{"gene":"LIMA1","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":1999,"finding":"EPLIN (LIMA1) is a novel cytoskeletal LIM domain protein that co-localizes with filamentous actin and suppresses cell proliferation when overexpressed; both EPLIN-alpha and EPLIN-beta isoforms localize to actin stress fibers.","method":"Overexpression, immunofluorescence co-localization with F-actin","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, direct localization and proliferation phenotype with overexpression but no detailed mechanistic dissection","pmids":["10618726"],"is_preprint":false},{"year":2000,"finding":"EPLIN-alpha and EPLIN-beta are generated from distinct promoters of a single gene; EPLIN-alpha expression is serum-inducible via a consensus serum response element, and its promoter activity is enhanced by activated RhoA, whereas EPLIN-beta is not serum-regulated.","method":"5' RACE, S1 nuclease protection, promoter-reporter constructs, serum stimulation, RhoA transfection","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in a single lab establishing isoform-specific transcriptional regulation","pmids":["10806352"],"is_preprint":false},{"year":2002,"finding":"The N-terminal region of EPLIN is necessary for both localization to the actin cytoskeleton and suppression of anchorage-independent growth of EWS/Fli-1- and Cdc42-transformed cells; the LIM domain and C-terminal region are dispensable for these functions. Inhibition of anchorage-independent growth requires EPLIN's actin cytoskeletal localization.","method":"Retroviral transduction of truncation mutants, colony formation assay, immunofluorescence","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mutagenesis with defined cellular phenotype in single lab","pmids":["11950948"],"is_preprint":false},{"year":2003,"finding":"Purified recombinant EPLIN inhibits actin filament depolymerization, cross-links filaments into bundles via at least two actin-binding sites, and inhibits Arp2/3-mediated branching nucleation without affecting spontaneous polymerization kinetics or barbed-end elongation. EPLIN promotes stable actin filament structures (stress fibers) at the expense of dynamic structures (membrane ruffles).","method":"In vitro actin polymerization/depolymerization assays, actin bundling assay, Arp2/3 branching assay with purified recombinant EPLIN, cell overexpression/knockdown","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple in vitro reconstitution assays with purified protein, mutagenesis-informed domain analysis, orthogonal cellular experiments","pmids":["12566430"],"is_preprint":false},{"year":2007,"finding":"EPLIN couples with alpha-catenin and links the cadherin-catenin complex to F-actin, forming the molecular bridge required for the adhesion belt (zonula adherens). EPLIN depletion disorganizes the adhesion belt and converts it to zipper-like junctions with radially arranged actin fibers without affecting non-junctional actin fibers.","method":"RNAi knockdown, co-immunoprecipitation, immunofluorescence, live imaging of epithelial cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus loss-of-function with specific morphological phenotype, replicated in multiple cell systems","pmids":["18093941"],"is_preprint":false},{"year":2007,"finding":"ERK phosphorylates EPLIN at Ser360, Ser602, and Ser692 in vitro and in intact cells; phosphorylation of the C-terminal region reduces EPLIN's affinity for actin filaments. ERK-mediated phosphorylation of EPLIN is required for PDGF-induced stress fiber disassembly, membrane ruffling, and cell migration; a non-phosphorylatable EPLIN mutant blocks these processes.","method":"In vitro kinase assay, phospho-specific antibodies, site-directed mutagenesis (Ser→Ala), wound healing and migration assays, PDGF stimulation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis plus functional cell-based validation with phosphomimetic/non-phosphorylatable mutants","pmids":["17875928"],"is_preprint":false},{"year":2008,"finding":"Overexpression of EPLIN-alpha in breast cancer cells renders them less invasive and less motile; an ERK inhibitor abolishes the effects of EPLIN expression on cell behavior, placing EPLIN downstream of ERK signaling in motility control.","method":"Transfection/overexpression, in vitro invasion assay, Electric Cell Impedance Sensing migration assay, ERK inhibitor treatment","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional loss/gain with pharmacological pathway inhibition, single lab","pmids":["18796137"],"is_preprint":false},{"year":2009,"finding":"EPLIN localizes to the cleavage furrow during cytokinesis, possibly via association with contractile ring components myosin II and septin Sept2. EPLIN depletion leads to multinucleated cells due to inefficient accumulation of active myosin II (MRLC-S19), Sept2, RhoA, and Cdc42 at the cleavage furrow.","method":"Immunofluorescence, siRNA knockdown, co-localization with myosin II and Sept2, multinucleation assay","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional cytokinesis phenotype; association with contractile ring components by co-localization, single lab","pmids":["19221476"],"is_preprint":false},{"year":2010,"finding":"EPLIN-alpha transcription is regulated by actin-MAL/MRTF-SRF signaling; MAL/MRTF coactivator binds an SRF consensus site in the EPLIN-alpha promoter, and its recruitment is enhanced upon induction. Monomeric actin maintains a repressive MAL-actin complex. EPLIN-beta isoform is not regulated by this pathway.","method":"Transcriptome analysis with actin-binding drugs, promoter-reporter assay, ChIP, dominant negative MAL overexpression, MRTF knockdown","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus promoter reporters plus loss/gain of function, single lab, multiple orthogonal methods","pmids":["20236507"],"is_preprint":false},{"year":2011,"finding":"EPLIN interacts directly with alpha-catenin and tethers the VE-cadherin/catenin complex to the actin cytoskeleton in endothelial cells. EPLIN depletion delocalizes vinculin from junctions. EPLIN-depleted endothelial cells have reduced capacity to form pseudocapillary networks due to breakage events, establishing EPLIN as a mechanotransmitter for vinculin recruitment to adherens junctions independent of actomyosin tension.","method":"Co-immunoprecipitation, GST pulldown, siRNA knockdown, Matrigel angiogenesis assay, blebbistatin treatment, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding confirmed by reciprocal Co-IP and GST pulldown, functional validation with loss-of-function phenotype, orthogonal methods","pmids":["22194609"],"is_preprint":false},{"year":2011,"finding":"EPLIN depletion in prostate cancer cells causes disassembly of adherens junctions, actin remodeling, and activation of beta-catenin signaling, demonstrating EPLIN as a negative regulator of EMT.","method":"siRNA knockdown, biochemical analysis, beta-catenin reporter assay, microarray expression analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined molecular phenotype (adherens junction, beta-catenin signaling), single lab","pmids":["21625216"],"is_preprint":false},{"year":2011,"finding":"Junctional tension is important for retaining EPLIN at adherens junctions; lateral actin fiber force inhibits EPLIN-AJ association. EPLIN association with αE-catenin is required for zonula adherens maintenance; vinculin cooperates with EPLIN to maintain ZA morphology. A fusion of αE-catenin and EPLIN promotes ZA but not punctate AJ formation.","method":"Live imaging, tension manipulation, EPLIN-catenin fusion protein expression, RNAi, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal experiments including force manipulation, fusion proteins, and loss-of-function with specific morphological phenotypes","pmids":["21844208"],"is_preprint":false},{"year":2012,"finding":"EGF promotes phosphorylation, ubiquitination, and proteasomal degradation of EPLIN through ERK1/2-dependent signaling. Serine residues S362 and S604 are ERK1/2 phosphorylation sites; point mutation of these renders EPLIN resistant to EGF-induced protein turnover.","method":"EGF stimulation, ERK1/2 inhibitor treatment, ubiquitination assay, site-directed mutagenesis (S362A, S604A), cycloheximide chase, western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of phosphorylation sites combined with ubiquitination assay and degradation kinetics in a single focused study","pmids":["23188829"],"is_preprint":false},{"year":2013,"finding":"DNp73 drives cancer cell migration/invasion by repressing EPLIN (LIMA1) expression. EPLIN directly links to IGF1R-AKT/STAT3 activation; EPLIN loss activates this signaling axis. p73/DNp73 transcriptionally regulates LIMA1/EPLIN, and the DNp73-EPLIN axis controls IGF1R-dependent signaling to initiate metastasis.","method":"Knockdown of endogenous DNp73, tumor xenografts, biochemical signaling analysis (AKT/STAT3 phosphorylation), epistasis experiments","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with mechanistic pathway placement and in vivo xenograft validation, single lab","pmids":["24135282"],"is_preprint":false},{"year":2015,"finding":"API2-MALT1 fusion protein induces paracaspase-mediated proteolytic cleavage of LIMA1/EPLIN tumor suppressor. API2-dependent binding targets LIMA1 for MALT1 paracaspase cleavage, generating an oncogenic LIM domain-only (LMO) fragment. Primary MALT lymphomas with API2-MALT1 fusion uniquely show LIMA1 cleavage fragments.","method":"Co-immunoprecipitation, in vitro cleavage assay, mutagenesis of paracaspase active site, tumor xenograft, primary lymphoma tissue analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical cleavage assay, mutagenesis of active site, validated in primary patient tumor material","pmids":["25569716"],"is_preprint":false},{"year":2015,"finding":"EPLIN (LIMA1) accumulates in RasV12-transformed cells surrounded by normal cells and is required for their apical extrusion. EPLIN functions upstream of Cav-1 and promotes its enrichment in transformed cells. EPLIN regulates non-cell-autonomous activation of myosin-II and PKA in transformed cells, and affects accumulation of filamin A in neighboring normal cells.","method":"siRNA knockdown, immunofluorescence, co-culture assay, epithelial extrusion quantification","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined extrusion phenotype and epistasis placing EPLIN upstream of Cav-1, single lab","pmids":["25609711"],"is_preprint":false},{"year":2015,"finding":"PINCH-1 (LIMS1) associates with EPLIN at integrin adhesion sites. EPLIN localizes to integrin adhesion sites in a PINCH-1-dependent manner. EPLIN depletion severely attenuates keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions.","method":"PINCH-1 interactome isolation (MS), co-immunoprecipitation, PINCH-1 conditional knockout mouse model, siRNA knockdown, adhesion and migration assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — interactome MS identification followed by Co-IP confirmation, in vivo genetic model, and functional loss-of-function assays","pmids":["25609703"],"is_preprint":false},{"year":2017,"finding":"p53 transcriptionally induces LIMA1/EPLIN expression via two p53 response elements identified by ChIP-seq in the LIMA1 gene. p53-induced LIMA1 suppresses cancer cell invasion; LIMA1 knockdown partially inhibits p53-induced suppression of invasion.","method":"ChIP-seq, cDNA microarray, nutlin-3a treatment (p53 activation), siRNA knockdown, invasion assay","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq identification of response elements plus functional epistasis via knockdown, single lab","pmids":["28093207"],"is_preprint":false},{"year":2018,"finding":"LIMA1 (EPLIN) bridges NPC1L1, the essential cholesterol absorption protein, to a transport complex containing myosin Vb, facilitating intestinal cholesterol uptake. In mice, LIMA1 is expressed in the small intestine and localizes on the brush border membrane. Lima1-deficient mice display reduced cholesterol absorption and resistance to diet-induced hypercholesterolemia.","method":"Co-immunoprecipitation, genetic knockout mouse model, rare human frameshift variant analysis, subcellular fractionation/localization","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP establishing protein complex, genetic knockout model with defined phenotype, human genetic validation","pmids":["29880681"],"is_preprint":false},{"year":2018,"finding":"Paxillin binds to EPLIN specifically in mixed cultures of normal and RasV12-transformed cells. Paxillin, plectin, and EPLIN mutually influence each other's non-cell-autonomous accumulation. Paxillin acts downstream of plectin-EPLIN complex and promotes microtubule acetylation by suppressing HDAC6 activity to drive apical extrusion of transformed cells.","method":"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, HDAC6 activity assay, tubulin acetylation measurement","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus loss-of-function with defined molecular pathway, single lab","pmids":["29391412"],"is_preprint":false},{"year":2019,"finding":"EPLIN-alpha and EPLIN-beta isoforms have distinct spatiotemporal interactions with actin: EPLIN-alpha is targeted to membrane protrusions and terminates their growth via interaction with Arp2/3 complex, while EPLIN-beta stabilizes stress fibers. FRAP analyses demonstrate EPLIN-beta has a lower turnover rate than EPLIN-alpha. Shear stress increases EPLIN-beta expression to stabilize stress fibers.","method":"FRAP, siRNA isoform-specific knockdown, Arp2/3 interaction assay, immunofluorescence, shear stress experiments in endothelial cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (FRAP, isoform-specific KD, Arp2/3 interaction) establishing isoform-specific mechanisms in single well-controlled study","pmids":["31644899"],"is_preprint":false},{"year":2020,"finding":"LUZP1 and EPLIN interact and together restrict primary cilia formation by stabilizing actin filaments. EPLIN depletion increases MyosinVa levels at the centrosome and promotes ciliogenesis. Both proteins mobilize ARP2 to centrosomes and interact with ciliogenesis/cilia-length regulators. LUZP1 and EPLIN represent novel players in actin-dependent centrosome to basal body conversion.","method":"Proximity-ligation (BioID), siRNA knockdown, co-immunoprecipitation, cilia formation quantification, immunofluorescence","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID plus Co-IP plus loss-of-function with cilia phenotype, single lab","pmids":["32496561"],"is_preprint":false},{"year":2021,"finding":"Rab40b-Cullin5 complex ubiquitylates and degrades EPLIN. Rab40b directly binds EPLIN. Rab40b-Cullin5-dependent localized ubiquitylation and degradation of EPLIN at the leading edge regulates focal adhesion and cytoskeletal dynamics, promoting cell migration and invasion.","method":"Co-immunoprecipitation, ubiquitylation assay, siRNA knockdown, focal adhesion dynamics assay, invasion assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus ubiquitylation assay plus loss-of-function with defined cytoskeletal and invasion phenotypes, multiple orthogonal methods","pmids":["33999101"],"is_preprint":false},{"year":2022,"finding":"Lima1 expression is controlled by the naive pluripotency circuit in mouse embryonic stem cells and is required for suppression of membrane blebbing and proper mitochondrial energetics. Forced Lima1 expression enables primed pluripotent stem cells to integrate into pre-implantation embryos.","method":"Lima1 knockout ESC lines, live imaging, mitochondrial function assays, chimera assay, embryo injection","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with specific cellular phenotypes (blebbing, mitochondrial function), single lab","pmids":["35105859"],"is_preprint":false},{"year":2022,"finding":"LIMA1 inhibits the Wnt/β-catenin signaling pathway in HCC cells by binding to BMI1 and inducing its destabilization. LIMA1 overexpression suppresses HCC cell proliferation and metastasis in vitro and in vivo.","method":"Co-immunoprecipitation, western blot, in vitro and in vivo functional assays, siRNA knockdown","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing LIMA1-BMI1 interaction with functional consequences, single lab","pmids":["36497115"],"is_preprint":false},{"year":2023,"finding":"EPLIN-beta (but not EPLIN-alpha) is a substrate of ornithine decarboxylase antizyme 1 (Az1/OAZ1). The interaction between EPLIN-beta and Az1 is indirect, and EPLIN-beta is degraded by Az1 in a ubiquitination-independent manner. Az1 absence leads to elevated EPLIN-beta levels and enhanced cellular migration.","method":"Quantitative proteomics, co-immunoprecipitation, ubiquitination assay, Az1 knockout, migration assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-based identification followed by Co-IP and ubiquitination assay plus functional migration phenotype, single lab","pmids":["37325974"],"is_preprint":false},{"year":2023,"finding":"MAD2 interferes with binding of the deubiquitinase USP44 to LIMA1 by sequestering USP44 in the nucleus, leading to impaired USP44/LIMA1 complex formation and enhanced K48-linked ubiquitination of LIMA1, promoting its degradation and activating the IGF1R/PI3K/AKT pathway.","method":"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), subcellular fractionation, siRNA knockdown, PI3K/AKT signaling analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing USP44-LIMA1 complex, ubiquitination linkage-specific assay, and MAD2-dependent disruption, single lab","pmids":["37752233"],"is_preprint":false},{"year":2024,"finding":"RNF40, an E3 ubiquitin ligase, directly mediates ubiquitination of LIMA1 and promotes its proteasome-dependent degradation in the cytoplasm. The 1-166 aa fragment of LIMA1 is required for interaction with RNF40. RNF40-mediated LIMA1 degradation suppresses lipid accumulation.","method":"Co-immunoprecipitation, ubiquitination assay, domain truncation mapping, proteasome inhibitor treatment, lipid accumulation assay","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay with domain mapping, single lab","pmids":["38909032"],"is_preprint":false},{"year":2024,"finding":"LIMA1 O-GlcNAcylation at T662, catalyzed by OGT (facilitated by HCF1 upregulation under steatosis), inhibits ubiquitin-dependent degradation of LIMA1 and enhances hepatocyte lipid deposition by activating β-catenin/FASn signaling. AAV-mediated T662 mutant LIMA1 (non-O-GlcNAcylatable) decreases steatosis, insulin resistance, inflammation and fibrosis in LIMA1 hepatocyte-specific knockout mice.","method":"O-GlcNAcylation mapping (site-specific), site-directed mutagenesis (T662A), AAV rescue in HKO mice, ubiquitination assay, western blot, metabolic phenotyping","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific PTM mutagenesis combined with in vivo rescue experiment and multiple orthogonal mechanistic assays","pmids":["39921472"],"is_preprint":false},{"year":2024,"finding":"Cytoplasmic p62 (SQSTM1) interacts with EPLIN via GST pulldown and stabilizes EPLIN protein, enhancing EPLIN-dependent migration and invasion of esophageal squamous cell carcinoma cells.","method":"GST pulldown, co-immunoprecipitation, siRNA knockdown, protein stability assay, migration/invasion assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GST pulldown plus Co-IP confirming interaction, functional rescue experiment, single lab","pmids":["38185251"],"is_preprint":false},{"year":2024,"finding":"STUB1 (E3 ubiquitin ligase) is recruited by BPNT1 to induce proteasomal degradation of LIMA1, promoting EMT and TNBC progression. Re-expression of LIMA1 in BPNT1-overexpressing cells partially attenuates BPNT1-driven EMT.","method":"Co-immunoprecipitation, ubiquitination/proteasomal degradation assay, re-expression rescue experiment, in vitro and in vivo functional assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing BPNT1-STUB1-LIMA1 axis with rescue experiment and in vivo validation, single lab","pmids":["41540000"],"is_preprint":false},{"year":2024,"finding":"SEPT9 directly interacts with the single LIM domain of EPLIN. SEPT9 depletion decreases cell adhesion and migration; these defects can be partly rescued by increased EPLIN levels. SEPT9 and EPLIN together regulate actin-related filopodia, stress fiber organization, and focal adhesion size.","method":"SEPT9 knockout fibroblasts, co-immunoprecipitation, LIM-domain interaction mapping, rescue by EPLIN overexpression, focal adhesion analysis, actin imaging","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with rescue plus domain-mapped interaction, single lab","pmids":["38719752"],"is_preprint":false},{"year":2025,"finding":"LIMA1 associates with IRBIT and the intestinal Cl-/HCO3- exchanger SLC26A3 (DRA). cAMP/ATP stimulation increases co-precipitation of LIMA1 with both IRBIT and DRA. Knockdown of LIMA1 reduces cAMP plus ATP stimulation of DRA but does not alter basal DRA activity, establishing a DRA-IRBIT-LIMA1 plasma membrane complex required for acute stimulation of DRA.","method":"Co-immunoprecipitation, siRNA knockdown, functional DRA transport assay (BCECF fluorometry), surface biotinylation","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing the complex plus loss-of-function with functional transporter phenotype, single lab","pmids":["40569378"],"is_preprint":false},{"year":2026,"finding":"CDK1 phosphorylates EPLIN at Ser203 during mitosis. This phosphorylation is required for EPLIN to maintain proper actin cortex architecture and correct spindle positioning. EPLIN-pSer203 enhances the ability of EPLIN to reorganize branched actin filaments into astral structures in vitro, sequestering actin bundles and Arp2/3 complex to prevent excessive de novo actin polymerization. EPLIN depletion causes spindle deviation and metaphase arrest with abnormal cortical actin accumulation and blebbing.","method":"In vitro kinase assay (CDK1), site-directed mutagenesis (Ser203), phospho-specific antibodies, siRNA depletion, live-cell imaging, in vitro actin reconstitution assay","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis, in vitro actin reconstitution, and multiple functional cellular phenotypes","pmids":["42237003"],"is_preprint":false},{"year":2026,"finding":"LIMA1 expression is sufficient to drive elongation of microvilli in epithelial cells and formation and stabilization of exaggerated filopodia in non-epithelial cells. Individual filopodia in LIMA1-overexpressing cells exhibit merging dynamics that enable formation of large core actin bundles from smaller precursors. In tuft cells, LIMA1 is restricted to the basal/rootlet ends of actin bundles.","method":"Live-cell and electron microscopy, LIMA1 overexpression, filopodia dynamics analysis, LIMA1-specific localization in tuft cells","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple microscopy modalities with overexpression and direct visualization of bundle dynamics, single lab","pmids":["42202079"],"is_preprint":false},{"year":2024,"finding":"EPLINα localizes to Rab21-positive recycling endosomes in an actin-binding-dependent manner and interacts with Rab21. EPLINα supports beta1-integrin recycling and cell migration. Coronin 1C was identified as an EPLINα proximal protein at Rab21-containing endosomes in an EPLINα-dependent manner. EPLINβ resides on actin stress fibers and does not show this endosomal localization.","method":"BioID proximity labeling, co-localization (confocal), co-immunoprecipitation, integrin recycling assay, migration assay, isoform-specific expression constructs","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID plus Co-IP plus functional recycling assay; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2024.06.27.600789"],"is_preprint":true}],"current_model":"LIMA1/EPLIN is an actin-binding and cross-linking protein with at least two actin-binding sites that inhibits filament depolymerization, suppresses Arp2/3-mediated branching nucleation, and bundles filaments; it links the cadherin-catenin (and VE-cadherin-catenin) complex to F-actin via α-catenin to maintain the epithelial adherens junction/adhesion belt, and is regulated by multiple post-translational modifications (ERK-mediated phosphorylation at Ser360/Ser602/Ser692 that reduces actin affinity, CDK1 phosphorylation at Ser203 required for mitotic cortex remodeling and spindle positioning, O-GlcNAcylation at T662 that stabilizes the protein and promotes lipid deposition, and ubiquitin-mediated proteasomal degradation by E3 ligases RNF40, STUB1, and the Rab40b-Cullin5 complex); it also bridges NPC1L1 to a myosin Vb transport complex for intestinal cholesterol absorption, participates in cytokinesis by stabilizing myosin II and Sept2 at the cleavage furrow, restricts primary ciliogenesis by stabilizing actin filaments, and can be proteolytically cleaved by the API2-MALT1 paracaspase to generate an oncogenic LIM-domain fragment."},"narrative":{"mechanistic_narrative":"LIMA1 (EPLIN) is an actin-binding and cross-linking protein that governs the stability and architecture of the actin cytoskeleton, acting broadly as a suppressor of cell motility, invasion, and proliferation [PMID:10618726, PMID:12566430]. Biochemically, purified LIMA1 uses at least two actin-binding sites to inhibit filament depolymerization, bundle filaments, and block Arp2/3-mediated branching nucleation, thereby favoring stable structures such as stress fibers over dynamic membrane ruffles [PMID:12566430]. Through direct binding to α-catenin it links the cadherin–catenin and VE-cadherin–catenin complexes to F-actin, forming the molecular bridge required to build and maintain the epithelial and endothelial adhesion belt (zonula adherens) and to recruit vinculin under junctional tension [PMID:18093941, PMID:22194609, PMID:21844208]. Its two isoforms are functionally and transcriptionally distinct: EPLIN-alpha is serum/RhoA- and MAL/MRTF-SRF-inducible and terminates membrane protrusion growth via Arp2/3, whereas the more stable EPLIN-beta stabilizes stress fibers [PMID:10806352, PMID:20236507, PMID:31644899]. LIMA1 abundance and actin affinity are tightly controlled by post-translational modification, including ERK phosphorylation that lowers actin affinity and triggers ubiquitin-proteasomal turnover to permit stress-fiber disassembly and migration [PMID:17875928, PMID:23188829], CDK1 phosphorylation at Ser203 that remodels the mitotic actin cortex for correct spindle positioning [PMID:42237003], O-GlcNAcylation at T662 that stabilizes the protein and drives hepatocyte lipid deposition [PMID:39921472], and degradation by multiple E3 ligases including the Rab40b-Cullin5 complex, RNF40, and STUB1 [PMID:33999101, PMID:38909032, PMID:41540000]. Beyond junctions, LIMA1 functions in cytokinesis by promoting accumulation of active myosin II and Sept2 at the cleavage furrow [PMID:19221476], restricts primary ciliogenesis together with LUZP1 by stabilizing actin [PMID:32496561], and bridges NPC1L1 to a myosin Vb transport complex to drive intestinal cholesterol absorption, with Lima1-deficient mice resistant to diet-induced hypercholesterolemia [PMID:29880681]. As a tumor suppressor it is transcriptionally induced by p53 and repressed by DNp73, and can be proteolytically inactivated by the API2-MALT1 paracaspase to generate an oncogenic LIM-domain fragment [PMID:25569716, PMID:28093207, PMID:24135282].","teleology":[{"year":1999,"claim":"Established LIMA1/EPLIN as a cytoskeleton-associated LIM domain protein whose overexpression restrains proliferation, framing it as a candidate growth suppressor tied to actin.","evidence":"Overexpression and immunofluorescence co-localization with F-actin in cultured cells","pmids":["10618726"],"confidence":"Medium","gaps":["No biochemical mechanism for actin interaction","Did not distinguish isoform-specific roles functionally"]},{"year":2000,"claim":"Showed the two isoforms arise from distinct promoters under different transcriptional control, explaining how EPLIN-alpha is coupled to Rho/serum signaling while EPLIN-beta is constitutive.","evidence":"5' RACE, S1 protection, promoter-reporter assays with serum and RhoA","pmids":["10806352"],"confidence":"Medium","gaps":["Did not define functional consequences of differential expression","Transcription factor identity at the SRE not resolved here"]},{"year":2002,"claim":"Mapped the growth-suppressive function to the N-terminal actin-localizing region, establishing that cytoskeletal targeting, not the LIM domain, drives suppression of anchorage-independent growth.","evidence":"Truncation mutant retroviral transduction with colony formation assays","pmids":["11950948"],"confidence":"Medium","gaps":["Biochemical activity of the N-terminus on actin not yet defined","LIM domain function left unexplained"]},{"year":2003,"claim":"Defined the core biochemical activity: LIMA1 cross-links and stabilizes actin filaments and inhibits Arp2/3 branching, providing the mechanistic basis for its bias toward stable cytoskeletal structures.","evidence":"In vitro reconstitution with purified protein (depolymerization, bundling, Arp2/3 branching) plus cellular gain/loss of function","pmids":["12566430"],"confidence":"High","gaps":["Did not identify in vivo binding partners beyond actin","Regulation of the two actin-binding sites unresolved"]},{"year":2007,"claim":"Identified LIMA1 as the α-catenin–F-actin bridge that builds the adhesion belt, answering how the cadherin-catenin complex couples to a stable actin ring.","evidence":"RNAi, reciprocal Co-IP, and live imaging in epithelial cells","pmids":["18093941"],"confidence":"High","gaps":["Mechanical regulation of the bridge not addressed","Did not test endothelial/VE-cadherin junctions"]},{"year":2007,"claim":"Placed LIMA1 under ERK control, showing phosphorylation lowers actin affinity to permit stress-fiber disassembly and migration, providing a switch between stable and dynamic cytoskeletal states.","evidence":"In vitro kinase assay, phospho-site mutagenesis, PDGF-stimulated migration assays","pmids":["17875928"],"confidence":"High","gaps":["Link between phosphorylation and protein turnover not yet made","Did not address junctional pools of LIMA1"]},{"year":2008,"claim":"Connected LIMA1 to cancer cell behavior, demonstrating it suppresses invasion/motility downstream of ERK signaling.","evidence":"Overexpression with invasion/migration assays and ERK inhibition in breast cancer cells","pmids":["18796137"],"confidence":"Medium","gaps":["Molecular target of ERK-LIMA1 axis in invasion not dissected","In vivo relevance not tested"]},{"year":2010,"claim":"Showed actin-MAL/MRTF-SRF signaling drives EPLIN-alpha transcription, establishing a feedback link between actin dynamics and isoform expression.","evidence":"Transcriptomics with actin drugs, promoter-reporter, ChIP, dominant-negative MAL","pmids":["20236507"],"confidence":"Medium","gaps":["Physiological trigger of this feedback in vivo unclear","EPLIN-beta regulatory inputs not identified"]},{"year":2009,"claim":"Extended LIMA1 function to cytokinesis, showing it concentrates contractile machinery (myosin II, Sept2, RhoA/Cdc42) at the cleavage furrow to prevent multinucleation.","evidence":"siRNA knockdown, co-localization, and multinucleation scoring","pmids":["19221476"],"confidence":"Medium","gaps":["Direct versus indirect interaction with furrow components not resolved","No biochemical binding data for myosin II/Sept2"]},{"year":2011,"claim":"Resolved the mechanics and partners of junctional LIMA1 across endothelium and epithelium, showing it tethers VE-cadherin/catenin to actin and that junctional tension retains it to recruit vinculin and maintain zonula adherens morphology.","evidence":"Co-IP/GST pulldown, tension manipulation, αE-catenin–EPLIN fusion, angiogenesis assays","pmids":["22194609","21844208","21625216"],"confidence":"High","gaps":["Force-sensing mechanism that ejects LIMA1 under tension not molecularly defined","Relationship between AJ and EMT roles incompletely integrated"]},{"year":2012,"claim":"Unified phosphorylation with degradation, showing EGF/ERK-dependent phosphorylation of LIMA1 triggers its ubiquitination and proteasomal turnover, explaining acute downregulation during motility induction.","evidence":"EGF stimulation, ERK inhibition, ubiquitination assay, phospho-site mutagenesis, cycloheximide chase","pmids":["23188829"],"confidence":"High","gaps":["E3 ligase responsible not identified here","Spatial control of degradation unaddressed"]},{"year":2017,"claim":"Established transcriptional tumor-suppressor circuitry, with p53 directly inducing LIMA1 to suppress invasion and DNp73 repressing it to activate pro-metastatic IGF1R-AKT/STAT3 signaling.","evidence":"ChIP-seq, nutlin-3a activation, knockdown epistasis, xenografts (with #24135282)","pmids":["28093207","24135282"],"confidence":"Medium","gaps":["Mechanism linking LIMA1 loss to IGF1R activation not biochemically defined","Contribution of cytoskeletal versus signaling roles to suppression unresolved"]},{"year":2015,"claim":"Demonstrated a non-cell-autonomous role in epithelial defense, with LIMA1 driving apical extrusion of RasV12-transformed cells upstream of Cav-1 and myosin-II/PKA.","evidence":"siRNA, co-culture extrusion assays, epistasis","pmids":["25609711"],"confidence":"Medium","gaps":["Direct molecular interactions in extrusion not fully mapped","Generality across oncogenes unclear"]},{"year":2015,"claim":"Linked LIMA1 to integrin adhesion, showing PINCH-1-dependent recruitment to adhesion sites is required for keratinocyte spreading and migration.","evidence":"PINCH-1 interactome MS, Co-IP, conditional knockout mouse, adhesion/migration assays","pmids":["25609703"],"confidence":"High","gaps":["Whether LIMA1 directly binds PINCH-1 not established","Relationship to junctional pool unclear"]},{"year":2015,"claim":"Revealed LIMA1 as a substrate of the API2-MALT1 paracaspase, showing oncogenic cleavage generates a LIM-domain-only fragment, a mechanism of tumor-suppressor inactivation in MALT lymphoma.","evidence":"Co-IP, in vitro cleavage with active-site mutagenesis, xenografts, primary tumor analysis","pmids":["25569716"],"confidence":"High","gaps":["Function of the liberated LIM fragment not fully characterized","Cleavage relevance outside MALT lymphoma untested"]},{"year":2018,"claim":"Uncovered a metabolic function, showing LIMA1 bridges NPC1L1 to a myosin Vb transport complex to drive intestinal cholesterol absorption, with knockout mice and a human variant validating the pathway.","evidence":"Co-IP, knockout mouse phenotyping, human frameshift variant, brush-border localization","pmids":["29880681"],"confidence":"High","gaps":["Structural basis of NPC1L1–LIMA1–myosin Vb assembly unknown","Tissue-specific regulation of this complex unresolved"]},{"year":2019,"claim":"Defined isoform-specific cytoskeletal mechanisms, showing EPLIN-alpha targets and terminates protrusions via Arp2/3 while the slower-turnover EPLIN-beta stabilizes stress fibers under shear stress.","evidence":"FRAP, isoform-specific knockdown, Arp2/3 interaction assays, shear stress in endothelial cells","pmids":["31644899"],"confidence":"High","gaps":["Structural determinants of isoform divergence not mapped","In vivo isoform-specific phenotypes not tested"]},{"year":2020,"claim":"Identified a ciliogenesis-restricting function, with LIMA1 and LUZP1 stabilizing actin at the centrosome to limit primary cilia formation.","evidence":"BioID, Co-IP, siRNA, cilia quantification","pmids":["32496561"],"confidence":"Medium","gaps":["Direct LIMA1–LUZP1 binding interface undefined","Mechanism of MyosinVa regulation at centrosome unclear"]},{"year":2021,"claim":"Showed spatially restricted degradation as a migration control mechanism, with the Rab40b-Cullin5 complex ubiquitylating LIMA1 at the leading edge to regulate focal adhesion and cytoskeletal dynamics.","evidence":"Co-IP, ubiquitylation assay, siRNA, focal adhesion and invasion assays","pmids":["33999101"],"confidence":"High","gaps":["Recognition determinants on LIMA1 for Rab40b not mapped","Integration with ERK-driven turnover unresolved"]},{"year":2022,"claim":"Connected LIMA1 to pluripotency and cell mechanics, showing the naive circuit requires Lima1 to suppress blebbing and support mitochondrial energetics, enabling embryo integration.","evidence":"Knockout ESCs, live imaging, mitochondrial assays, chimera/embryo injection","pmids":["35105859"],"confidence":"Medium","gaps":["Mechanistic link to mitochondrial function unexplained","Whether actin-bundling activity underlies the phenotype untested"]},{"year":2022,"claim":"Added a signaling-suppressor function, showing LIMA1 binds BMI1 to destabilize it and inhibit Wnt/β-catenin signaling in HCC.","evidence":"Co-IP, western blot, in vitro/in vivo functional assays","pmids":["36497115"],"confidence":"Medium","gaps":["Direct versus adaptor-mediated BMI1 destabilization unclear","Reconciliation with cytoskeletal role not addressed"]},{"year":2023,"claim":"Expanded post-translational regulation, identifying ubiquitin-independent degradation of EPLIN-beta by antizyme Az1/OAZ1 that constrains migration.","evidence":"Quantitative proteomics, Co-IP, ubiquitination assay, Az1 knockout migration assays","pmids":["37325974"],"confidence":"Medium","gaps":["Indirect interaction adaptor not identified","Isoform-selectivity mechanism undefined"]},{"year":2023,"claim":"Revealed a deubiquitination axis, showing USP44 stabilizes LIMA1 and MAD2 sequesters USP44 to promote K48-ubiquitination and degradation, activating IGF1R/PI3K/AKT.","evidence":"Co-IP, K48-linkage ubiquitination assay, fractionation, signaling analysis","pmids":["37752233"],"confidence":"Medium","gaps":["E3 ligase opposing USP44 not identified","Physiological context of MAD2-USP44 competition unclear"]},{"year":2024,"claim":"Consolidated a multi-ligase degradation network and a metabolic-PTM axis, identifying RNF40 and STUB1 as LIMA1 E3 ligases and O-GlcNAcylation at T662 as a stabilizing modification driving hepatic lipid deposition.","evidence":"Co-IP, ubiquitination assays, domain mapping, site-specific O-GlcNAc mutagenesis with AAV rescue in knockout mice","pmids":["38909032","41540000","39921472"],"confidence":"High","gaps":["Hierarchy/competition among the multiple E3 ligases unresolved","How O-GlcNAc and ubiquitin sites interplay structurally unknown"]},{"year":2024,"claim":"Added stabilizing partners and new actin/transport functions, including p62/SQSTM1 stabilization promoting invasion, SEPT9 binding the LIM domain to organize adhesions, EPLINα-Rab21 endosomal control of integrin recycling, and LIMA1-driven microvillus/filopodia elongation.","evidence":"GST pulldown, Co-IP, LIM-domain mapping, SEPT9 knockout rescue, BioID, integrin recycling and microscopy assays","pmids":["38185251","38719752","42202079","bio_10.1101_2024.06.27.600789"],"confidence":"Medium","gaps":["Direct structural interactions for several partners undefined","Endosomal recycling role from preprint awaits peer review"]},{"year":2025,"claim":"Identified a transporter-regulatory role, showing LIMA1 forms a plasma-membrane DRA-IRBIT-LIMA1 complex required for acute cAMP/ATP stimulation of SLC26A3 anion exchange.","evidence":"Co-IP, siRNA, BCECF transport assays, surface biotinylation","pmids":["40569378"],"confidence":"Medium","gaps":["Direct binding interfaces within the complex not mapped","In vivo relevance to intestinal physiology untested"]},{"year":2026,"claim":"Defined a mitotic regulatory mechanism, showing CDK1 phosphorylation at Ser203 reorganizes branched actin into astral structures to maintain cortical architecture and correct spindle positioning.","evidence":"In vitro CDK1 kinase assay, Ser203 mutagenesis, phospho-antibodies, live imaging, in vitro actin reconstitution","pmids":["42237003"],"confidence":"High","gaps":["How pSer203 alters the two actin-binding sites structurally unresolved","Integration with cytokinesis role not addressed"]},{"year":null,"claim":"How LIMA1's distinct functional pools — adherens junctions, focal/integrin adhesions, recycling endosomes, brush-border transport complexes, and the mitotic cortex — are coordinately partitioned and switched by its dense network of post-translational modifications remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model of how PTMs gate actin-binding-site usage","Competition/hierarchy among multiple E3 ligases and deubiquitinases unmapped","Mechanistic basis for isoform-specific localization to distinct compartments incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3,20,33,34]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,9,11,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,33]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,3,20]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,9,18,32]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[35]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[21]}],"pathway":[{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[4,9,11]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,33]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[18,28]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,13,24]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[12,22,27,30]}],"complexes":["cadherin-catenin adherens junction complex","NPC1L1-myosin Vb cholesterol transport complex","DRA-IRBIT-LIMA1 plasma membrane complex","Rab40b-Cullin5 ubiquitin ligase complex"],"partners":["CTNNA1","ACTB","LIMS1","SEPT9","NPC1L1","RAB40B","RNF40","USP44"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UHB6","full_name":"LIM domain and actin-binding protein 1","aliases":["Epithelial protein lost in neoplasm"],"length_aa":759,"mass_kda":85.2,"function":"Actin-binding protein involved in actin cytoskeleton regulation and dynamics. Increases the number and size of actin stress fibers and inhibits membrane ruffling. Inhibits actin filament depolymerization. Bundles actin filaments, delays filament nucleation and reduces formation of branched filaments (PubMed:12566430, PubMed:33999101). Acts as a negative regulator of primary cilium formation (PubMed:32496561). Plays a role in cholesterol homeostasis. Influences plasma cholesterol levels through regulation of intestinal cholesterol absorption. May act as a scaffold protein by regulating NPC1L1 transportation, an essential protein for cholesterol absorption, to the plasma membrane by recruiting MYO5B to NPC1L1, and thus facilitates cholesterol uptake (By similarity)","subcellular_location":"Cytoplasm; Cell junction, focal adhesion; Cytoplasm, cytoskeleton; Cytoplasm, cytoskeleton, stress fiber; Cell membrane; Cell projection, ruffle; Cell projection, lamellipodium","url":"https://www.uniprot.org/uniprotkb/Q9UHB6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LIMA1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CALD1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/LIMA1","total_profiled":1310},"omim":[{"mim_id":"618079","title":"LOW DENSITY LIPOPROTEIN CHOLESTEROL LEVEL QUANTITATIVE TRAIT LOCUS 8; LDLCQ8","url":"https://www.omim.org/entry/618079"},{"mim_id":"608364","title":"LIM DOMAIN AND ACTIN-BINDING PROTEIN 1; LIMA1","url":"https://www.omim.org/entry/608364"},{"mim_id":"601422","title":"LEUCINE ZIPPER PROTEIN 1; LUZP1","url":"https://www.omim.org/entry/601422"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Actin filaments","reliability":"Approved"},{"location":"Focal adhesion sites","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/LIMA1"},"hgnc":{"alias_symbol":["EPLIN"],"prev_symbol":[]},"alphafold":{"accession":"Q9UHB6","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHB6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHB6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHB6-F1-predicted_aligned_error_v6.png","plddt_mean":51.72},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LIMA1","jax_strain_url":"https://www.jax.org/strain/search?query=LIMA1"},"sequence":{"accession":"Q9UHB6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UHB6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UHB6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHB6"}},"corpus_meta":[{"pmid":"18093941","id":"PMC_18093941","title":"EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin 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both EPLIN-alpha and EPLIN-beta isoforms localize to actin stress fibers.\",\n      \"method\": \"Overexpression, immunofluorescence co-localization with F-actin\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, direct localization and proliferation phenotype with overexpression but no detailed mechanistic dissection\",\n      \"pmids\": [\"10618726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"EPLIN-alpha and EPLIN-beta are generated from distinct promoters of a single gene; EPLIN-alpha expression is serum-inducible via a consensus serum response element, and its promoter activity is enhanced by activated RhoA, whereas EPLIN-beta is not serum-regulated.\",\n      \"method\": \"5' RACE, S1 nuclease protection, promoter-reporter constructs, serum stimulation, RhoA transfection\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in a single lab establishing isoform-specific transcriptional regulation\",\n      \"pmids\": [\"10806352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The N-terminal region of EPLIN is necessary for both localization to the actin cytoskeleton and suppression of anchorage-independent growth of EWS/Fli-1- and Cdc42-transformed cells; the LIM domain and C-terminal region are dispensable for these functions. Inhibition of anchorage-independent growth requires EPLIN's actin cytoskeletal localization.\",\n      \"method\": \"Retroviral transduction of truncation mutants, colony formation assay, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutagenesis with defined cellular phenotype in single lab\",\n      \"pmids\": [\"11950948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Purified recombinant EPLIN inhibits actin filament depolymerization, cross-links filaments into bundles via at least two actin-binding sites, and inhibits Arp2/3-mediated branching nucleation without affecting spontaneous polymerization kinetics or barbed-end elongation. EPLIN promotes stable actin filament structures (stress fibers) at the expense of dynamic structures (membrane ruffles).\",\n      \"method\": \"In vitro actin polymerization/depolymerization assays, actin bundling assay, Arp2/3 branching assay with purified recombinant EPLIN, cell overexpression/knockdown\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple in vitro reconstitution assays with purified protein, mutagenesis-informed domain analysis, orthogonal cellular experiments\",\n      \"pmids\": [\"12566430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EPLIN couples with alpha-catenin and links the cadherin-catenin complex to F-actin, forming the molecular bridge required for the adhesion belt (zonula adherens). EPLIN depletion disorganizes the adhesion belt and converts it to zipper-like junctions with radially arranged actin fibers without affecting non-junctional actin fibers.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation, immunofluorescence, live imaging of epithelial cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus loss-of-function with specific morphological phenotype, replicated in multiple cell systems\",\n      \"pmids\": [\"18093941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ERK phosphorylates EPLIN at Ser360, Ser602, and Ser692 in vitro and in intact cells; phosphorylation of the C-terminal region reduces EPLIN's affinity for actin filaments. ERK-mediated phosphorylation of EPLIN is required for PDGF-induced stress fiber disassembly, membrane ruffling, and cell migration; a non-phosphorylatable EPLIN mutant blocks these processes.\",\n      \"method\": \"In vitro kinase assay, phospho-specific antibodies, site-directed mutagenesis (Ser→Ala), wound healing and migration assays, PDGF stimulation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis plus functional cell-based validation with phosphomimetic/non-phosphorylatable mutants\",\n      \"pmids\": [\"17875928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Overexpression of EPLIN-alpha in breast cancer cells renders them less invasive and less motile; an ERK inhibitor abolishes the effects of EPLIN expression on cell behavior, placing EPLIN downstream of ERK signaling in motility control.\",\n      \"method\": \"Transfection/overexpression, in vitro invasion assay, Electric Cell Impedance Sensing migration assay, ERK inhibitor treatment\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional loss/gain with pharmacological pathway inhibition, single lab\",\n      \"pmids\": [\"18796137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EPLIN localizes to the cleavage furrow during cytokinesis, possibly via association with contractile ring components myosin II and septin Sept2. EPLIN depletion leads to multinucleated cells due to inefficient accumulation of active myosin II (MRLC-S19), Sept2, RhoA, and Cdc42 at the cleavage furrow.\",\n      \"method\": \"Immunofluorescence, siRNA knockdown, co-localization with myosin II and Sept2, multinucleation assay\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional cytokinesis phenotype; association with contractile ring components by co-localization, single lab\",\n      \"pmids\": [\"19221476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EPLIN-alpha transcription is regulated by actin-MAL/MRTF-SRF signaling; MAL/MRTF coactivator binds an SRF consensus site in the EPLIN-alpha promoter, and its recruitment is enhanced upon induction. Monomeric actin maintains a repressive MAL-actin complex. EPLIN-beta isoform is not regulated by this pathway.\",\n      \"method\": \"Transcriptome analysis with actin-binding drugs, promoter-reporter assay, ChIP, dominant negative MAL overexpression, MRTF knockdown\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus promoter reporters plus loss/gain of function, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"20236507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EPLIN interacts directly with alpha-catenin and tethers the VE-cadherin/catenin complex to the actin cytoskeleton in endothelial cells. EPLIN depletion delocalizes vinculin from junctions. EPLIN-depleted endothelial cells have reduced capacity to form pseudocapillary networks due to breakage events, establishing EPLIN as a mechanotransmitter for vinculin recruitment to adherens junctions independent of actomyosin tension.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, siRNA knockdown, Matrigel angiogenesis assay, blebbistatin treatment, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding confirmed by reciprocal Co-IP and GST pulldown, functional validation with loss-of-function phenotype, orthogonal methods\",\n      \"pmids\": [\"22194609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EPLIN depletion in prostate cancer cells causes disassembly of adherens junctions, actin remodeling, and activation of beta-catenin signaling, demonstrating EPLIN as a negative regulator of EMT.\",\n      \"method\": \"siRNA knockdown, biochemical analysis, beta-catenin reporter assay, microarray expression analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined molecular phenotype (adherens junction, beta-catenin signaling), single lab\",\n      \"pmids\": [\"21625216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Junctional tension is important for retaining EPLIN at adherens junctions; lateral actin fiber force inhibits EPLIN-AJ association. EPLIN association with αE-catenin is required for zonula adherens maintenance; vinculin cooperates with EPLIN to maintain ZA morphology. A fusion of αE-catenin and EPLIN promotes ZA but not punctate AJ formation.\",\n      \"method\": \"Live imaging, tension manipulation, EPLIN-catenin fusion protein expression, RNAi, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal experiments including force manipulation, fusion proteins, and loss-of-function with specific morphological phenotypes\",\n      \"pmids\": [\"21844208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EGF promotes phosphorylation, ubiquitination, and proteasomal degradation of EPLIN through ERK1/2-dependent signaling. Serine residues S362 and S604 are ERK1/2 phosphorylation sites; point mutation of these renders EPLIN resistant to EGF-induced protein turnover.\",\n      \"method\": \"EGF stimulation, ERK1/2 inhibitor treatment, ubiquitination assay, site-directed mutagenesis (S362A, S604A), cycloheximide chase, western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of phosphorylation sites combined with ubiquitination assay and degradation kinetics in a single focused study\",\n      \"pmids\": [\"23188829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DNp73 drives cancer cell migration/invasion by repressing EPLIN (LIMA1) expression. EPLIN directly links to IGF1R-AKT/STAT3 activation; EPLIN loss activates this signaling axis. p73/DNp73 transcriptionally regulates LIMA1/EPLIN, and the DNp73-EPLIN axis controls IGF1R-dependent signaling to initiate metastasis.\",\n      \"method\": \"Knockdown of endogenous DNp73, tumor xenografts, biochemical signaling analysis (AKT/STAT3 phosphorylation), epistasis experiments\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with mechanistic pathway placement and in vivo xenograft validation, single lab\",\n      \"pmids\": [\"24135282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"API2-MALT1 fusion protein induces paracaspase-mediated proteolytic cleavage of LIMA1/EPLIN tumor suppressor. API2-dependent binding targets LIMA1 for MALT1 paracaspase cleavage, generating an oncogenic LIM domain-only (LMO) fragment. Primary MALT lymphomas with API2-MALT1 fusion uniquely show LIMA1 cleavage fragments.\",\n      \"method\": \"Co-immunoprecipitation, in vitro cleavage assay, mutagenesis of paracaspase active site, tumor xenograft, primary lymphoma tissue analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical cleavage assay, mutagenesis of active site, validated in primary patient tumor material\",\n      \"pmids\": [\"25569716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EPLIN (LIMA1) accumulates in RasV12-transformed cells surrounded by normal cells and is required for their apical extrusion. EPLIN functions upstream of Cav-1 and promotes its enrichment in transformed cells. EPLIN regulates non-cell-autonomous activation of myosin-II and PKA in transformed cells, and affects accumulation of filamin A in neighboring normal cells.\",\n      \"method\": \"siRNA knockdown, immunofluorescence, co-culture assay, epithelial extrusion quantification\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined extrusion phenotype and epistasis placing EPLIN upstream of Cav-1, single lab\",\n      \"pmids\": [\"25609711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PINCH-1 (LIMS1) associates with EPLIN at integrin adhesion sites. EPLIN localizes to integrin adhesion sites in a PINCH-1-dependent manner. EPLIN depletion severely attenuates keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions.\",\n      \"method\": \"PINCH-1 interactome isolation (MS), co-immunoprecipitation, PINCH-1 conditional knockout mouse model, siRNA knockdown, adhesion and migration assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — interactome MS identification followed by Co-IP confirmation, in vivo genetic model, and functional loss-of-function assays\",\n      \"pmids\": [\"25609703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"p53 transcriptionally induces LIMA1/EPLIN expression via two p53 response elements identified by ChIP-seq in the LIMA1 gene. p53-induced LIMA1 suppresses cancer cell invasion; LIMA1 knockdown partially inhibits p53-induced suppression of invasion.\",\n      \"method\": \"ChIP-seq, cDNA microarray, nutlin-3a treatment (p53 activation), siRNA knockdown, invasion assay\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq identification of response elements plus functional epistasis via knockdown, single lab\",\n      \"pmids\": [\"28093207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LIMA1 (EPLIN) bridges NPC1L1, the essential cholesterol absorption protein, to a transport complex containing myosin Vb, facilitating intestinal cholesterol uptake. In mice, LIMA1 is expressed in the small intestine and localizes on the brush border membrane. Lima1-deficient mice display reduced cholesterol absorption and resistance to diet-induced hypercholesterolemia.\",\n      \"method\": \"Co-immunoprecipitation, genetic knockout mouse model, rare human frameshift variant analysis, subcellular fractionation/localization\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP establishing protein complex, genetic knockout model with defined phenotype, human genetic validation\",\n      \"pmids\": [\"29880681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Paxillin binds to EPLIN specifically in mixed cultures of normal and RasV12-transformed cells. Paxillin, plectin, and EPLIN mutually influence each other's non-cell-autonomous accumulation. Paxillin acts downstream of plectin-EPLIN complex and promotes microtubule acetylation by suppressing HDAC6 activity to drive apical extrusion of transformed cells.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, HDAC6 activity assay, tubulin acetylation measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus loss-of-function with defined molecular pathway, single lab\",\n      \"pmids\": [\"29391412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPLIN-alpha and EPLIN-beta isoforms have distinct spatiotemporal interactions with actin: EPLIN-alpha is targeted to membrane protrusions and terminates their growth via interaction with Arp2/3 complex, while EPLIN-beta stabilizes stress fibers. FRAP analyses demonstrate EPLIN-beta has a lower turnover rate than EPLIN-alpha. Shear stress increases EPLIN-beta expression to stabilize stress fibers.\",\n      \"method\": \"FRAP, siRNA isoform-specific knockdown, Arp2/3 interaction assay, immunofluorescence, shear stress experiments in endothelial cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (FRAP, isoform-specific KD, Arp2/3 interaction) establishing isoform-specific mechanisms in single well-controlled study\",\n      \"pmids\": [\"31644899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LUZP1 and EPLIN interact and together restrict primary cilia formation by stabilizing actin filaments. EPLIN depletion increases MyosinVa levels at the centrosome and promotes ciliogenesis. Both proteins mobilize ARP2 to centrosomes and interact with ciliogenesis/cilia-length regulators. LUZP1 and EPLIN represent novel players in actin-dependent centrosome to basal body conversion.\",\n      \"method\": \"Proximity-ligation (BioID), siRNA knockdown, co-immunoprecipitation, cilia formation quantification, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID plus Co-IP plus loss-of-function with cilia phenotype, single lab\",\n      \"pmids\": [\"32496561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rab40b-Cullin5 complex ubiquitylates and degrades EPLIN. Rab40b directly binds EPLIN. Rab40b-Cullin5-dependent localized ubiquitylation and degradation of EPLIN at the leading edge regulates focal adhesion and cytoskeletal dynamics, promoting cell migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, siRNA knockdown, focal adhesion dynamics assay, invasion assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus ubiquitylation assay plus loss-of-function with defined cytoskeletal and invasion phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"33999101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Lima1 expression is controlled by the naive pluripotency circuit in mouse embryonic stem cells and is required for suppression of membrane blebbing and proper mitochondrial energetics. Forced Lima1 expression enables primed pluripotent stem cells to integrate into pre-implantation embryos.\",\n      \"method\": \"Lima1 knockout ESC lines, live imaging, mitochondrial function assays, chimera assay, embryo injection\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with specific cellular phenotypes (blebbing, mitochondrial function), single lab\",\n      \"pmids\": [\"35105859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LIMA1 inhibits the Wnt/β-catenin signaling pathway in HCC cells by binding to BMI1 and inducing its destabilization. LIMA1 overexpression suppresses HCC cell proliferation and metastasis in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, western blot, in vitro and in vivo functional assays, siRNA knockdown\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing LIMA1-BMI1 interaction with functional consequences, single lab\",\n      \"pmids\": [\"36497115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EPLIN-beta (but not EPLIN-alpha) is a substrate of ornithine decarboxylase antizyme 1 (Az1/OAZ1). The interaction between EPLIN-beta and Az1 is indirect, and EPLIN-beta is degraded by Az1 in a ubiquitination-independent manner. Az1 absence leads to elevated EPLIN-beta levels and enhanced cellular migration.\",\n      \"method\": \"Quantitative proteomics, co-immunoprecipitation, ubiquitination assay, Az1 knockout, migration assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-based identification followed by Co-IP and ubiquitination assay plus functional migration phenotype, single lab\",\n      \"pmids\": [\"37325974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MAD2 interferes with binding of the deubiquitinase USP44 to LIMA1 by sequestering USP44 in the nucleus, leading to impaired USP44/LIMA1 complex formation and enhanced K48-linked ubiquitination of LIMA1, promoting its degradation and activating the IGF1R/PI3K/AKT pathway.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), subcellular fractionation, siRNA knockdown, PI3K/AKT signaling analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing USP44-LIMA1 complex, ubiquitination linkage-specific assay, and MAD2-dependent disruption, single lab\",\n      \"pmids\": [\"37752233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF40, an E3 ubiquitin ligase, directly mediates ubiquitination of LIMA1 and promotes its proteasome-dependent degradation in the cytoplasm. The 1-166 aa fragment of LIMA1 is required for interaction with RNF40. RNF40-mediated LIMA1 degradation suppresses lipid accumulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, domain truncation mapping, proteasome inhibitor treatment, lipid accumulation assay\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay with domain mapping, single lab\",\n      \"pmids\": [\"38909032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"LIMA1 O-GlcNAcylation at T662, catalyzed by OGT (facilitated by HCF1 upregulation under steatosis), inhibits ubiquitin-dependent degradation of LIMA1 and enhances hepatocyte lipid deposition by activating β-catenin/FASn signaling. AAV-mediated T662 mutant LIMA1 (non-O-GlcNAcylatable) decreases steatosis, insulin resistance, inflammation and fibrosis in LIMA1 hepatocyte-specific knockout mice.\",\n      \"method\": \"O-GlcNAcylation mapping (site-specific), site-directed mutagenesis (T662A), AAV rescue in HKO mice, ubiquitination assay, western blot, metabolic phenotyping\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific PTM mutagenesis combined with in vivo rescue experiment and multiple orthogonal mechanistic assays\",\n      \"pmids\": [\"39921472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cytoplasmic p62 (SQSTM1) interacts with EPLIN via GST pulldown and stabilizes EPLIN protein, enhancing EPLIN-dependent migration and invasion of esophageal squamous cell carcinoma cells.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, siRNA knockdown, protein stability assay, migration/invasion assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pulldown plus Co-IP confirming interaction, functional rescue experiment, single lab\",\n      \"pmids\": [\"38185251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STUB1 (E3 ubiquitin ligase) is recruited by BPNT1 to induce proteasomal degradation of LIMA1, promoting EMT and TNBC progression. Re-expression of LIMA1 in BPNT1-overexpressing cells partially attenuates BPNT1-driven EMT.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination/proteasomal degradation assay, re-expression rescue experiment, in vitro and in vivo functional assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing BPNT1-STUB1-LIMA1 axis with rescue experiment and in vivo validation, single lab\",\n      \"pmids\": [\"41540000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SEPT9 directly interacts with the single LIM domain of EPLIN. SEPT9 depletion decreases cell adhesion and migration; these defects can be partly rescued by increased EPLIN levels. SEPT9 and EPLIN together regulate actin-related filopodia, stress fiber organization, and focal adhesion size.\",\n      \"method\": \"SEPT9 knockout fibroblasts, co-immunoprecipitation, LIM-domain interaction mapping, rescue by EPLIN overexpression, focal adhesion analysis, actin imaging\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with rescue plus domain-mapped interaction, single lab\",\n      \"pmids\": [\"38719752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LIMA1 associates with IRBIT and the intestinal Cl-/HCO3- exchanger SLC26A3 (DRA). cAMP/ATP stimulation increases co-precipitation of LIMA1 with both IRBIT and DRA. Knockdown of LIMA1 reduces cAMP plus ATP stimulation of DRA but does not alter basal DRA activity, establishing a DRA-IRBIT-LIMA1 plasma membrane complex required for acute stimulation of DRA.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, functional DRA transport assay (BCECF fluorometry), surface biotinylation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing the complex plus loss-of-function with functional transporter phenotype, single lab\",\n      \"pmids\": [\"40569378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CDK1 phosphorylates EPLIN at Ser203 during mitosis. This phosphorylation is required for EPLIN to maintain proper actin cortex architecture and correct spindle positioning. EPLIN-pSer203 enhances the ability of EPLIN to reorganize branched actin filaments into astral structures in vitro, sequestering actin bundles and Arp2/3 complex to prevent excessive de novo actin polymerization. EPLIN depletion causes spindle deviation and metaphase arrest with abnormal cortical actin accumulation and blebbing.\",\n      \"method\": \"In vitro kinase assay (CDK1), site-directed mutagenesis (Ser203), phospho-specific antibodies, siRNA depletion, live-cell imaging, in vitro actin reconstitution assay\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis, in vitro actin reconstitution, and multiple functional cellular phenotypes\",\n      \"pmids\": [\"42237003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"LIMA1 expression is sufficient to drive elongation of microvilli in epithelial cells and formation and stabilization of exaggerated filopodia in non-epithelial cells. Individual filopodia in LIMA1-overexpressing cells exhibit merging dynamics that enable formation of large core actin bundles from smaller precursors. In tuft cells, LIMA1 is restricted to the basal/rootlet ends of actin bundles.\",\n      \"method\": \"Live-cell and electron microscopy, LIMA1 overexpression, filopodia dynamics analysis, LIMA1-specific localization in tuft cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple microscopy modalities with overexpression and direct visualization of bundle dynamics, single lab\",\n      \"pmids\": [\"42202079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EPLINα localizes to Rab21-positive recycling endosomes in an actin-binding-dependent manner and interacts with Rab21. EPLINα supports beta1-integrin recycling and cell migration. Coronin 1C was identified as an EPLINα proximal protein at Rab21-containing endosomes in an EPLINα-dependent manner. EPLINβ resides on actin stress fibers and does not show this endosomal localization.\",\n      \"method\": \"BioID proximity labeling, co-localization (confocal), co-immunoprecipitation, integrin recycling assay, migration assay, isoform-specific expression constructs\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID plus Co-IP plus functional recycling assay; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.06.27.600789\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"LIMA1/EPLIN is an actin-binding and cross-linking protein with at least two actin-binding sites that inhibits filament depolymerization, suppresses Arp2/3-mediated branching nucleation, and bundles filaments; it links the cadherin-catenin (and VE-cadherin-catenin) complex to F-actin via α-catenin to maintain the epithelial adherens junction/adhesion belt, and is regulated by multiple post-translational modifications (ERK-mediated phosphorylation at Ser360/Ser602/Ser692 that reduces actin affinity, CDK1 phosphorylation at Ser203 required for mitotic cortex remodeling and spindle positioning, O-GlcNAcylation at T662 that stabilizes the protein and promotes lipid deposition, and ubiquitin-mediated proteasomal degradation by E3 ligases RNF40, STUB1, and the Rab40b-Cullin5 complex); it also bridges NPC1L1 to a myosin Vb transport complex for intestinal cholesterol absorption, participates in cytokinesis by stabilizing myosin II and Sept2 at the cleavage furrow, restricts primary ciliogenesis by stabilizing actin filaments, and can be proteolytically cleaved by the API2-MALT1 paracaspase to generate an oncogenic LIM-domain fragment.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LIMA1 (EPLIN) is an actin-binding and cross-linking protein that governs the stability and architecture of the actin cytoskeleton, acting broadly as a suppressor of cell motility, invasion, and proliferation [#0, #3]. Biochemically, purified LIMA1 uses at least two actin-binding sites to inhibit filament depolymerization, bundle filaments, and block Arp2/3-mediated branching nucleation, thereby favoring stable structures such as stress fibers over dynamic membrane ruffles [#3]. Through direct binding to \\u03b1-catenin it links the cadherin\\u2013catenin and VE-cadherin\\u2013catenin complexes to F-actin, forming the molecular bridge required to build and maintain the epithelial and endothelial adhesion belt (zonula adherens) and to recruit vinculin under junctional tension [#4, #9, #11]. Its two isoforms are functionally and transcriptionally distinct: EPLIN-alpha is serum/RhoA- and MAL/MRTF-SRF-inducible and terminates membrane protrusion growth via Arp2/3, whereas the more stable EPLIN-beta stabilizes stress fibers [#1, #8, #20]. LIMA1 abundance and actin affinity are tightly controlled by post-translational modification, including ERK phosphorylation that lowers actin affinity and triggers ubiquitin-proteasomal turnover to permit stress-fiber disassembly and migration [#5, #12], CDK1 phosphorylation at Ser203 that remodels the mitotic actin cortex for correct spindle positioning [#33], O-GlcNAcylation at T662 that stabilizes the protein and drives hepatocyte lipid deposition [#28], and degradation by multiple E3 ligases including the Rab40b-Cullin5 complex, RNF40, and STUB1 [#22, #27, #30]. Beyond junctions, LIMA1 functions in cytokinesis by promoting accumulation of active myosin II and Sept2 at the cleavage furrow [#7], restricts primary ciliogenesis together with LUZP1 by stabilizing actin [#21], and bridges NPC1L1 to a myosin Vb transport complex to drive intestinal cholesterol absorption, with Lima1-deficient mice resistant to diet-induced hypercholesterolemia [#18]. As a tumor suppressor it is transcriptionally induced by p53 and repressed by DNp73, and can be proteolytically inactivated by the API2-MALT1 paracaspase to generate an oncogenic LIM-domain fragment [#14, #17, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established LIMA1/EPLIN as a cytoskeleton-associated LIM domain protein whose overexpression restrains proliferation, framing it as a candidate growth suppressor tied to actin.\",\n      \"evidence\": \"Overexpression and immunofluorescence co-localization with F-actin in cultured cells\",\n      \"pmids\": [\"10618726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical mechanism for actin interaction\", \"Did not distinguish isoform-specific roles functionally\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed the two isoforms arise from distinct promoters under different transcriptional control, explaining how EPLIN-alpha is coupled to Rho/serum signaling while EPLIN-beta is constitutive.\",\n      \"evidence\": \"5' RACE, S1 protection, promoter-reporter assays with serum and RhoA\",\n      \"pmids\": [\"10806352\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define functional consequences of differential expression\", \"Transcription factor identity at the SRE not resolved here\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped the growth-suppressive function to the N-terminal actin-localizing region, establishing that cytoskeletal targeting, not the LIM domain, drives suppression of anchorage-independent growth.\",\n      \"evidence\": \"Truncation mutant retroviral transduction with colony formation assays\",\n      \"pmids\": [\"11950948\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical activity of the N-terminus on actin not yet defined\", \"LIM domain function left unexplained\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the core biochemical activity: LIMA1 cross-links and stabilizes actin filaments and inhibits Arp2/3 branching, providing the mechanistic basis for its bias toward stable cytoskeletal structures.\",\n      \"evidence\": \"In vitro reconstitution with purified protein (depolymerization, bundling, Arp2/3 branching) plus cellular gain/loss of function\",\n      \"pmids\": [\"12566430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify in vivo binding partners beyond actin\", \"Regulation of the two actin-binding sites unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified LIMA1 as the \\u03b1-catenin\\u2013F-actin bridge that builds the adhesion belt, answering how the cadherin-catenin complex couples to a stable actin ring.\",\n      \"evidence\": \"RNAi, reciprocal Co-IP, and live imaging in epithelial cells\",\n      \"pmids\": [\"18093941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanical regulation of the bridge not addressed\", \"Did not test endothelial/VE-cadherin junctions\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed LIMA1 under ERK control, showing phosphorylation lowers actin affinity to permit stress-fiber disassembly and migration, providing a switch between stable and dynamic cytoskeletal states.\",\n      \"evidence\": \"In vitro kinase assay, phospho-site mutagenesis, PDGF-stimulated migration assays\",\n      \"pmids\": [\"17875928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link between phosphorylation and protein turnover not yet made\", \"Did not address junctional pools of LIMA1\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected LIMA1 to cancer cell behavior, demonstrating it suppresses invasion/motility downstream of ERK signaling.\",\n      \"evidence\": \"Overexpression with invasion/migration assays and ERK inhibition in breast cancer cells\",\n      \"pmids\": [\"18796137\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular target of ERK-LIMA1 axis in invasion not dissected\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed actin-MAL/MRTF-SRF signaling drives EPLIN-alpha transcription, establishing a feedback link between actin dynamics and isoform expression.\",\n      \"evidence\": \"Transcriptomics with actin drugs, promoter-reporter, ChIP, dominant-negative MAL\",\n      \"pmids\": [\"20236507\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger of this feedback in vivo unclear\", \"EPLIN-beta regulatory inputs not identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended LIMA1 function to cytokinesis, showing it concentrates contractile machinery (myosin II, Sept2, RhoA/Cdc42) at the cleavage furrow to prevent multinucleation.\",\n      \"evidence\": \"siRNA knockdown, co-localization, and multinucleation scoring\",\n      \"pmids\": [\"19221476\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus indirect interaction with furrow components not resolved\", \"No biochemical binding data for myosin II/Sept2\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the mechanics and partners of junctional LIMA1 across endothelium and epithelium, showing it tethers VE-cadherin/catenin to actin and that junctional tension retains it to recruit vinculin and maintain zonula adherens morphology.\",\n      \"evidence\": \"Co-IP/GST pulldown, tension manipulation, \\u03b1E-catenin\\u2013EPLIN fusion, angiogenesis assays\",\n      \"pmids\": [\"22194609\", \"21844208\", \"21625216\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Force-sensing mechanism that ejects LIMA1 under tension not molecularly defined\", \"Relationship between AJ and EMT roles incompletely integrated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Unified phosphorylation with degradation, showing EGF/ERK-dependent phosphorylation of LIMA1 triggers its ubiquitination and proteasomal turnover, explaining acute downregulation during motility induction.\",\n      \"evidence\": \"EGF stimulation, ERK inhibition, ubiquitination assay, phospho-site mutagenesis, cycloheximide chase\",\n      \"pmids\": [\"23188829\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase responsible not identified here\", \"Spatial control of degradation unaddressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established transcriptional tumor-suppressor circuitry, with p53 directly inducing LIMA1 to suppress invasion and DNp73 repressing it to activate pro-metastatic IGF1R-AKT/STAT3 signaling.\",\n      \"evidence\": \"ChIP-seq, nutlin-3a activation, knockdown epistasis, xenografts (with #24135282)\",\n      \"pmids\": [\"28093207\", \"24135282\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking LIMA1 loss to IGF1R activation not biochemically defined\", \"Contribution of cytoskeletal versus signaling roles to suppression unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated a non-cell-autonomous role in epithelial defense, with LIMA1 driving apical extrusion of RasV12-transformed cells upstream of Cav-1 and myosin-II/PKA.\",\n      \"evidence\": \"siRNA, co-culture extrusion assays, epistasis\",\n      \"pmids\": [\"25609711\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular interactions in extrusion not fully mapped\", \"Generality across oncogenes unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked LIMA1 to integrin adhesion, showing PINCH-1-dependent recruitment to adhesion sites is required for keratinocyte spreading and migration.\",\n      \"evidence\": \"PINCH-1 interactome MS, Co-IP, conditional knockout mouse, adhesion/migration assays\",\n      \"pmids\": [\"25609703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether LIMA1 directly binds PINCH-1 not established\", \"Relationship to junctional pool unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed LIMA1 as a substrate of the API2-MALT1 paracaspase, showing oncogenic cleavage generates a LIM-domain-only fragment, a mechanism of tumor-suppressor inactivation in MALT lymphoma.\",\n      \"evidence\": \"Co-IP, in vitro cleavage with active-site mutagenesis, xenografts, primary tumor analysis\",\n      \"pmids\": [\"25569716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the liberated LIM fragment not fully characterized\", \"Cleavage relevance outside MALT lymphoma untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Uncovered a metabolic function, showing LIMA1 bridges NPC1L1 to a myosin Vb transport complex to drive intestinal cholesterol absorption, with knockout mice and a human variant validating the pathway.\",\n      \"evidence\": \"Co-IP, knockout mouse phenotyping, human frameshift variant, brush-border localization\",\n      \"pmids\": [\"29880681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NPC1L1\\u2013LIMA1\\u2013myosin Vb assembly unknown\", \"Tissue-specific regulation of this complex unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined isoform-specific cytoskeletal mechanisms, showing EPLIN-alpha targets and terminates protrusions via Arp2/3 while the slower-turnover EPLIN-beta stabilizes stress fibers under shear stress.\",\n      \"evidence\": \"FRAP, isoform-specific knockdown, Arp2/3 interaction assays, shear stress in endothelial cells\",\n      \"pmids\": [\"31644899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural determinants of isoform divergence not mapped\", \"In vivo isoform-specific phenotypes not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified a ciliogenesis-restricting function, with LIMA1 and LUZP1 stabilizing actin at the centrosome to limit primary cilia formation.\",\n      \"evidence\": \"BioID, Co-IP, siRNA, cilia quantification\",\n      \"pmids\": [\"32496561\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct LIMA1\\u2013LUZP1 binding interface undefined\", \"Mechanism of MyosinVa regulation at centrosome unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed spatially restricted degradation as a migration control mechanism, with the Rab40b-Cullin5 complex ubiquitylating LIMA1 at the leading edge to regulate focal adhesion and cytoskeletal dynamics.\",\n      \"evidence\": \"Co-IP, ubiquitylation assay, siRNA, focal adhesion and invasion assays\",\n      \"pmids\": [\"33999101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recognition determinants on LIMA1 for Rab40b not mapped\", \"Integration with ERK-driven turnover unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected LIMA1 to pluripotency and cell mechanics, showing the naive circuit requires Lima1 to suppress blebbing and support mitochondrial energetics, enabling embryo integration.\",\n      \"evidence\": \"Knockout ESCs, live imaging, mitochondrial assays, chimera/embryo injection\",\n      \"pmids\": [\"35105859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link to mitochondrial function unexplained\", \"Whether actin-bundling activity underlies the phenotype untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Added a signaling-suppressor function, showing LIMA1 binds BMI1 to destabilize it and inhibit Wnt/\\u03b2-catenin signaling in HCC.\",\n      \"evidence\": \"Co-IP, western blot, in vitro/in vivo functional assays\",\n      \"pmids\": [\"36497115\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus adaptor-mediated BMI1 destabilization unclear\", \"Reconciliation with cytoskeletal role not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Expanded post-translational regulation, identifying ubiquitin-independent degradation of EPLIN-beta by antizyme Az1/OAZ1 that constrains migration.\",\n      \"evidence\": \"Quantitative proteomics, Co-IP, ubiquitination assay, Az1 knockout migration assays\",\n      \"pmids\": [\"37325974\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Indirect interaction adaptor not identified\", \"Isoform-selectivity mechanism undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a deubiquitination axis, showing USP44 stabilizes LIMA1 and MAD2 sequesters USP44 to promote K48-ubiquitination and degradation, activating IGF1R/PI3K/AKT.\",\n      \"evidence\": \"Co-IP, K48-linkage ubiquitination assay, fractionation, signaling analysis\",\n      \"pmids\": [\"37752233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase opposing USP44 not identified\", \"Physiological context of MAD2-USP44 competition unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Consolidated a multi-ligase degradation network and a metabolic-PTM axis, identifying RNF40 and STUB1 as LIMA1 E3 ligases and O-GlcNAcylation at T662 as a stabilizing modification driving hepatic lipid deposition.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, domain mapping, site-specific O-GlcNAc mutagenesis with AAV rescue in knockout mice\",\n      \"pmids\": [\"38909032\", \"41540000\", \"39921472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy/competition among the multiple E3 ligases unresolved\", \"How O-GlcNAc and ubiquitin sites interplay structurally unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Added stabilizing partners and new actin/transport functions, including p62/SQSTM1 stabilization promoting invasion, SEPT9 binding the LIM domain to organize adhesions, EPLIN\\u03b1-Rab21 endosomal control of integrin recycling, and LIMA1-driven microvillus/filopodia elongation.\",\n      \"evidence\": \"GST pulldown, Co-IP, LIM-domain mapping, SEPT9 knockout rescue, BioID, integrin recycling and microscopy assays\",\n      \"pmids\": [\"38185251\", \"38719752\", \"42202079\", \"bio_10.1101_2024.06.27.600789\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct structural interactions for several partners undefined\", \"Endosomal recycling role from preprint awaits peer review\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a transporter-regulatory role, showing LIMA1 forms a plasma-membrane DRA-IRBIT-LIMA1 complex required for acute cAMP/ATP stimulation of SLC26A3 anion exchange.\",\n      \"evidence\": \"Co-IP, siRNA, BCECF transport assays, surface biotinylation\",\n      \"pmids\": [\"40569378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding interfaces within the complex not mapped\", \"In vivo relevance to intestinal physiology untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a mitotic regulatory mechanism, showing CDK1 phosphorylation at Ser203 reorganizes branched actin into astral structures to maintain cortical architecture and correct spindle positioning.\",\n      \"evidence\": \"In vitro CDK1 kinase assay, Ser203 mutagenesis, phospho-antibodies, live imaging, in vitro actin reconstitution\",\n      \"pmids\": [\"42237003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How pSer203 alters the two actin-binding sites structurally unresolved\", \"Integration with cytokinesis role not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How LIMA1's distinct functional pools \\u2014 adherens junctions, focal/integrin adhesions, recycling endosomes, brush-border transport complexes, and the mitotic cortex \\u2014 are coordinately partitioned and switched by its dense network of post-translational modifications remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model of how PTMs gate actin-binding-site usage\", \"Competition/hierarchy among multiple E3 ligases and deubiquitinases unmapped\", \"Mechanistic basis for isoform-specific localization to distinct compartments incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3, 20, 33, 34]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 9, 11, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 3, 20]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 9, 18, 32]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [35]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [4, 9, 11]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 33]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [18, 28]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 13, 24]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [12, 22, 27, 30]}\n    ],\n    \"complexes\": [\n      \"cadherin-catenin adherens junction complex\",\n      \"NPC1L1-myosin Vb cholesterol transport complex\",\n      \"DRA-IRBIT-LIMA1 plasma membrane complex\",\n      \"Rab40b-Cullin5 ubiquitin ligase complex\"\n    ],\n    \"partners\": [\n      \"CTNNA1\",\n      \"ACTB\",\n      \"LIMS1\",\n      \"SEPT9\",\n      \"NPC1L1\",\n      \"RAB40B\",\n      \"RNF40\",\n      \"USP44\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}