin polymerization, recruits and regulates the dissociation kinetics of CLIP-170/CLIP-115, directly binds drebrin on F-actin to coordinate microtubule-actin coupling in growth cones and dendritic spines, interacts with ankyrin G at the axon initial segment, binds IP3R3 via an SxIP motif to amplify Ca2+ signaling in endothelial cells, is stabilized during mitosis by Aurora A/B phosphorylation at Ser-176 (which disrupts SIAH-1-mediated ubiquitination and proteasomal degradation), and is epigenetically repressed by EZH2-mediated H3K27 trimethylation; collectively these interactions place EB3 as a context-specific scaffold that links microtubule dynamics to adhesion, signaling, neuronal polarity, and cellular morphogenesis."},"narrative":{"mechanistic_narrative":"MAPRE3/EB3 is a microtubule plus-end tracking protein (+TIP) that autonomously marks growing microtubule ends and acts as a context-specific scaffold linking microtubule dynamics to actin, adhesion, calcium signaling, and cellular morphogenesis [PMID:12684451, PMID:16148041]. It directly binds CLIP-170/CLIP-115 through their C-terminal tyrosine residues and controls their dissociation kinetics from microtubule tips [PMID:16148041], targets the catastrophe factor MCAK to growing ends [PMID:20850319], and undergoes liquid-liquid phase separation that recruits tubulin and nucleates polymerization more efficiently than EB1 [PMID:41135671]. EB3 bridges the microtubule and F-actin networks by binding drebrin on F-actin, a coupling required for growth cone formation, neurite extension, and apical epithelial elongation [PMID:18806788, PMID:22275434, PMID:34478582], and it associates with structural scaffolds including ankyrin G at the axon initial segment, myomegalin/CAMSAP2 tethers at Golgi membranes, and BPAG1 [PMID:21551097, PMID:28814570, PMID:25244344]. Through an SxIP/TxIP motif EB3 binds IP3R3 and STIM2 to amplify ER calcium release: in endothelial cells the EB3-IP3R3 interaction drives thrombin- and inflammation-induced Ca2+ flux, junction disassembly, and vascular permeability, which a peptide targeting the EB3-IP3R3 interface (CIPRI) reverses in vivo [PMID:26119739, PMID:37290041, PMID:29247211]. EB3 abundance and activity are tightly regulated: Aurora A/B phosphorylation at Ser-176 stabilizes EB3 during mitosis by disrupting SIAH-1-mediated ubiquitination and degradation to support cell cycle progression and cytokinesis [PMID:19696028, PMID:23712260], calcineurin-driven Ser-162 phosphorylation destabilizes the dimer to permit adherens junction assembly [PMID:23159740], and EZH2-mediated H3K27 trimethylation epigenetically represses MAPRE3, with re-expression inducing cell cycle arrest and apoptosis in ovarian cancer [PMID:38199479]. EB3 is also selectively required for myoblast fusion and primary cilium assembly [PMID:17658256, PMID:21768326].","teleology":[{"year":2000,"claim":"Established EB3 as a microtubule-associated EB1-family protein with a defined binding partner, framing it as a candidate microtubule regulator in the CNS.","evidence":"Yeast two-hybrid and confocal co-localization identifying binding to APCL/APC2","pmids":["10644998"],"confidence":"Medium","gaps":["No direct demonstration of plus-end tracking","Functional consequence of APCL binding not established"]},{"year":2003,"claim":"Showed EB3 autonomously tracks growing microtubule plus-ends in living neurons, reporting microtubule polarity and growth in dendrites.","evidence":"Live-cell EB3-GFP imaging with quantitative directional analysis in cultured neurons","pmids":["12684451"],"confidence":"High","gaps":["Molecular basis of end recognition not defined","Does not address how EB3 differs functionally from EB1"]},{"year":2005,"claim":"Defined EB3 as a recruitment hub that controls partner residence at microtubule tips, answering how +TIP networks are organized.","evidence":"Direct binding assays plus RNAi depletion and rescue measuring CLIP-170/CLIP-115 dissociation kinetics","pmids":["16148041"],"confidence":"High","gaps":["Stoichiometry of EB3-CLIP complexes unresolved","EB1 vs EB3 differential roles not fully separated"]},{"year":2007,"claim":"Revealed cell-type-specific, non-redundant EB3 functions in myoblast fusion and beta-catenin signaling, distinguishing it from EB1.","evidence":"RNAi knockdown, EB1/EB3 chimeras, and C2C12 differentiation; separate Y2H/pull-down with DDA3 plus reporter assays","pmids":["17658256","17310996"],"confidence":"High","gaps":["Calponin-domain residues driving fusion not mechanistically explained","DDA3 cooperation only Medium confidence"]},{"year":2008,"claim":"Identified direct EB3-drebrin binding as a molecular coupler between dynamic microtubule tips and F-actin, explaining microtubule guidance in growth cones.","evidence":"In vitro direct binding, co-localization, and dominant-negative neurite outgrowth assays","pmids":["18806788"],"confidence":"High","gaps":["Regulation of the EB3-drebrin interaction not defined"]},{"year":2009,"claim":"Established that EB3 protein levels are cell-cycle regulated through a kinase/ubiquitin-ligase switch, linking +TIP abundance to mitotic progression.","evidence":"In vitro Aurora-A/B kinase assays, co-IP of SIAH-1 complex disruption, RNAi and proteasome inhibition","pmids":["19696028"],"confidence":"High","gaps":["Structural basis of Ser-176 phospho-control of SIAH-1 binding unresolved"]},{"year":2010,"claim":"Demonstrated with purified components that EB3 both rescues microtubule growth against a catastrophe factor and targets that factor to ends, defining a dual regulatory activity.","evidence":"In vitro reconstitution of microtubule dynamics with TIRF microscopy and MCAK","pmids":["20850319"],"confidence":"High","gaps":["Physiological context of EB3-MCAK regulation in cells not addressed"]},{"year":2011,"claim":"Placed EB3 at specialized cellular landmarks—the axon initial segment, primary cilia base, and dendritic branch points—through scaffold interactions controlling its localization and comet lifetime.","evidence":"Direct interaction/co-IP with ankyrin G and PSD-95, GST pull-down/MS at the cilium base, knockdown and EM phenotypes","pmids":["21551097","21768326","21248129"],"confidence":"High","gaps":["PSD-95 interaction only Medium confidence","How EB3 is stabilized at the AIS mechanistically unresolved"]},{"year":2012,"claim":"Showed EB3 dimer stability is a regulated node coupling calcium/calcineurin signaling and actin-MT crosstalk to junction and epithelial morphogenesis.","evidence":"Ser-162 phospho-mutagenesis with Ca2+ imaging and permeability assays; co-IP of drebrin/myosin IIB/spectrin complex with knockdown","pmids":["23159740","22275434"],"confidence":"High","gaps":["Epithelial drebrin-EB3 complex only Medium confidence","Kinase acting downstream of calcineurin on Ser-162 not identified"]},{"year":2013,"claim":"Defined a distinct EB3 role in late mitosis—midbody microtubule stability, focal adhesion control, and cytokinesis—via Aurora-B phosphorylation at Ser-176.","evidence":"Phospho-mutagenesis, live-cell imaging, and RNAi in dividing cells","pmids":["23712260"],"confidence":"High","gaps":["How the same Ser-176 site governs both stability and cortical MT growth not integrated"]},{"year":2015,"claim":"Established EB3 as a direct amplifier of IP3R3 calcium signaling in endothelium, providing in vivo evidence for its role in vascular permeability.","evidence":"Motif mutagenesis, siRNA, endothelial conditional knockout mouse, Ca2+ imaging, permeability assays","pmids":["26119739"],"confidence":"High","gaps":["Mechanism by which EB3 promotes IP3R3 clustering not structurally defined"]},{"year":2017,"claim":"Extended EB3 SxIP-motif calcium coupling to neurons, linking STIM2-EB3 to dendritic spine maintenance and Alzheimer's-model spine rescue.","evidence":"Co-IP, SxIP motif mutagenesis, knockdown/overexpression rescue, spine morphometry; co-IP of Golgi myomegalin tether with CRISPR knockout","pmids":["29247211","28814570"],"confidence":"High","gaps":["STIM2-EB3 finding only Medium confidence","Selectivity of EB3 for distinct SxIP partners in vivo unresolved"]},{"year":2018,"claim":"Suggested EB3 forms a phosphoregulated complex with the kinase ITPKA that dynamically cycles during synaptic plasticity.","evidence":"Co-IP with phospho-mutant and chemical LTP induction","pmids":["30466786"],"confidence":"Low","gaps":["Single co-IP without reciprocal or in vitro binding validation","Functional consequence of complex cycling not established"]},{"year":2021,"claim":"Resolved EB3 vs EB1 spatial specialization at growth cone tips, explaining why EB3 specifically drives neuritogenesis through the drebrin/F-actin zippering pathway.","evidence":"CRISPR editing, pharmacological MT perturbation, and live imaging of plus-end position","pmids":["34478582"],"confidence":"High","gaps":["Molecular basis of EB3's more proximal tip localization not defined"]},{"year":2023,"claim":"Identified EB3 as a regulator of the labile microtubule pool via the severing enzyme fidgetin and developed a peptide therapeutic against the EB3-IP3R3 interface for lung injury.","evidence":"RNAi/epistasis with fidgetin in an SCI model; CIPRI peptide competition with in vitro and in vivo endotoxemia/sepsis validation","pmids":["37449637","37290041"],"confidence":"High","gaps":["Fidgetin study only Medium confidence","Whether CIPRI affects EB3's other SxIP interactions unaddressed"]},{"year":2024,"claim":"Defined biophysical and transcriptional layers of EB3 control—its higher LLPS propensity and tubulin-nucleating capacity versus EB1, GTP-cap-independent comet behavior, and EZH2/H3K27me3 epigenetic repression with tumor-suppressive re-expression.","evidence":"In vitro LLPS/FRAP/polymerization assays, reconstitution with microfabricated barriers and modeling, and ChIP/knockdown/xenograft in ovarian cancer","pmids":["41135671","39604262","38199479"],"confidence":"High","gaps":["EZH2/ovarian cancer link only Medium confidence","Comet-modeling study only Medium confidence","How LLPS relates to physiological +TIP recruitment in cells not established"]},{"year":null,"claim":"How EB3's many context-specific partner interactions are coordinated and prioritized within a single cell—and how its phase-separation behavior governs partner recruitment in vivo—remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No integrated model selecting among CLIP-170, drebrin, IP3R3, STIM2, and Golgi/AIS scaffold engagements","Structural basis of SxIP-partner discrimination not solved","In vivo relevance of LLPS to plus-end function untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,2,5,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,5,15,16]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,7,15]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,2,7]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[9]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[9]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[16,14]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11,15,22]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,5,20]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[9,16]}],"complexes":[],"partners":["CLIP1","DRD2","ANK3","ITPR3","STIM2","MCAK","SIAH1","PDE4DIP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UPY8","full_name":"Microtubule-associated protein RP/EB family member 3","aliases":["EB1 protein family member 3","EBF3","End-binding protein 3","EB3","RP3"],"length_aa":281,"mass_kda":32.0,"function":"Plus-end tracking protein (+TIP) that binds to the plus-end of microtubules and regulates the dynamics of the microtubule cytoskeleton (PubMed:19255245, PubMed:28814570). Promotes microtubule growth (PubMed:19255245, PubMed:28814570). May be involved in spindle function by stabilizing microtubules and anchoring them at centrosomes (PubMed:19255245, PubMed:28814570). Also acts as a regulator of minus-end microtubule organization: interacts with the complex formed by AKAP9 and PDE4DIP, leading to recruit CAMSAP2 to the Golgi apparatus, thereby tethering non-centrosomal minus-end microtubules to the Golgi, an important step for polarized cell movement (PubMed:28814570). Promotes elongation of CAMSAP2-decorated microtubule stretches on the minus-end of microtubules (PubMed:28814570)","subcellular_location":"Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q9UPY8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAPRE3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAPRE3","total_profiled":1310},"omim":[{"mim_id":"616734","title":"SKIN CREASES, CONGENITAL SYMMETRIC CIRCUMFERENTIAL, 2; CSCSC2","url":"https://www.omim.org/entry/616734"},{"mim_id":"610674","title":"SPERM FLAGELLAR PROTEIN 1; SPEF1","url":"https://www.omim.org/entry/610674"},{"mim_id":"605789","title":"MICROTUBULE-ASSOCIATED PROTEIN, RP/EB FAMILY, MEMBER 2; MAPRE2","url":"https://www.omim.org/entry/605789"},{"mim_id":"605788","title":"MICROTUBULE-ASSOCIATED PROTEIN, RP/EB FAMILY, MEMBER 3; MAPRE3","url":"https://www.omim.org/entry/605788"},{"mim_id":"603108","title":"MICROTUBULE-ASSOCIATED PROTEIN, RP/EB FAMILY, MEMBER 1; MAPRE1","url":"https://www.omim.org/entry/603108"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":161.0},{"tissue":"skeletal muscle","ntpm":139.6}],"url":"https://www.proteinatlas.org/search/MAPRE3"},"hgnc":{"alias_symbol":["RP3","EB3"],"prev_symbol":[]},"alphafold":{"accession":"Q9UPY8","domains":[{"cath_id":"1.10.418.10","chopping":"16-129","consensus_level":"high","plddt":95.8861,"start":16,"end":129},{"cath_id":"1.20.5.1430","chopping":"201-255","consensus_level":"high","plddt":90.6073,"start":201,"end":255}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPY8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UPY8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UPY8-F1-predicted_aligned_error_v6.png","plddt_mean":79.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAPRE3","jax_strain_url":"https://www.jax.org/strain/search?query=MAPRE3"},"sequence":{"accession":"Q9UPY8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UPY8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UPY8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPY8"}},"corpus_meta":[{"pmid":"12684451","id":"PMC_12684451","title":"Visualization 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\"Yeast two-hybrid, confocal microscopy, sequence analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid and co-localization, single lab, two methods but no in vitro reconstitution\",\n      \"pmids\": [\"10644998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EB3-GFP tracks exclusively to the growing plus-ends of microtubules in live neurons, marking microtubule growth events; microtubules grow slower in neurons than in glia/COS-1 cells, and ~65% of EB3-GFP movements in proximal dendrites are directed distally vs. ~35% toward the cell body, reflecting the mixed microtubule polarity of dendrites.\",\n      \"method\": \"Live-cell fluorescence microscopy with EB3-GFP in cultured neurons\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct live-cell imaging with quantitative analysis, replicated across multiple cell types and compartments, foundational study\",\n      \"pmids\": [\"12684451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EB1 and EB3 directly bind CLIP-170 and CLIP-115 through their C-terminal tyrosine residues, and control CLIP dissociation kinetics from microtubule plus-ends; RNAi depletion of EB1 and EB3 accelerates CLIP dissociation from tips, which is rescued by EB1 but not EB2 expression.\",\n      \"method\": \"RNA interference, direct binding assay, live-cell imaging of CLIP dynamics\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays plus RNAi rescue experiments, multiple orthogonal methods in single study\",\n      \"pmids\": [\"16148041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EB3 is specifically upregulated upon myogenic differentiation; knockdown of EB3 (but not EB1) prevents myoblast elongation and fusion, impairs microtubule capture at the cell cortex, and disrupts microtubule dynamics; two specific amino acids in the calponin-like domain of EB3 are required for myoblast fusion.\",\n      \"method\": \"RNAi knockdown, live-cell imaging, EB1/EB3 chimera expression, C2C12 myoblast differentiation assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined phenotype, domain-mapping with chimeras, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"17658256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EB3 interacts with the p53-target DDA3 protein; the interaction requires intact microtubules, maps to aa 118-329 of DDA3 and both N- and C-termini of EB3; DDA3 and EB3 cooperate for microtubule binding in vitro; ectopic expression of DDA3 and EB3 enhances beta-catenin-dependent transactivation and cyclin D1 production.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, in vitro microtubule-binding assay, immunofluorescence, reporter assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in single lab, direct binding confirmed in vitro\",\n      \"pmids\": [\"17310996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EB3 directly binds drebrin (an F-actin-associated protein); in growth cones this interaction occurs specifically when drebrin is on F-actin in the proximal region of filopodia and EB3 is at microtubule tips invading filopodia; disruption of this interaction impairs growth cone formation and neurite extension.\",\n      \"method\": \"Direct binding assay (in vitro), co-localization, dominant-negative disruption, neurite outgrowth assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding established in vitro, functional loss-of-interaction phenotype, multiple methods\",\n      \"pmids\": [\"18806788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EB3 stability is regulated during mitosis by Aurora-A and Aurora-B kinases that phosphorylate EB3 at Ser-176, disrupting the EB3-SIAH-1 complex; SIAH-1 ubiquitin ligase mediates EB3 polyubiquitination and proteasomal degradation during G1; phosphorylation-induced stabilization of EB3 during mitosis facilitates cell cycle progression at prometaphase.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, RNAi knockdown, proteasome inhibition, cell cycle analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase phosphorylation, co-IP of complex disruption, RNAi knockdown, multiple methods in single study\",\n      \"pmids\": [\"19696028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In vitro reconstitution demonstrated that EB3 restores robust microtubule growth in the presence of MCAK (which alone blocks assembly) and targets MCAK to growing microtubule ends by increasing its association rate; the EB3-dependent targeting requires direct EB3-MCAK interaction and enhances MCAK's capacity to induce catastrophes without affecting growth/shortening velocities.\",\n      \"method\": \"In vitro reconstitution of microtubule dynamics, TIRF microscopy, quantitative analysis of dynamics\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified components, quantitative dynamics measurements, multiple conditions tested\",\n      \"pmids\": [\"20850319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EB3 (and EB1) directly interact with the AIS scaffold protein ankyrin G (ankG); EB3 is concentrated and stabilized at the axon initial segment (AIS) in mature neurons; knockdown of ankG leads to cell-wide upregulation of EB3 comets; EB3/EB1 participate in AIS maintenance.\",\n      \"method\": \"Direct interaction assay, live-cell imaging, immunofluorescence, ankG knockdown, fractionation/localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction identified, subcellular localization with functional consequence via knockdown, multiple orthogonal methods\",\n      \"pmids\": [\"21551097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EB3 (and EB1) are required for assembly of primary cilia; cells lacking EB1 or EB3 have defective MT minus-end anchoring at centrosome/basal body and short cilia stumps; GST pull-down and mass spectrometry showed EB1/EB3 interact with proteins implicated in MT minus-end anchoring and vesicular trafficking to cilia base; EB3 localizes to the tip of motile cilia and affects centriole-associated rootlet filament formation.\",\n      \"method\": \"Protein depletion (siRNA), dominant-negative expression, electron microscopy, GST pull-down, mass spectrometry, immunoprecipitation, live imaging\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined structural phenotype, protein interactions confirmed by multiple methods, direct localization\",\n      \"pmids\": [\"21768326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The SH3 domain of PSD-95 interacts with a proline-rich region within EB3; overexpression of PSD-95 decreases the lifetime of EB3 comets in dendrites, leading to less organized microtubules at dendritic branch points and decreased dendritic branching.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging (EB3 comet analysis), overexpression in neurons\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus live imaging of comet dynamics, single lab, two orthogonal methods\",\n      \"pmids\": [\"21248129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VE-cadherin outside-in signaling activates Src and PLCγ2, causing Ca2+ release from ER stores, activating calcineurin (CaN); CaN downregulation leads to phosphorylation of EB3 at Ser-162, destabilizing the EB3 dimer, suppressing microtubule growth, and enabling adherens junction assembly; phospho-defective S162A EB3 mutant induces MT growth in confluent monolayers and disassembles AJs.\",\n      \"method\": \"Phospho-specific mutagenesis, calcium imaging, pharmacological inhibition, co-immunoprecipitation, endothelial permeability assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific phospho-mutagenesis with defined functional consequence, pharmacological pathway dissection, multiple orthogonal methods in single study\",\n      \"pmids\": [\"23159740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Drebrin E and EB3 form a complex with myosin IIB and βII-spectrin at the apical domain of columnar epithelial cells; depletion of drebrin E disrupts apical accumulation of EB3 and impairs cell elongation; EB3 depletion produces a similar elongation defect; the complex connects F-actin and microtubule networks apically during epithelial morphogenesis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence, morphometric analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP of complex, loss-of-function phenotype, single lab, multiple components tested\",\n      \"pmids\": [\"22275434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Aurora B phosphorylates EB3 at Ser-176 at the midbody to control cortical microtubule growth; EB3 stabilizes focal adhesions and coordinates daughter cell spreading during mitotic exit, promotes midbody microtubule stability, and is required for efficient cytokinesis; EB1 and EB3 play temporally distinct roles in cell division, with EB1 involved in spindle orientation before anaphase.\",\n      \"method\": \"Phospho-specific mutagenesis, live-cell imaging, RNAi knockdown, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific phospho-mutagenesis with functional readouts, live-cell imaging, loss-of-function, multiple orthogonal methods\",\n      \"pmids\": [\"23712260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"BPAG1a/b C-terminal isoform-specific tails bind both EB1 and EB3 and are sufficient to bundle microtubules; knockdown of BPAG1a/b in C2.7 myoblasts impairs directness of cell migration and disrupts Golgi structure.\",\n      \"method\": \"GST pull-down, co-immunoprecipitation, microtubule bundling assay, siRNA knockdown, migration assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct binding demonstrated, loss-of-function phenotype, single lab\",\n      \"pmids\": [\"25244344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EB3 binds to IP3 receptors (IP3R3) through an S/TxIP EB-binding motif; in endothelial cells, EB3 depletion or mutation of the TxIP motif of IP3R3 prevents α-thrombin-induced IP3R3 clustering, Ca2+ increase, myosin light chain phosphorylation, and vascular permeability increase; selective EB3 gene deletion in mouse endothelial cells abrogates α-thrombin-induced endothelial permeability.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis of binding motif, siRNA knockdown, conditional knockout mouse, Ca2+ imaging, permeability assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding via motif mutagenesis, in vitro and in vivo loss-of-function, multiple orthogonal methods, in vivo validation\",\n      \"pmids\": [\"26119739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EB3 (and EB1) form a complex with myomegalin that acts as a membrane-microtubule tether at Golgi membranes; CRISPR/Cas9 knockout of EB2/EB3 and C-terminal half of EB1 reduces CAMSAP2-decorated microtubule minus end lengths, detaches microtubules from Golgi membranes, compacts the Golgi complex, and disrupts cell migration, polarity, and focal adhesion distribution.\",\n      \"method\": \"CRISPR/Cas9 knockout, co-immunoprecipitation, electron microscopy, live-cell imaging, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR knockout with defined structural phenotypes, interaction validated by co-IP, multiple orthogonal methods, rigorous controls\",\n      \"pmids\": [\"28814570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"STIM2 forms a Ca2+-dependent complex with EB3 via a Ser-x-Ile-Pro (SxIP) amino acid motif; disruption of STIM2-EB3 interaction results in loss of mushroom dendritic spines; EB3 overexpression rescues mushroom spine loss in a PS1-M146V knock-in Alzheimer's disease model, while STIM2 overexpression fails to rescue spines after EB3 knockdown.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis of SxIP motif, siRNA knockdown, overexpression, spine morphometry in hippocampal neurons\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP with motif identification, epistasis via rescue experiments, single lab\",\n      \"pmids\": [\"29247211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IP3K-A (ITPKA) binds to EB3, and this interaction is regulated by PKA-dependent phosphorylation of IP3K-A at Ser119; the IP3K-A/EB3 complex dissociates and reassociates rapidly during chemically-induced LTP conditions.\",\n      \"method\": \"Co-immunoprecipitation, phospho-specific mutagenesis, chemical LTP induction\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP with phospho-mutant, single lab, single method per finding\",\n      \"pmids\": [\"30466786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EB3 comet intensity profiles in mouse fibroblasts frequently exhibit secondary EB3-binding peaks (EB3-islands) on 56% of growing microtubule comets, which are stationary and originate from EB3 comets moving with growing MT tips; modeling suggests additional protein factors beyond GTP hydrolysis contribute to EB3 residence time on MTs.\",\n      \"method\": \"Live-cell fluorescence microscopy (EB3-RFP), quantitative image analysis, computational modeling\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct live-cell imaging with quantitative analysis and modeling, single lab\",\n      \"pmids\": [\"31141458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of EB3 (but not EB1) inhibits neuritogenesis in embryonic cortical neurons; EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia, enabling continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin; pharmacological blockade of microtubule dynamics is associated with selective loss of EB3 (not EB1) from plus-ends.\",\n      \"method\": \"Gene editing (CRISPR), pharmacological inhibition, overexpression, live-cell imaging, morphometric analysis\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function by gene editing plus live imaging of plus-end dynamics, multiple orthogonal approaches, EB1 vs EB3 specificity established\",\n      \"pmids\": [\"34478582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Fidgetin (microtubule-severing enzyme) trims tyrosinated (labile) microtubules by interacting with EB3; fidgetin depletion increases EB3 at neurite ends, elongates the labile microtubule domain, and increases axon length and branching; EB3 knockdown does not change fidgetin expression but fidgetin deletion markedly increases EB3 levels.\",\n      \"method\": \"RNA interference, overexpression, rat SCI model, immunofluorescence, EB3 localization analysis\",\n      \"journal\": \"Neural regeneration research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function for both proteins with epistatic analysis, in vivo model, single lab\",\n      \"pmids\": [\"37449637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A 14-amino acid peptide (CIPRI) targeting the EB3-IP3R3 interface disrupts EB3-IP3R3 interaction in vitro and in mouse lungs; disruption mitigates Ca2+ release from ER stores, prevents VE-cadherin junction disassembly, and reduces inflammation-induced lung injury and microvascular leakage in mice.\",\n      \"method\": \"Peptide competition assay, co-immunoprecipitation, Ca2+ imaging, in vivo endotoxemia/sepsis mouse model, vascular permeability assay\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct interface disruption with peptide, both in vitro and in vivo validation, multiple functional readouts\",\n      \"pmids\": [\"37290041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EZH2-mediated H3K27 trimethylation epigenetically represses MAPRE3 (EB3) expression at its promoter in ovarian cancer; EZH2 knockdown decreases H3K27me3 enrichment at the MAPRE3 promoter and increases EB3 expression; MAPRE3 overexpression induces cell cycle arrest and apoptosis in ovarian cancer cells.\",\n      \"method\": \"ChIP assay (H3K27me3 at MAPRE3 promoter), EZH2 knockdown, overexpression, xenograft tumor model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP showing epigenetic repression with functional consequences, single lab, in vitro and in vivo validation\",\n      \"pmids\": [\"38199479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EB3 undergoes liquid-liquid phase separation (LLPS) with significantly higher propensity than EB1 despite 67% sequence identity; this difference involves contributions from multiple protein regions with histidine residues in the N-terminal domain playing a key role; EB3 condensates have lower dynamics (less fluid) than EB1 and higher capacity to recruit tubulin and nucleate polymerization; EB3/CLIP-170 droplets show higher tubulin polymerization capacity than EB1/CLIP-170.\",\n      \"method\": \"In vitro LLPS assay, fluorescence recovery after photobleaching (FRAP), mutagenesis, in vitro tubulin polymerization assay, cell-based condensate imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of LLPS, mutagenesis, functional tubulin polymerization assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"41135671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In vitro reconstitution showed that EB3-dependent comet size at microtubule ends is independent of GTP-cap size; a phenomenological model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate fully describes stalled and freely growing microtubule lifetime distributions; modulation of microtubule growth velocity in cells does not consistently alter EB3 comet length.\",\n      \"method\": \"In vitro reconstitution with microfabricated barriers, GFP-EB3 live imaging, mathematical modeling\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with modeling, single lab, novel mechanistic insight about cap independence\",\n      \"pmids\": [\"39604262\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAPRE3/EB3 is a microtubule plus-end tracking protein (+TIP) that autonomously recognizes the GTP/GDP-Pi cap at growing microtubule ends, undergoes liquid-liquid phase separation to nucleate tubulin polymerization, recruits and regulates the dissociation kinetics of CLIP-170/CLIP-115, directly binds drebrin on F-actin to coordinate microtubule-actin coupling in growth cones and dendritic spines, interacts with ankyrin G at the axon initial segment, binds IP3R3 via an SxIP motif to amplify Ca2+ signaling in endothelial cells, is stabilized during mitosis by Aurora A/B phosphorylation at Ser-176 (which disrupts SIAH-1-mediated ubiquitination and proteasomal degradation), and is epigenetically repressed by EZH2-mediated H3K27 trimethylation; collectively these interactions place EB3 as a context-specific scaffold that links microtubule dynamics to adhesion, signaling, neuronal polarity, and cellular morphogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAPRE3/EB3 is a microtubule plus-end tracking protein (+TIP) that autonomously marks growing microtubule ends and acts as a context-specific scaffold linking microtubule dynamics to actin, adhesion, calcium signaling, and cellular morphogenesis [#1, #2]. It directly binds CLIP-170/CLIP-115 through their C-terminal tyrosine residues and controls their dissociation kinetics from microtubule tips [#2], targets the catastrophe factor MCAK to growing ends [#7], and undergoes liquid-liquid phase separation that recruits tubulin and nucleates polymerization more efficiently than EB1 [#24]. EB3 bridges the microtubule and F-actin networks by binding drebrin on F-actin, a coupling required for growth cone formation, neurite extension, and apical epithelial elongation [#5, #12, #20], and it associates with structural scaffolds including ankyrin G at the axon initial segment, myomegalin/CAMSAP2 tethers at Golgi membranes, and BPAG1 [#8, #16, #14]. Through an SxIP/TxIP motif EB3 binds IP3R3 and STIM2 to amplify ER calcium release: in endothelial cells the EB3-IP3R3 interaction drives thrombin- and inflammation-induced Ca2+ flux, junction disassembly, and vascular permeability, which a peptide targeting the EB3-IP3R3 interface (CIPRI) reverses in vivo [#15, #22, #17]. EB3 abundance and activity are tightly regulated: Aurora A/B phosphorylation at Ser-176 stabilizes EB3 during mitosis by disrupting SIAH-1-mediated ubiquitination and degradation to support cell cycle progression and cytokinesis [#6, #13], calcineurin-driven Ser-162 phosphorylation destabilizes the dimer to permit adherens junction assembly [#11], and EZH2-mediated H3K27 trimethylation epigenetically represses MAPRE3, with re-expression inducing cell cycle arrest and apoptosis in ovarian cancer [#23]. EB3 is also selectively required for myoblast fusion and primary cilium assembly [#3, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established EB3 as a microtubule-associated EB1-family protein with a defined binding partner, framing it as a candidate microtubule regulator in the CNS.\",\n      \"evidence\": \"Yeast two-hybrid and confocal co-localization identifying binding to APCL/APC2\",\n      \"pmids\": [\"10644998\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct demonstration of plus-end tracking\", \"Functional consequence of APCL binding not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed EB3 autonomously tracks growing microtubule plus-ends in living neurons, reporting microtubule polarity and growth in dendrites.\",\n      \"evidence\": \"Live-cell EB3-GFP imaging with quantitative directional analysis in cultured neurons\",\n      \"pmids\": [\"12684451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of end recognition not defined\", \"Does not address how EB3 differs functionally from EB1\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined EB3 as a recruitment hub that controls partner residence at microtubule tips, answering how +TIP networks are organized.\",\n      \"evidence\": \"Direct binding assays plus RNAi depletion and rescue measuring CLIP-170/CLIP-115 dissociation kinetics\",\n      \"pmids\": [\"16148041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of EB3-CLIP complexes unresolved\", \"EB1 vs EB3 differential roles not fully separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Revealed cell-type-specific, non-redundant EB3 functions in myoblast fusion and beta-catenin signaling, distinguishing it from EB1.\",\n      \"evidence\": \"RNAi knockdown, EB1/EB3 chimeras, and C2C12 differentiation; separate Y2H/pull-down with DDA3 plus reporter assays\",\n      \"pmids\": [\"17658256\", \"17310996\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Calponin-domain residues driving fusion not mechanistically explained\", \"DDA3 cooperation only Medium confidence\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified direct EB3-drebrin binding as a molecular coupler between dynamic microtubule tips and F-actin, explaining microtubule guidance in growth cones.\",\n      \"evidence\": \"In vitro direct binding, co-localization, and dominant-negative neurite outgrowth assays\",\n      \"pmids\": [\"18806788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of the EB3-drebrin interaction not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that EB3 protein levels are cell-cycle regulated through a kinase/ubiquitin-ligase switch, linking +TIP abundance to mitotic progression.\",\n      \"evidence\": \"In vitro Aurora-A/B kinase assays, co-IP of SIAH-1 complex disruption, RNAi and proteasome inhibition\",\n      \"pmids\": [\"19696028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Ser-176 phospho-control of SIAH-1 binding unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated with purified components that EB3 both rescues microtubule growth against a catastrophe factor and targets that factor to ends, defining a dual regulatory activity.\",\n      \"evidence\": \"In vitro reconstitution of microtubule dynamics with TIRF microscopy and MCAK\",\n      \"pmids\": [\"20850319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context of EB3-MCAK regulation in cells not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed EB3 at specialized cellular landmarks—the axon initial segment, primary cilia base, and dendritic branch points—through scaffold interactions controlling its localization and comet lifetime.\",\n      \"evidence\": \"Direct interaction/co-IP with ankyrin G and PSD-95, GST pull-down/MS at the cilium base, knockdown and EM phenotypes\",\n      \"pmids\": [\"21551097\", \"21768326\", \"21248129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PSD-95 interaction only Medium confidence\", \"How EB3 is stabilized at the AIS mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed EB3 dimer stability is a regulated node coupling calcium/calcineurin signaling and actin-MT crosstalk to junction and epithelial morphogenesis.\",\n      \"evidence\": \"Ser-162 phospho-mutagenesis with Ca2+ imaging and permeability assays; co-IP of drebrin/myosin IIB/spectrin complex with knockdown\",\n      \"pmids\": [\"23159740\", \"22275434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Epithelial drebrin-EB3 complex only Medium confidence\", \"Kinase acting downstream of calcineurin on Ser-162 not identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined a distinct EB3 role in late mitosis—midbody microtubule stability, focal adhesion control, and cytokinesis—via Aurora-B phosphorylation at Ser-176.\",\n      \"evidence\": \"Phospho-mutagenesis, live-cell imaging, and RNAi in dividing cells\",\n      \"pmids\": [\"23712260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the same Ser-176 site governs both stability and cortical MT growth not integrated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established EB3 as a direct amplifier of IP3R3 calcium signaling in endothelium, providing in vivo evidence for its role in vascular permeability.\",\n      \"evidence\": \"Motif mutagenesis, siRNA, endothelial conditional knockout mouse, Ca2+ imaging, permeability assays\",\n      \"pmids\": [\"26119739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which EB3 promotes IP3R3 clustering not structurally defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended EB3 SxIP-motif calcium coupling to neurons, linking STIM2-EB3 to dendritic spine maintenance and Alzheimer's-model spine rescue.\",\n      \"evidence\": \"Co-IP, SxIP motif mutagenesis, knockdown/overexpression rescue, spine morphometry; co-IP of Golgi myomegalin tether with CRISPR knockout\",\n      \"pmids\": [\"29247211\", \"28814570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"STIM2-EB3 finding only Medium confidence\", \"Selectivity of EB3 for distinct SxIP partners in vivo unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Suggested EB3 forms a phosphoregulated complex with the kinase ITPKA that dynamically cycles during synaptic plasticity.\",\n      \"evidence\": \"Co-IP with phospho-mutant and chemical LTP induction\",\n      \"pmids\": [\"30466786\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP without reciprocal or in vitro binding validation\", \"Functional consequence of complex cycling not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved EB3 vs EB1 spatial specialization at growth cone tips, explaining why EB3 specifically drives neuritogenesis through the drebrin/F-actin zippering pathway.\",\n      \"evidence\": \"CRISPR editing, pharmacological MT perturbation, and live imaging of plus-end position\",\n      \"pmids\": [\"34478582\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of EB3's more proximal tip localization not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified EB3 as a regulator of the labile microtubule pool via the severing enzyme fidgetin and developed a peptide therapeutic against the EB3-IP3R3 interface for lung injury.\",\n      \"evidence\": \"RNAi/epistasis with fidgetin in an SCI model; CIPRI peptide competition with in vitro and in vivo endotoxemia/sepsis validation\",\n      \"pmids\": [\"37449637\", \"37290041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Fidgetin study only Medium confidence\", \"Whether CIPRI affects EB3's other SxIP interactions unaddressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined biophysical and transcriptional layers of EB3 control—its higher LLPS propensity and tubulin-nucleating capacity versus EB1, GTP-cap-independent comet behavior, and EZH2/H3K27me3 epigenetic repression with tumor-suppressive re-expression.\",\n      \"evidence\": \"In vitro LLPS/FRAP/polymerization assays, reconstitution with microfabricated barriers and modeling, and ChIP/knockdown/xenograft in ovarian cancer\",\n      \"pmids\": [\"41135671\", \"39604262\", \"38199479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"EZH2/ovarian cancer link only Medium confidence\", \"Comet-modeling study only Medium confidence\", \"How LLPS relates to physiological +TIP recruitment in cells not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EB3's many context-specific partner interactions are coordinated and prioritized within a single cell—and how its phase-separation behavior governs partner recruitment in vivo—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No integrated model selecting among CLIP-170, drebrin, IP3R3, STIM2, and Golgi/AIS scaffold engagements\", \"Structural basis of SxIP-partner discrimination not solved\", \"In vivo relevance of LLPS to plus-end function untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 2, 5, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 5, 15, 16]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 7, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 2, 7]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [16, 14]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 15, 22]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 5, 20]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [9, 16]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CLIP1\", \"DRD2\", \"ANK3\", \"ITPR3\", \"STIM2\", \"MCAK\", \"SIAH1\", \"PDE4DIP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}