{"gene":"CFL1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1998,"finding":"LIM-kinase 1 (LIMK1) directly phosphorylates cofilin (CFL1) at Serine 3, both in vitro and in vivo. This phosphorylation abolishes cofilin's actin-depolymerizing activity. LIMK1 expression induces actin stress fiber formation, while an inactive LIMK1 suppresses Rac-induced lamellipodium formation. Rac and insulin activate LIMK1, placing LIMK1-mediated cofilin phosphorylation downstream of Rac in actin cytoskeletal reorganization.","method":"In vitro kinase assay, site-directed mutagenesis (Ser3), overexpression and dominant-negative LIMK1 in cultured cells, actin depolymerization assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro phosphorylation with mutagenesis validation, multiple cell-based phenotypic readouts, foundational paper","pmids":["9655398"],"is_preprint":false},{"year":1999,"finding":"The Rho-associated kinase ROCK phosphorylates and activates LIM-kinase, which in turn phosphorylates cofilin (CFL1) to inactivate its actin-depolymerizing activity. This ROCK→LIMK→cofilin cascade mediates Rho-induced actin cytoskeletal reorganization (stress fiber formation) and neurite retraction. ROCK does not phosphorylate cofilin directly; the Y-27632 ROCK inhibitor blocks cofilin phosphorylation in cells.","method":"Pharmacological inhibition (Y-27632), overexpression of LIMK1 in HeLa cells, in vivo phosphorylation assays in neuroblastoma cells, epistasis with dominant-negative constructs","journal":"Science","confidence":"High","confidence_rationale":"Tier 1–2 — cell-based epistasis with pharmacological inhibitor and overexpression, replicated across labs","pmids":["10436159"],"is_preprint":false},{"year":2002,"finding":"The Slingshot (SSH) family of phosphatases dephosphorylate phospho-cofilin (p-CFL1) to reactivate its actin-depolymerizing function. SSH binds F-actin and dephosphorylates P-cofilin both in cultured cells and in cell-free assays. Loss of SSH in Drosophila dramatically increases F-actin levels and phospho-cofilin, disrupting epidermal cell morphogenesis. Human SSH homologs (hSSH1) suppress LIMK1-induced actin reorganization by reactivating cofilin.","method":"Drosophila loss-of-function genetics, cell-free phosphatase assays, overexpression of hSSH in mammalian cells, F-actin binding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — cell-free dephosphorylation assay plus in vivo genetic validation in Drosophila and mammalian cells","pmids":["11832213"],"is_preprint":false},{"year":1996,"finding":"Human non-muscle cofilin (CFL1) was cloned from a promyelocytic cDNA library and mapped to chromosome 11q13 by PCR in somatic cell hybrids and FISH. Muscle-type cofilin (CFL2) was mapped to chromosome 14. CFL1 encodes an actin-binding protein involved in translocation of the actin-cofilin complex from cytoplasm to nucleus.","method":"cDNA cloning, PCR mapping in rodent-human somatic cell hybrids, FISH with genomic cosmid clones, irradiation hybrid panel mapping","journal":"Annals of human genetics","confidence":"High","confidence_rationale":"Tier 2 — direct chromosomal mapping with multiple orthogonal methods","pmids":["8800436"],"is_preprint":false},{"year":2007,"finding":"Genetic variants (SNPs) in the human CFL1 gene are associated with increased spina bifida risk, consistent with the essential role of CFL1 in actin depolymerization during neural tube closure. Cfl1 knockout mice fail to close the neural tube at E10.5 and die in utero, establishing CFL1 as essential for neural tube development.","method":"Population-based case-control SNP association study, reference to Cfl1 knockout mouse phenotype","journal":"BMC medical genetics","confidence":"Medium","confidence_rationale":"Tier 3 — genetic association with mechanistic reference to KO mouse; KO data from prior work","pmids":["17352815"],"is_preprint":false},{"year":2017,"finding":"miR-200b-3p and miR-429-5p directly target LIMK1, reducing LIMK1 expression and consequently decreasing phosphorylation (inactivation) of cofilin-1 (CFL1). Reduced LIMK1 activity allows CFL1 to remain active, altering F-actin/G-actin dynamics. This LIMK1/CFL1 pathway mediates the suppressive effects of these miRNAs on triple-negative breast cancer cell proliferation, migration, and invasion.","method":"Luciferase reporter assay for direct miRNA targeting of LIMK1, Western blotting for CFL1 and p-CFL1, migration/invasion assays, cell cycle analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct target validation plus downstream phosphorylation readout, single lab","pmids":["29156719"],"is_preprint":false},{"year":2019,"finding":"In contracting zebrafish cardiomyocytes, mechanical forces regulate vinculin (VCL) localization and activation. VCL recruits the phosphatase SSH1 and its effector cofilin (CFL) to regulate F-actin rearrangement and promote myofilament maturation. The VCL-SSH1-CFL axis is essential: loss of VCL or disruption of this pathway impairs myofilament maturation in response to cardiac contractility.","method":"Zebrafish genetic knockouts, interactome analysis (contracting vs. non-contracting cardiomyocytes by MS), co-immunoprecipitation, live imaging","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP/interactome with genetic loss-of-function and defined cellular phenotype in vivo","pmids":["31495694"],"is_preprint":false},{"year":2020,"finding":"SSH1, the canonical cofilin (CFL1) phosphatase, also dephosphorylates phospho-Ser403-SQSTM1/p62, thereby impairing autophagic cargo clearance. This action of SSH1 on SQSTM1 is separable from SSH1-mediated CFL1 activation and independent of CFL1, revealing a bifurcated SSH1 function. SSH1-mediated inhibition of SQSTM1 impairs clearance of phospho-MAPT/tau.","method":"RNAi knockdown, overexpression, defined phospho-mutant constructs, fluorescent autophagy reporters, experiments in cell lines, primary neurons, and mouse brains","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (genetic, biochemical, reporter) in multiple cell systems clarifying CFL1-independent SSH1 action","pmids":["33044112"],"is_preprint":false},{"year":2021,"finding":"Hhex (hematopoietically expressed homeobox) inhibits CFL1 phosphorylation, keeping CFL1 in its active F-actin-severing form, thereby suppressing cell migration and protrusion formation in lung cancer cells. Mechanistically, Hhex enhances the interaction of RHOGDIA with RHOA/CDC42, maintaining these GTPases in their inactive state and blocking the RHOA/CDC42→p-CFL1 signaling cascade. This prevents filopodium and lamellipodium formation.","method":"Western blot, co-immunoprecipitation, wound-healing scratch assay, laser confocal microscopy, overexpression/knockdown in NSCLC and HEK293FT cells","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-IP and phosphorylation assays with functional readout, single lab","pmids":["34321041"],"is_preprint":false},{"year":2021,"finding":"E2F4-induced lncRNA AGAP2-AS1 acts as a competing endogenous RNA (ceRNA) to sponge miR-182-5p, thereby upregulating CFL1 expression. CFL1 restoration counteracts the suppression of CRC cell growth, migration, invasion, and EMT caused by AGAP2-AS1 depletion, placing CFL1 as a downstream effector of this AGAP2-AS1/miR-182-5p axis in colorectal cancer progression.","method":"RT-qPCR, Western blot, luciferase reporter assays, rescue experiments in vitro and in vivo (xenograft)","journal":"Digestive and liver disease","confidence":"Medium","confidence_rationale":"Tier 3 — functional rescue experiment plus reporter validation, single lab","pmids":["34838479"],"is_preprint":false},{"year":2022,"finding":"Computational analysis of nonsynonymous SNPs in CFL1 identifies L84P and L99A as the most damaging variants. Molecular docking shows these variants reduce cofilin-1 binding affinity for actin, and molecular dynamics simulations confirm protein structure destabilization. The actin-binding regions of cofilin-1 are highly conserved and critical for function.","method":"In silico prediction tools (SIFT, PolyPhen-2, DynaMut/DUET), molecular docking, molecular dynamics simulation","journal":"Gene","confidence":"Low","confidence_rationale":"Tier 4 — computational prediction only, no experimental validation of variants","pmids":["35092861"],"is_preprint":false},{"year":2022,"finding":"Estradiol (E2) inhibits HIV-1 infection in PBMCs and endocervical tissue and simultaneously increases total CFL1 and phosphorylated CFL1 (p-CFL1) protein expression, raising the p-CFL1/CFL1 ratio. LIM kinase inhibitor (LIMKi3) abrogates both the anti-HIV effect and E2-induced CFL1/p-CFL1 upregulation, and CFL1 knockdown partially restores HIV infection, indicating that LIMK-mediated CFL1 phosphorylation is part of the mechanism by which E2 restricts HIV-1 entry/spread.","method":"HIV infection assay, siRNA knockdown of CFL1, pharmacological LIMK inhibition (LIMKi3), Western blotting for total and p-CFL1, dose-response experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 — siRNA knockdown + pharmacological inhibition with functional readout, single lab","pmids":["35418661"],"is_preprint":false},{"year":2023,"finding":"CFL1 promotes transcription of phosphoglycerate dehydrogenase (PHGDH), enhancing serine synthesis and metabolism to increase antioxidant production, thereby scavenging ROS induced by sorafenib and reducing sorafenib sensitivity in hepatocellular carcinoma. siRNA-mediated CFL1 silencing re-sensitizes HCC cells to sorafenib.","method":"Transcriptome sequencing comparison of sorafenib-sensitive vs. insensitive patients, CFL1 siRNA knockdown via nanoparticles, PHGDH promoter assays, ROS measurement, in vivo tumor growth assays","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic pathway identified by transcriptomics plus functional siRNA knockdown with multiple readouts in vitro and in vivo","pmids":["37203277"],"is_preprint":false},{"year":2023,"finding":"HUNK kinase directly phosphorylates GEF-H1 at Serine 645, which activates RhoA and triggers a phosphorylation cascade of LIMK-1 and CFL-1, thereby stabilizing F-actin and inhibiting EMT and metastasis in colorectal cancer cells. This places CFL-1 phosphorylation as a downstream effector of the HUNK→GEF-H1→RhoA→LIMK-1 cascade.","method":"In vitro kinase assay (HUNK phosphorylating GEF-H1), site-directed mutagenesis (S645), Western blotting for LIMK-1/p-CFL-1, migration/invasion assays, clinical tissue analysis","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay with mutagenesis plus cell-based epistasis and clinical validation","pmids":["37193711"],"is_preprint":false},{"year":2024,"finding":"CFL1 overexpression in pSS bone marrow mesenchymal stem cells rescues their impaired migration and proliferation. RNA-seq identifies CCR1 as a downstream target gene of CFL1, and inhibition of CCR1 with BX431 suppresses the CFL1-induced increase in migration/proliferation, establishing that CFL1 promotes BM-MSC motility via upregulation of the CCL5/CCR1 axis.","method":"Lentivirus-mediated CFL1 overexpression, RNA-seq, Transwell migration assay, wound healing assay, CCR1 inhibitor rescue experiment (BX431), NOD mouse therapeutic model","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2–3 — RNA-seq plus pharmacological rescue with functional assays, single lab","pmids":["38183912"],"is_preprint":false},{"year":2024,"finding":"c-Myc directly transactivates the CFL1 promoter by binding to E-box elements (particularly middle and proximal E-boxes), inducing cofilin-1 expression. Cofilin-1 upregulation is required for c-Myc-induced oncogene-induced senescence (OIS): cofilin-1 knockdown suppresses cMIS. Physical interaction between c-Myc and cofilin-1 was detected, enhanced by H2O2. The conditioned medium from cMIS cells promotes migration and proliferation of other NSCLC cells, an effect abrogated by cofilin-1 silencing.","method":"ChIP-qPCR, luciferase reporter assay, siRNA knockdown, co-immunoprecipitation (c-Myc and cofilin-1), senescence assays, conditioned medium transfer experiments","journal":"Cell death discovery","confidence":"High","confidence_rationale":"Tier 1–2 — ChIP-qPCR demonstrating direct promoter binding, Co-IP showing physical interaction, functional knockdown with defined phenotypic readout","pmids":["41888102"],"is_preprint":false},{"year":2024,"finding":"Conditional knockout of neuronal CFL1 (cofilin-1) impedes tumor-nerve interactions in head and neck squamous cell carcinoma. HNSCC cells induce CFL1 expression in adjacent neurons, and it is neuronal (not tumoral) CFL1 that drives cancer-nerve crosstalk and perineural invasion, as demonstrated by multiplex fluorescent immunohistochemistry localizing CFL1 to nerves and by conditional KO experiments.","method":"Multiplex fluorescent immunohistochemistry, conditional neuronal CFL1 knockout, Gene Ontology/GSEA analysis","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 — conditional KO with defined cellular phenotype, localization data, single lab","pmids":["38353363"],"is_preprint":false},{"year":2024,"finding":"miR-342-5p targets CFL1 (cofilin-1) mRNA in HDACi-resistant HCC cells; overexpression of miR-342-5p decreases cofilin-1 protein expression and increases ROS-mediated apoptosis, sensitizing cells to HDACi treatment. This identifies the miR-342-5p/CFL1 axis as a mediator of HDACi resistance.","method":"miRNA microarray, qRT-PCR, gain/loss-of-function studies, Western blot, apoptosis assays (Bax, caspase-3, Bcl-2), ROS measurement","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 3 — functional miRNA/target relationship with biochemical readouts, single lab","pmids":["39152428"],"is_preprint":false},{"year":2024,"finding":"CFL1 (cofilin-1) is required for mast cell development: expression of a non-functional form of Cfl1 in connective tissue mast cells (CTMCs) using Mcpt5-Cre results in complete absence of CTMCs without affecting basophils. CTMCs lacking Cfl1 function show impaired systemic anaphylaxis but normal susceptibility to contact hypersensitivity and psoriasis-like dermatitis, demonstrating that cofilin-1-mediated actin dynamics are essential specifically for CTMC generation.","method":"Conditional knock-in mouse (Mcpt5-Cre-nf-Cfl1fl/fl), mast cell/basophil enumeration, anaphylaxis model, contact hypersensitivity model, imiquimod model, vaccinia virus infection","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 — conditional genetic KO in vivo with multiple disease model readouts showing specific CTMC requirement","pmids":["41684538"],"is_preprint":false},{"year":2025,"finding":"Overexpression of cofilin (Cfl1) in the polymorphic layer of the hippocampal dentate gyrus increases motivation to seek alcohol and sucrose rewards, impairs extinction of alcohol seeking, and inhibits ML-DG synapses; reducing Cfl1 has opposite effects. Three SNPs in human CFL1 (rs369270402, rs2376005, rs36124259) are associated with increased AUD risk, and CFL1 mRNA blood levels correlate with alcohol-related hospital admissions. AUD-prone mice show differential hippocampal Cfl expression linked to actin cytoskeleton and synaptic function genes.","method":"RNA sequencing, local viral vector Cfl overexpression/knockdown in DG, IntelliCage behavioral model, electrophysiology (ML-DG synapse recording), human genetic association study","journal":"Molecular psychiatry","confidence":"High","confidence_rationale":"Tier 2 — in vivo gain/loss-of-function with electrophysiology and behavioral readouts plus human genetic validation","pmids":["40931167"],"is_preprint":false},{"year":2024,"finding":"Deficiency of ADF and Cofilin1 (Cfl1) in microglia causes profound morphological changes, reduces microglial fine process motility and migration toward laser-induced lesions in vivo, and increases stabilized F-actin with altered microtubule dynamics. Microglial ADF/Cfl1 deficiency also impairs learning and memory, linking microglial cytoskeletal dynamics to neuronal cognitive function.","method":"Conditional microglial ADF/Cfl1 knockout, in vivo two-photon imaging, F-actin immunostaining, microtubule dynamics assays, behavioral learning/memory tests","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with in vivo imaging and behavioral readouts, preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.09.27.615114"],"is_preprint":true}],"current_model":"CFL1 (cofilin-1) is an actin-depolymerizing/severing protein whose activity is primarily regulated by reversible phosphorylation at Serine 3: LIM-kinase 1 (downstream of Rac→LIMK1 and Rho→ROCK→LIMK1 signaling) phosphorylates and inactivates CFL1, while the Slingshot (SSH1) phosphatase family reactivates it by dephosphorylation; active CFL1 severs and depolymerizes actin filaments to drive cytoskeletal remodeling essential for cell migration, lamellipodium formation, neural tube closure, cardiac myofilament maturation, mast cell development, microglial motility, synaptic plasticity, and hippocampal-dependent cognition, and its dysregulation is implicated in cancer invasion (via LIMK1/CFL1, HUNK→GEF-H1→RhoA→LIMK-1→CFL-1, and Hhex→RHOGDIA→RHOA/CDC42→CFL1 axes), sorafenib resistance (via CFL1-driven PHGDH/serine metabolism), senescence-associated secretion (via direct c-Myc transactivation of the CFL1 promoter), and alcohol use disorder (via hippocampal Cfl1 regulation of reward circuitry)."},"narrative":{"teleology":[{"year":1996,"claim":"Cloning and chromosomal mapping of human CFL1 to 11q13 established its identity as a non-muscle actin-binding protein distinct from muscle-type CFL2, providing the molecular entry point for functional studies.","evidence":"cDNA cloning from promyelocytic library, somatic cell hybrid PCR, and FISH mapping","pmids":["8800436"],"confidence":"High","gaps":["No functional assay performed at this stage","Regulation of CFL1 activity was unknown"]},{"year":1998,"claim":"Identification of LIM-kinase 1 as the direct Ser3 kinase for CFL1 resolved how Rac signaling controls actin depolymerization, establishing the foundational LIMK1→p-CFL1 inactivation mechanism.","evidence":"In vitro kinase assay with site-directed Ser3 mutagenesis, actin depolymerization assay, dominant-negative LIMK1 in cultured cells","pmids":["9655398"],"confidence":"High","gaps":["Upstream activation of LIMK1 by Rho-family effectors not yet mapped","No phosphatase for CFL1 reactivation had been identified"]},{"year":1999,"claim":"Placing ROCK upstream of LIMK in a Rho→ROCK→LIMK→CFL1 cascade explained how Rho-induced stress fiber formation and neurite retraction converge on cofilin inactivation.","evidence":"Pharmacological ROCK inhibition (Y-27632), epistasis with dominant-negative constructs in HeLa and neuroblastoma cells","pmids":["10436159"],"confidence":"High","gaps":["Mechanism of CFL1 reactivation still unknown","Whether additional kinases target CFL1-Ser3 remained open"]},{"year":2002,"claim":"Discovery of the Slingshot (SSH) phosphatase family as the CFL1-Ser3 phosphatase completed the phospho-cycling circuit, showing that F-actin-bound SSH dephosphorylates p-CFL1 to restore severing activity.","evidence":"Cell-free phosphatase assay, Drosophila loss-of-function genetics showing elevated F-actin and p-cofilin, mammalian overexpression rescuing LIMK1-induced phenotypes","pmids":["11832213"],"confidence":"High","gaps":["Spatial and temporal regulation of SSH1 in specific tissues was uncharacterized","Whether SSH has CFL1-independent substrates was unknown"]},{"year":2007,"claim":"Association of CFL1 SNPs with spina bifida, combined with the neural tube closure failure in Cfl1-knockout mice, established CFL1 as a developmental disease gene and demonstrated its in vivo essentiality.","evidence":"Population-based case-control SNP association study referencing prior Cfl1 KO lethality at E10.5","pmids":["17352815"],"confidence":"Medium","gaps":["Specific CFL1 variants were not functionally validated","Contribution of CFL1 versus other actin regulators to neural tube closure was not dissected"]},{"year":2019,"claim":"Identification of a VCL→SSH1→CFL axis in contracting zebrafish cardiomyocytes revealed that mechanical force-dependent cofilin activation drives myofilament maturation, extending CFL1 function beyond migration into cardiac development.","evidence":"Zebrafish VCL knockout, interactome mass spectrometry, co-immunoprecipitation, live imaging of myofilament assembly","pmids":["31495694"],"confidence":"High","gaps":["Whether this axis operates identically in mammalian cardiomyocytes was not tested","Structural basis of VCL-SSH1 recruitment was not resolved"]},{"year":2020,"claim":"Demonstration that SSH1 dephosphorylates SQSTM1/p62 independently of CFL1 clarified that CFL1 is not the sole physiological substrate of its activating phosphatase, defining the boundary of CFL1 involvement in autophagy.","evidence":"RNAi knockdown, phospho-mutant constructs, fluorescent autophagy reporters in primary neurons and cell lines","pmids":["33044112"],"confidence":"High","gaps":["Whether CFL1 itself has any direct role in autophagy remained unclear","Other potential SSH1 substrates besides CFL1 and SQSTM1 were not surveyed"]},{"year":2021,"claim":"Two independent studies placed CFL1 phosphorylation status as a convergence node for upstream oncogenic signaling: the Hhex→RHOGDIA→RHOA/CDC42 axis suppresses CFL1 phosphorylation to restrict lung cancer migration, while the AGAP2-AS1/miR-182-5p ceRNA axis upregulates CFL1 expression to promote colorectal cancer EMT.","evidence":"Co-IP, wound-healing assays, luciferase miRNA-target reporters, xenograft rescue experiments","pmids":["34321041","34838479"],"confidence":"Medium","gaps":["Neither study demonstrated direct CFL1-actin severing in the cancer context biochemically","Whether CFL1 expression level versus phosphorylation status is the more critical variable in vivo was not resolved"]},{"year":2023,"claim":"Identification of HUNK→GEF-H1(pSer645)→RhoA→LIMK-1→p-CFL1 as a metastasis-suppressive cascade in CRC extended the kinase hierarchy upstream of CFL1, while CFL1-driven PHGDH transcription and serine metabolism explained sorafenib resistance in HCC, revealing a non-cytoskeletal transcriptional function for CFL1.","evidence":"In vitro HUNK kinase assay with Ser645 mutagenesis, epistasis in CRC cells; CFL1 siRNA nanoparticle delivery with PHGDH promoter assays and ROS measurement in HCC xenografts","pmids":["37193711","37203277"],"confidence":"High","gaps":["How CFL1 activates PHGDH transcription mechanistically (as a cytoskeletal protein) was not explained","Whether CFL1 enters the nucleus to regulate transcription directly was not tested"]},{"year":2024,"claim":"Multiple 2024 studies broadened CFL1's physiological scope: conditional knockout showed CFL1 is absolutely required for connective-tissue mast cell development; c-Myc was identified as a direct transcriptional activator of CFL1 required for oncogene-induced senescence; and neuronal CFL1 was shown to mediate tumor-nerve crosstalk in HNSCC.","evidence":"Mcpt5-Cre conditional knock-in mouse with anaphylaxis models; ChIP-qPCR and Co-IP showing c-Myc–CFL1 promoter binding and physical interaction; conditional neuronal CFL1 KO in HNSCC model","pmids":["41684538","41888102","38353363"],"confidence":"High","gaps":["How CFL1 loss specifically prevents CTMC but not basophil development was not mechanistically resolved","Whether c-Myc–CFL1 physical interaction has a function beyond transcriptional regulation is unknown","Neuronal CFL1 contribution to perineural invasion needs replication"]},{"year":2025,"claim":"Gain- and loss-of-function manipulation of Cfl1 in hippocampal dentate gyrus demonstrated that CFL1 regulates synaptic transmission and reward-seeking behavior, and human CFL1 SNPs were associated with alcohol use disorder risk, establishing CFL1 as a neuromodulatory factor in addiction circuitry.","evidence":"Viral vector Cfl overexpression/knockdown in DG, electrophysiology at ML-DG synapses, IntelliCage behavioral paradigm, human SNP association and mRNA correlation analysis","pmids":["40931167"],"confidence":"High","gaps":["Specific CFL1-dependent actin substrates at hippocampal synapses were not identified","Whether CFL1 phosphorylation or total expression drives the AUD-related phenotype is unresolved"]},{"year":null,"claim":"Key unresolved questions include how CFL1 activates transcription of genes such as PHGDH (whether through nuclear translocation or indirect signaling), the structural basis of the CFL1-actin severing/depolymerization mechanism at atomic resolution in a mammalian context, and the cell-type-specific determinants that make certain lineages (CTMCs, neural tube, hippocampal neurons) uniquely dependent on CFL1 despite ubiquitous expression.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of human CFL1 bound to F-actin during severing","Nuclear function of CFL1 is mechanistically undefined","Redundancy with ADF/CFL2 across tissues is poorly mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,2,6,8,18,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12,15]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,1,2,6,8,13,18,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,8,13]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[18]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[19,20]}],"complexes":[],"partners":["LIMK1","SSH1","ROCK1","VCL","MYC","ACTA1","RHOA"],"other_free_text":[]},"mechanistic_narrative":"Cofilin-1 (CFL1) is an essential actin-severing and depolymerizing protein whose regulated cycling between active (dephosphorylated) and inactive (Ser3-phosphorylated) states drives actin cytoskeletal remodeling in virtually all non-muscle cell types. LIM-kinase 1 phosphorylates CFL1-Ser3 downstream of Rac and Rho/ROCK signaling to inactivate it, while the Slingshot phosphatase SSH1 dephosphorylates and reactivates it, and this toggle controls lamellipodium dynamics, cell migration, neural tube closure, connective-tissue mast cell development, cardiac myofilament maturation, and hippocampal synaptic function [PMID:9655398, PMID:10436159, PMID:11832213, PMID:31495694, PMID:41684538, PMID:40931167]. Beyond cytoskeletal regulation, CFL1 is transcriptionally induced by c-Myc at E-box elements in its promoter and is required for oncogene-induced senescence and the associated secretory phenotype, and it promotes PHGDH-dependent serine metabolism to confer sorafenib resistance in hepatocellular carcinoma [PMID:41888102, PMID:37203277]. Genetic variants in CFL1 are associated with increased risk of spina bifida and alcohol use disorder, consistent with its indispensable roles in neural tube closure and hippocampal reward circuitry [PMID:17352815, PMID:40931167]."},"prefetch_data":{"uniprot":{"accession":"P23528","full_name":"Cofilin-1","aliases":["18 kDa phosphoprotein","p18","Cofilin, non-muscle isoform"],"length_aa":166,"mass_kda":18.5,"function":"Binds to F-actin and exhibits pH-sensitive F-actin depolymerizing activity (PubMed:11812157). In conjunction with the subcortical maternal complex (SCMC), plays an essential role for zygotes to progress beyond the first embryonic cell divisions via regulation of actin dynamics (PubMed:15580268). Required for the centralization of the mitotic spindle and symmetric division of zygotes (By similarity). Plays a role in the regulation of cell morphology and cytoskeletal organization in epithelial cells (PubMed:21834987). Required for the up-regulation of atypical chemokine receptor ACKR2 from endosomal compartment to cell membrane, increasing its efficiency in chemokine uptake and degradation (PubMed:23633677). Required for neural tube morphogenesis and neural crest cell migration (By similarity)","subcellular_location":"Nucleus matrix; Cytoplasm, cytoskeleton; Cell projection, ruffle membrane; Cell projection, lamellipodium membrane; Cell projection, lamellipodium; Cell projection, growth cone; Cell projection, axon","url":"https://www.uniprot.org/uniprotkb/P23528/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CFL1","classification":"Common Essential","n_dependent_lines":745,"n_total_lines":1208,"dependency_fraction":0.6167218543046358},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ACTB","stoichiometry":10.0},{"gene":"ACTG1","stoichiometry":0.2},{"gene":"CALM3","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CTTN","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CFL1","total_profiled":1310},"omim":[{"mim_id":"621464","title":"LIMB- AND CNS-EXPRESSED GENE 1-LIKE; LIX1L","url":"https://www.omim.org/entry/621464"},{"mim_id":"621425","title":"ABRA C-TERMINAL-LIKE PROTEIN; ABRACL","url":"https://www.omim.org/entry/621425"},{"mim_id":"616450","title":"EF-HAND DOMAIN FAMILY, MEMBER D2; EFHD2","url":"https://www.omim.org/entry/616450"},{"mim_id":"616128","title":"FAMILY WITH SEQUENCE SIMILARITY 89, MEMBER B; FAM89B","url":"https://www.omim.org/entry/616128"},{"mim_id":"610932","title":"TWINFILIN ACTIN-BINDING PROTEIN 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ADF/cofilin.","date":"2002","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/11832213","citation_count":563,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28302793","id":"PMC_28302793","title":"Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15.","date":"2017","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/28302793","citation_count":533,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17110338","id":"PMC_17110338","title":"Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/17110338","citation_count":517,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21423176","id":"PMC_21423176","title":"Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation.","date":"2011","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21423176","citation_count":490,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12665801","id":"PMC_12665801","title":"Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.","date":"2003","source":"Nature biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/12665801","citation_count":485,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":25283,"output_tokens":3464,"usd":0.063905},"stage2":{"model":"claude-opus-4-6","input_tokens":6848,"output_tokens":3123,"usd":0.168472},"total_usd":0.539231,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":37044,"output_tokens":7397,"usd":0.111044},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":9758,"output_tokens":3270,"usd":0.19581}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"LIMK1 directly phosphorylates and inactivates CFL1, reducing its F-actin severing activity; miR-200b-3p and miR-429-5p suppress this pathway by targeting LIMK1, thereby decreasing CFL1 phosphorylation and inhibiting actin cytoskeleton reorganization, cell migration, and invasion in triple-negative breast cancer cells.\",\n      \"method\": \"Western blot for CFL1 phosphorylation after LIMK1 inhibition; miRNA mimic transfection with functional migration/invasion assays; luciferase reporter confirming LIMK1 as direct miRNA target\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (reporter assay, KD, functional assays) in single lab\",\n      \"pmids\": [\"29156719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In cardiomyocytes, vinculin (VCL) recruits the phosphatase SSH1, which dephosphorylates and activates cofilin (CFL), promoting F-actin rearrangement and myofilament maturation; mechanical forces from cardiac contractility regulate this VCL-SSH1-CFL axis.\",\n      \"method\": \"Interactome analysis (MS) of VCL in contracting vs. non-contracting cardiomyocytes; co-immunoprecipitation; zebrafish loss-of-function with myofilament phenotype readout; live imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, MS interactome, clean KO with defined cellular phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"31495694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SSH1, the canonical CFL1 phosphatase, dephosphorylates phospho-Ser403 of the autophagy receptor SQSTM1/p62, impairing its autophagic flux; this action is separable from SSH1-mediated CFL1 activation, demonstrating CFL1-independent SSH1 substrates.\",\n      \"method\": \"RNAi knockdown and overexpression of SSH1; defined mutant constructs; fluorescent autophagy flux reporters in cell lines and primary neurons; epistasis with SQSTM1 Ser403 phospho-mutants\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD/OE with mechanistic epistasis, multiple cell systems; single lab\",\n      \"pmids\": [\"33044112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Hhex enhances the interaction of RHOGDIA with RHOA/CDC42, maintaining them in an inactive state, which reduces downstream phosphorylation of CFL1 by LIMK, thereby preserving CFL1 F-actin-severing activity and inhibiting filopodium/lamellipodium formation and lung cancer cell migration.\",\n      \"method\": \"Co-immunoprecipitation of RHOGDIA with RHOA/CDC42; Western blot for p-CFL1; wound-healing and laser confocal imaging; knockdown/overexpression in NSCLC cells\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, phosphorylation readout, and functional migration assays in single lab\",\n      \"pmids\": [\"34321041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HUNK kinase directly phosphorylates GEF-H1 at Ser645, activating RhoA and triggering a cascade of LIMK-1/CFL-1 phosphorylation that stabilizes F-actin and inhibits EMT and metastasis in colorectal cancer cells.\",\n      \"method\": \"In vitro kinase assay demonstrating direct HUNK phosphorylation of GEF-H1; phospho-specific Western blot for CFL-1; KD/KO with EMT phenotype readout\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — direct kinase assay plus downstream phosphorylation cascade validated in cells; single lab\",\n      \"pmids\": [\"37193711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CFL1 promotes PHGDH (phosphoglycerate dehydrogenase) transcription, enhancing serine synthesis and antioxidant production, thereby scavenging ROS induced by sorafenib and reducing sorafenib sensitivity in hepatocellular carcinoma cells.\",\n      \"method\": \"Transcriptome sequencing of sorafenib-sensitive vs. insensitive HCC; siRNA-mediated CFL1 knockdown with metabolic (serine synthesis) and ROS readouts; nanoparticle co-delivery in vivo tumor model\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transcriptomic + functional KD with defined metabolic mechanism; single lab with in vivo validation\",\n      \"pmids\": [\"37203277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human non-muscle cofilin (CFL1) was cloned and mapped to chromosome 11q13; it functions as an actin-binding protein involved in translocation of the actin-cofilin complex from cytoplasm to nucleus.\",\n      \"method\": \"PCR amplification in rodent-human somatic cell hybrid panels; FISH with genomic cosmid clones; irradiation hybrid mapping\",\n      \"journal\": \"Annals of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal mapping with multiple orthogonal methods; functional assignment based on protein family characterization\",\n      \"pmids\": [\"8800436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CFL1 silencing in gastric cancer (AGS) cells suppresses cell migration and increases apoptosis, demonstrating CFL1 is required for actin-dependent motility in cancer cells.\",\n      \"method\": \"siRNA knockdown delivered by cationic nanoparticles; wound-healing migration assay; apoptosis assay; RT-qPCR confirmation of knockdown\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — KD with functional phenotype but no direct pathway placement beyond actin reorganization; single lab\",\n      \"pmids\": [\"31990066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CFL1 overexpression in pSS bone marrow mesenchymal stem cells rescues their migratory deficiency by upregulating CCR1, functioning through the CCL5/CCR1 axis downstream of CFL1.\",\n      \"method\": \"Lentiviral CFL1 overexpression; RNA-seq identifying CCR1 as downstream target; Transwell and wound-healing migration assays; CCR1 inhibitor rescue experiments; in vivo NOD mouse therapeutic model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function + RNA-seq + pharmacological rescue identify downstream pathway; single lab\",\n      \"pmids\": [\"38183912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Estradiol increases total CFL1 and phosphorylated CFL1 (pCFL1) protein levels in PBMCs and endocervical mucosa via LIM kinase activation; LIMK inhibition or CFL1 knockdown abolishes estradiol-mediated inhibition of HIV-1 infection, placing CFL1 in the E2-LIMK-CFL1 axis controlling HIV-1 susceptibility.\",\n      \"method\": \"Western blot for total and pCFL1 after E2 treatment; LIMKi3 pharmacological inhibition; siRNA CFL1 knockdown; HIV-1 infection assays in PBMCs and tissue explants\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological and genetic loss-of-function with defined infection phenotype; single lab, multiple cell/tissue systems\",\n      \"pmids\": [\"35418661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"c-Myc transcriptionally activates CFL1 by binding E-box elements in the CFL1 promoter; cofilin-1 is required for c-Myc-induced senescence (increased F-actin and nuclear G-actin), and a physical interaction between c-Myc and cofilin-1 proteins was detected, enhanced by H2O2.\",\n      \"method\": \"ChIP-qPCR identifying c-Myc binding to E-boxes in CFL1 promoter; cofilin-1 knockdown rescuing senescence phenotype; co-immunoprecipitation of c-Myc and cofilin-1; conditioned medium bystander migration assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP-qPCR for direct transcriptional activation, Co-IP for protein interaction, functional KD; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41888102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Expression of a non-functional form of CFL1 (nf-Cfl1) specifically in connective tissue mast cells (CTMCs) via Mcpt5-Cre causes complete absence of CTMCs, establishing that CFL1-dependent actin depolymerization is required for CTMC development; loss of CTMCs impairs systemic anaphylaxis induction.\",\n      \"method\": \"Conditional knock-in mouse (Mcpt5-Cre-nf-Cfl1fl/fl); cell counting of mast cells and basophils; anaphylaxis and inflammatory disease models\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with defined cellular and physiological phenotype; single lab\",\n      \"pmids\": [\"41684538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In a mouse model, overexpression of Cfl1 in the polymorphic layer of the hippocampal dentate gyrus increases motivation to seek alcohol rewards, impairs extinction of reward seeking, and inhibits molecular layer synapses; conversely, reducing Cfl1 has opposite effects, placing CFL1 as a regulator of hippocampal synaptic function and AUD-related behaviors.\",\n      \"method\": \"Local AAV-mediated Cfl1 overexpression and knockdown in hippocampal DG; two-photon imaging; behavioral assays (IntelliCage); electrophysiology of ML-DG synapses; RNA sequencing\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional gain/loss-of-function with defined synaptic and behavioral phenotypes; single lab\",\n      \"pmids\": [\"40931167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ADF and Cofilin1 (Cfl1) are required for microglia cytoskeletal dynamics; conditional knockout of ADF/Cfl1 in microglia causes increased stabilized F-actin accumulation, reduced microglial process motility, impaired migration toward lesions in vivo, and decreased learning and memory.\",\n      \"method\": \"Conditional microglial ADF/Cfl1 KO mice; in vivo two-photon imaging of microglial motility and lesion response; phalloidin staining for F-actin; behavioral cognition tests\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean conditional KO with in vivo imaging and defined cellular/behavioral phenotype; single lab; preprint\",\n      \"pmids\": [\"bio_10.1101_2024.09.27.615114\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CFL1 (cofilin-1) is an actin-severing/depolymerizing protein whose activity is primarily regulated by phosphorylation: LIMK1/2 phosphorylates and inactivates CFL1, while the phosphatase SSH1 (recruited to the membrane by vinculin under mechanical force) reactivates it; active CFL1 severs F-actin to drive actin cytoskeleton dynamics required for cell migration, CTMC development, microglial motility, cardiomyocyte myofilament maturation, hippocampal synaptic function, and chemoresistance, and its expression is transcriptionally activated by c-Myc via E-box elements in its promoter.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll systematically classify each paper before extracting discoveries.\n\n**Classification:**\n\nKEEP (canonical human/mammalian CFL1 = cofilin-1):\n- [2] LIMK1/CFL1 pathway in breast cancer — KEEP\n- [5] VCL-SSH1-CFL axis in zebrafish cardiomyocytes — KEEP (ortholog, consistent function)\n- [7] CFL1 promotes PHGDH transcription, serine synthesis in HCC — KEEP\n- [8] Chromosomal mapping of human CFL1 — KEEP\n- [13] CFL1 gene polymorphisms and spina bifida — KEEP\n- [14] Hhex/RHOA/CDC42-CFL1 axis in lung cancer — KEEP\n- [16] AGAP2-AS1/miR-182-5p/CFL1 axis in CRC — KEEP\n- [17] HUNK/GEF-H1/RhoA/LIMK-1/CFL-1 in CRC — KEEP\n- [18] SSH1 dephosphorylates SQSTM1 independent of CFL — KEEP (describes CFL1's phosphatase SSH1)\n- [20] CFL1 in inflammatory response (review) — KEEP\n- [21] nsSNPs in CFL1 affecting cofilin-1 structure — KEEP\n- [22] CFL1 silencing in gastric cancer — KEEP\n- [25] CFL1 restores BM-MSC migration via CCR1 — KEEP\n- [26] Neuronal CFL1 in HNSCC — KEEP\n- [29] Estradiol inhibits HIV-1 via CFL1 expression — KEEP\n- [37] Hippocampal Cfl/CFL1 in AUD — KEEP\n- [40] c-Myc transactivates CFL1, senescence — KEEP\n- [42] CFL1 in CML imatinib therapy — KEEP\n- [43] Cfl1 in mast cells (mouse KO) — KEEP\n- [47] ADF/Cfl1 in microglia (preprint) — KEEP\n- [48] CFL1 in EVs/ASD (preprint) — mentioned incidentally, EXCLUDE (not about CFL1 mechanism)\n- [49] RhoA/ROCK1/LIMK1/CFL1 in cervical cancer (preprint) — KEEP\n\nGene2pubmed curated:\n- [g6] ROCK→LIMK→cofilin phosphorylation — KEEP\n- [g10] LIMK1 phosphorylates cofilin at Ser3 — KEEP\n- [g25] Slingshot phosphatases dephosphorylate ADF/cofilin — KEEP\n\nEXCLUDE (symbol collisions or alt-locus):\n- [1] Plant CFL1 (WW domain, rice/Arabidopsis cuticle) — EXCLUDE (plant gene, incompatible)\n- [3] Expression correlation only — EXCLUDE (no mechanism)\n- [4] Candida albicans CFL1 (ferric reductase) — EXCLUDE (fungal collision)\n- [6] Lactobacillus CFL1 strain name — EXCLUDE\n- [9] C. albicans CFL1 ferric reductase — EXCLUDE\n- [10] Immunogenic antigen panel — EXCLUDE (no mechanism)\n- [11] Plant lectin CFL (Cratylia argentea) — EXCLUDE\n- [12] C. albicans Cfl1 ferric reductase — EXCLUDE\n- [15] Cucumber CFL (LFY homolog) — EXCLUDE\n- [19] ADF/CFL phylogenetics (computational) — EXCLUDE (no direct experiment on CFL1)\n- [23] Cryptococcus Cfl1 adhesin — EXCLUDE\n- [24] CFL bulb bioremediation — EXCLUDE\n- [27] CFL1-AS1 lncRNA (alt-locus product) — EXCLUDE\n- [28] Laccase P-CFL (fungal enzyme) — EXCLUDE\n- [30] Cucumber CFL gene — EXCLUDE\n- [31] miR-342-5p/CFL1 in HCC — KEEP (describes CFL1 mechanism)\n- [32] Cratylia argentea lectin CFL — EXCLUDE\n- [33] Lactobacillus CFL1 strain — EXCLUDE\n- [34] Cryptococcus Cfl1p adhesin — EXCLUDE\n- [35] C. elegans CFL-1 (F-box protein, FBXL20 homolog) — EXCLUDE (incompatible function)\n- [36] Lactobacillus CFL1 strain — EXCLUDE\n- [38] Anti-CFL1 autoantibody diagnostic — EXCLUDE (no mechanism)\n- [39] Calcaneofibular ligament (CFL) injury — EXCLUDE\n- [41] CFL number (math/fluid dynamics) — EXCLUDE\n- [44] Calcaneofibular ligament repair (preprint) — EXCLUDE\n- [45] Cell-free layer CFL in blood flow (preprint) — EXCLUDE\n- [46] Cell-free layer CFL in microfluidics (preprint) — EXCLUDE\n\nGene2pubmed:\n- [g1-g5, g7-g9, g11-g24, g26-g30] Large proteomics/interactome surveys — EXCLUDE (no specific CFL1 mechanism established; global phosphoproteomics screens don't constitute a specific mechanistic discovery for CFL1)\n- [g6] ROCK→LIMK→cofilin — KEEP\n- [g10] LIMK1 phosphorylates cofilin Ser3 — KEEP\n- [g25] SSH/Slingshot dephosphorylates cofilin — KEEP\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"LIM-kinase 1 (LIMK1) directly phosphorylates cofilin (CFL1) at Serine 3, both in vitro and in vivo. This phosphorylation abolishes cofilin's actin-depolymerizing activity. LIMK1 expression induces actin stress fiber formation, while an inactive LIMK1 suppresses Rac-induced lamellipodium formation. Rac and insulin activate LIMK1, placing LIMK1-mediated cofilin phosphorylation downstream of Rac in actin cytoskeletal reorganization.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (Ser3), overexpression and dominant-negative LIMK1 in cultured cells, actin depolymerization assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro phosphorylation with mutagenesis validation, multiple cell-based phenotypic readouts, foundational paper\",\n      \"pmids\": [\"9655398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The Rho-associated kinase ROCK phosphorylates and activates LIM-kinase, which in turn phosphorylates cofilin (CFL1) to inactivate its actin-depolymerizing activity. This ROCK→LIMK→cofilin cascade mediates Rho-induced actin cytoskeletal reorganization (stress fiber formation) and neurite retraction. ROCK does not phosphorylate cofilin directly; the Y-27632 ROCK inhibitor blocks cofilin phosphorylation in cells.\",\n      \"method\": \"Pharmacological inhibition (Y-27632), overexpression of LIMK1 in HeLa cells, in vivo phosphorylation assays in neuroblastoma cells, epistasis with dominant-negative constructs\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — cell-based epistasis with pharmacological inhibitor and overexpression, replicated across labs\",\n      \"pmids\": [\"10436159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The Slingshot (SSH) family of phosphatases dephosphorylate phospho-cofilin (p-CFL1) to reactivate its actin-depolymerizing function. SSH binds F-actin and dephosphorylates P-cofilin both in cultured cells and in cell-free assays. Loss of SSH in Drosophila dramatically increases F-actin levels and phospho-cofilin, disrupting epidermal cell morphogenesis. Human SSH homologs (hSSH1) suppress LIMK1-induced actin reorganization by reactivating cofilin.\",\n      \"method\": \"Drosophila loss-of-function genetics, cell-free phosphatase assays, overexpression of hSSH in mammalian cells, F-actin binding assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cell-free dephosphorylation assay plus in vivo genetic validation in Drosophila and mammalian cells\",\n      \"pmids\": [\"11832213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human non-muscle cofilin (CFL1) was cloned from a promyelocytic cDNA library and mapped to chromosome 11q13 by PCR in somatic cell hybrids and FISH. Muscle-type cofilin (CFL2) was mapped to chromosome 14. CFL1 encodes an actin-binding protein involved in translocation of the actin-cofilin complex from cytoplasm to nucleus.\",\n      \"method\": \"cDNA cloning, PCR mapping in rodent-human somatic cell hybrids, FISH with genomic cosmid clones, irradiation hybrid panel mapping\",\n      \"journal\": \"Annals of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal mapping with multiple orthogonal methods\",\n      \"pmids\": [\"8800436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Genetic variants (SNPs) in the human CFL1 gene are associated with increased spina bifida risk, consistent with the essential role of CFL1 in actin depolymerization during neural tube closure. Cfl1 knockout mice fail to close the neural tube at E10.5 and die in utero, establishing CFL1 as essential for neural tube development.\",\n      \"method\": \"Population-based case-control SNP association study, reference to Cfl1 knockout mouse phenotype\",\n      \"journal\": \"BMC medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — genetic association with mechanistic reference to KO mouse; KO data from prior work\",\n      \"pmids\": [\"17352815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"miR-200b-3p and miR-429-5p directly target LIMK1, reducing LIMK1 expression and consequently decreasing phosphorylation (inactivation) of cofilin-1 (CFL1). Reduced LIMK1 activity allows CFL1 to remain active, altering F-actin/G-actin dynamics. This LIMK1/CFL1 pathway mediates the suppressive effects of these miRNAs on triple-negative breast cancer cell proliferation, migration, and invasion.\",\n      \"method\": \"Luciferase reporter assay for direct miRNA targeting of LIMK1, Western blotting for CFL1 and p-CFL1, migration/invasion assays, cell cycle analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct target validation plus downstream phosphorylation readout, single lab\",\n      \"pmids\": [\"29156719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In contracting zebrafish cardiomyocytes, mechanical forces regulate vinculin (VCL) localization and activation. VCL recruits the phosphatase SSH1 and its effector cofilin (CFL) to regulate F-actin rearrangement and promote myofilament maturation. The VCL-SSH1-CFL axis is essential: loss of VCL or disruption of this pathway impairs myofilament maturation in response to cardiac contractility.\",\n      \"method\": \"Zebrafish genetic knockouts, interactome analysis (contracting vs. non-contracting cardiomyocytes by MS), co-immunoprecipitation, live imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP/interactome with genetic loss-of-function and defined cellular phenotype in vivo\",\n      \"pmids\": [\"31495694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SSH1, the canonical cofilin (CFL1) phosphatase, also dephosphorylates phospho-Ser403-SQSTM1/p62, thereby impairing autophagic cargo clearance. This action of SSH1 on SQSTM1 is separable from SSH1-mediated CFL1 activation and independent of CFL1, revealing a bifurcated SSH1 function. SSH1-mediated inhibition of SQSTM1 impairs clearance of phospho-MAPT/tau.\",\n      \"method\": \"RNAi knockdown, overexpression, defined phospho-mutant constructs, fluorescent autophagy reporters, experiments in cell lines, primary neurons, and mouse brains\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (genetic, biochemical, reporter) in multiple cell systems clarifying CFL1-independent SSH1 action\",\n      \"pmids\": [\"33044112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Hhex (hematopoietically expressed homeobox) inhibits CFL1 phosphorylation, keeping CFL1 in its active F-actin-severing form, thereby suppressing cell migration and protrusion formation in lung cancer cells. Mechanistically, Hhex enhances the interaction of RHOGDIA with RHOA/CDC42, maintaining these GTPases in their inactive state and blocking the RHOA/CDC42→p-CFL1 signaling cascade. This prevents filopodium and lamellipodium formation.\",\n      \"method\": \"Western blot, co-immunoprecipitation, wound-healing scratch assay, laser confocal microscopy, overexpression/knockdown in NSCLC and HEK293FT cells\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP and phosphorylation assays with functional readout, single lab\",\n      \"pmids\": [\"34321041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"E2F4-induced lncRNA AGAP2-AS1 acts as a competing endogenous RNA (ceRNA) to sponge miR-182-5p, thereby upregulating CFL1 expression. CFL1 restoration counteracts the suppression of CRC cell growth, migration, invasion, and EMT caused by AGAP2-AS1 depletion, placing CFL1 as a downstream effector of this AGAP2-AS1/miR-182-5p axis in colorectal cancer progression.\",\n      \"method\": \"RT-qPCR, Western blot, luciferase reporter assays, rescue experiments in vitro and in vivo (xenograft)\",\n      \"journal\": \"Digestive and liver disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — functional rescue experiment plus reporter validation, single lab\",\n      \"pmids\": [\"34838479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Computational analysis of nonsynonymous SNPs in CFL1 identifies L84P and L99A as the most damaging variants. Molecular docking shows these variants reduce cofilin-1 binding affinity for actin, and molecular dynamics simulations confirm protein structure destabilization. The actin-binding regions of cofilin-1 are highly conserved and critical for function.\",\n      \"method\": \"In silico prediction tools (SIFT, PolyPhen-2, DynaMut/DUET), molecular docking, molecular dynamics simulation\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational prediction only, no experimental validation of variants\",\n      \"pmids\": [\"35092861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Estradiol (E2) inhibits HIV-1 infection in PBMCs and endocervical tissue and simultaneously increases total CFL1 and phosphorylated CFL1 (p-CFL1) protein expression, raising the p-CFL1/CFL1 ratio. LIM kinase inhibitor (LIMKi3) abrogates both the anti-HIV effect and E2-induced CFL1/p-CFL1 upregulation, and CFL1 knockdown partially restores HIV infection, indicating that LIMK-mediated CFL1 phosphorylation is part of the mechanism by which E2 restricts HIV-1 entry/spread.\",\n      \"method\": \"HIV infection assay, siRNA knockdown of CFL1, pharmacological LIMK inhibition (LIMKi3), Western blotting for total and p-CFL1, dose-response experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — siRNA knockdown + pharmacological inhibition with functional readout, single lab\",\n      \"pmids\": [\"35418661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CFL1 promotes transcription of phosphoglycerate dehydrogenase (PHGDH), enhancing serine synthesis and metabolism to increase antioxidant production, thereby scavenging ROS induced by sorafenib and reducing sorafenib sensitivity in hepatocellular carcinoma. siRNA-mediated CFL1 silencing re-sensitizes HCC cells to sorafenib.\",\n      \"method\": \"Transcriptome sequencing comparison of sorafenib-sensitive vs. insensitive patients, CFL1 siRNA knockdown via nanoparticles, PHGDH promoter assays, ROS measurement, in vivo tumor growth assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway identified by transcriptomics plus functional siRNA knockdown with multiple readouts in vitro and in vivo\",\n      \"pmids\": [\"37203277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HUNK kinase directly phosphorylates GEF-H1 at Serine 645, which activates RhoA and triggers a phosphorylation cascade of LIMK-1 and CFL-1, thereby stabilizing F-actin and inhibiting EMT and metastasis in colorectal cancer cells. This places CFL-1 phosphorylation as a downstream effector of the HUNK→GEF-H1→RhoA→LIMK-1 cascade.\",\n      \"method\": \"In vitro kinase assay (HUNK phosphorylating GEF-H1), site-directed mutagenesis (S645), Western blotting for LIMK-1/p-CFL-1, migration/invasion assays, clinical tissue analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay with mutagenesis plus cell-based epistasis and clinical validation\",\n      \"pmids\": [\"37193711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CFL1 overexpression in pSS bone marrow mesenchymal stem cells rescues their impaired migration and proliferation. RNA-seq identifies CCR1 as a downstream target gene of CFL1, and inhibition of CCR1 with BX431 suppresses the CFL1-induced increase in migration/proliferation, establishing that CFL1 promotes BM-MSC motility via upregulation of the CCL5/CCR1 axis.\",\n      \"method\": \"Lentivirus-mediated CFL1 overexpression, RNA-seq, Transwell migration assay, wound healing assay, CCR1 inhibitor rescue experiment (BX431), NOD mouse therapeutic model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — RNA-seq plus pharmacological rescue with functional assays, single lab\",\n      \"pmids\": [\"38183912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"c-Myc directly transactivates the CFL1 promoter by binding to E-box elements (particularly middle and proximal E-boxes), inducing cofilin-1 expression. Cofilin-1 upregulation is required for c-Myc-induced oncogene-induced senescence (OIS): cofilin-1 knockdown suppresses cMIS. Physical interaction between c-Myc and cofilin-1 was detected, enhanced by H2O2. The conditioned medium from cMIS cells promotes migration and proliferation of other NSCLC cells, an effect abrogated by cofilin-1 silencing.\",\n      \"method\": \"ChIP-qPCR, luciferase reporter assay, siRNA knockdown, co-immunoprecipitation (c-Myc and cofilin-1), senescence assays, conditioned medium transfer experiments\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP-qPCR demonstrating direct promoter binding, Co-IP showing physical interaction, functional knockdown with defined phenotypic readout\",\n      \"pmids\": [\"41888102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Conditional knockout of neuronal CFL1 (cofilin-1) impedes tumor-nerve interactions in head and neck squamous cell carcinoma. HNSCC cells induce CFL1 expression in adjacent neurons, and it is neuronal (not tumoral) CFL1 that drives cancer-nerve crosstalk and perineural invasion, as demonstrated by multiplex fluorescent immunohistochemistry localizing CFL1 to nerves and by conditional KO experiments.\",\n      \"method\": \"Multiplex fluorescent immunohistochemistry, conditional neuronal CFL1 knockout, Gene Ontology/GSEA analysis\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — conditional KO with defined cellular phenotype, localization data, single lab\",\n      \"pmids\": [\"38353363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-342-5p targets CFL1 (cofilin-1) mRNA in HDACi-resistant HCC cells; overexpression of miR-342-5p decreases cofilin-1 protein expression and increases ROS-mediated apoptosis, sensitizing cells to HDACi treatment. This identifies the miR-342-5p/CFL1 axis as a mediator of HDACi resistance.\",\n      \"method\": \"miRNA microarray, qRT-PCR, gain/loss-of-function studies, Western blot, apoptosis assays (Bax, caspase-3, Bcl-2), ROS measurement\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — functional miRNA/target relationship with biochemical readouts, single lab\",\n      \"pmids\": [\"39152428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CFL1 (cofilin-1) is required for mast cell development: expression of a non-functional form of Cfl1 in connective tissue mast cells (CTMCs) using Mcpt5-Cre results in complete absence of CTMCs without affecting basophils. CTMCs lacking Cfl1 function show impaired systemic anaphylaxis but normal susceptibility to contact hypersensitivity and psoriasis-like dermatitis, demonstrating that cofilin-1-mediated actin dynamics are essential specifically for CTMC generation.\",\n      \"method\": \"Conditional knock-in mouse (Mcpt5-Cre-nf-Cfl1fl/fl), mast cell/basophil enumeration, anaphylaxis model, contact hypersensitivity model, imiquimod model, vaccinia virus infection\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional genetic KO in vivo with multiple disease model readouts showing specific CTMC requirement\",\n      \"pmids\": [\"41684538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Overexpression of cofilin (Cfl1) in the polymorphic layer of the hippocampal dentate gyrus increases motivation to seek alcohol and sucrose rewards, impairs extinction of alcohol seeking, and inhibits ML-DG synapses; reducing Cfl1 has opposite effects. Three SNPs in human CFL1 (rs369270402, rs2376005, rs36124259) are associated with increased AUD risk, and CFL1 mRNA blood levels correlate with alcohol-related hospital admissions. AUD-prone mice show differential hippocampal Cfl expression linked to actin cytoskeleton and synaptic function genes.\",\n      \"method\": \"RNA sequencing, local viral vector Cfl overexpression/knockdown in DG, IntelliCage behavioral model, electrophysiology (ML-DG synapse recording), human genetic association study\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain/loss-of-function with electrophysiology and behavioral readouts plus human genetic validation\",\n      \"pmids\": [\"40931167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Deficiency of ADF and Cofilin1 (Cfl1) in microglia causes profound morphological changes, reduces microglial fine process motility and migration toward laser-induced lesions in vivo, and increases stabilized F-actin with altered microtubule dynamics. Microglial ADF/Cfl1 deficiency also impairs learning and memory, linking microglial cytoskeletal dynamics to neuronal cognitive function.\",\n      \"method\": \"Conditional microglial ADF/Cfl1 knockout, in vivo two-photon imaging, F-actin immunostaining, microtubule dynamics assays, behavioral learning/memory tests\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with in vivo imaging and behavioral readouts, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.09.27.615114\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CFL1 (cofilin-1) is an actin-depolymerizing/severing protein whose activity is primarily regulated by reversible phosphorylation at Serine 3: LIM-kinase 1 (downstream of Rac→LIMK1 and Rho→ROCK→LIMK1 signaling) phosphorylates and inactivates CFL1, while the Slingshot (SSH1) phosphatase family reactivates it by dephosphorylation; active CFL1 severs and depolymerizes actin filaments to drive cytoskeletal remodeling essential for cell migration, lamellipodium formation, neural tube closure, cardiac myofilament maturation, mast cell development, microglial motility, synaptic plasticity, and hippocampal-dependent cognition, and its dysregulation is implicated in cancer invasion (via LIMK1/CFL1, HUNK→GEF-H1→RhoA→LIMK-1→CFL-1, and Hhex→RHOGDIA→RHOA/CDC42→CFL1 axes), sorafenib resistance (via CFL1-driven PHGDH/serine metabolism), senescence-associated secretion (via direct c-Myc transactivation of the CFL1 promoter), and alcohol use disorder (via hippocampal Cfl1 regulation of reward circuitry).\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CFL1 (cofilin-1) is an actin-severing and depolymerizing protein whose regulated cycling between active and inactive states controls actin cytoskeleton dynamics critical for cell migration, immune cell development, synaptic function, and stress responses. CFL1 is inactivated by LIMK1/2-mediated phosphorylation downstream of RhoA/CDC42 signaling and reactivated by the phosphatase SSH1, which is recruited to sites of mechanical force by vinculin in cardiomyocytes to drive F-actin rearrangement and myofilament maturation [PMID:29156719, PMID:31495694, PMID:34321041, PMID:37193711]. CFL1-dependent actin depolymerization is essential for connective tissue mast cell development, microglial process motility and lesion response, and hippocampal synaptic plasticity regulating reward-seeking behavior [PMID:41684538, PMID:40931167]. CFL1 expression is transcriptionally activated by c-Myc via E-box elements in its promoter and contributes to c-Myc-induced cellular senescence, while in cancer cells CFL1 promotes chemoresistance by upregulating PHGDH-dependent serine synthesis and ROS scavenging [PMID:41888102, PMID:37203277].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Cloning and chromosomal mapping of human CFL1 established it as a non-muscle actin-binding protein at 11q13 capable of translocating the actin-cofilin complex from cytoplasm to nucleus, providing the first molecular identity for this gene in humans.\",\n      \"evidence\": \"PCR-based somatic cell hybrid mapping, FISH, and irradiation hybrid mapping\",\n      \"pmids\": [\"8800436\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of nuclear translocation of the actin-cofilin complex was not characterized\", \"Functional distinction from cofilin-2 (muscle isoform) was not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstration that LIMK1 directly phosphorylates and inactivates CFL1's F-actin severing activity established the key inhibitory kinase and linked the LIMK1-CFL1 axis to actin-dependent cell migration and invasion.\",\n      \"evidence\": \"Western blot for CFL1 phosphorylation after LIMK1 inhibition; miRNA mimic transfection with luciferase reporter and migration/invasion assays in TNBC cells\",\n      \"pmids\": [\"29156719\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro kinase assay for LIMK1-CFL1 was not shown in this study\", \"Whether other kinases contribute to CFL1 phosphorylation in this context was not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of the VCL-SSH1-CFL axis in cardiomyocytes revealed how mechanical forces reactivate CFL1: vinculin recruits the phosphatase SSH1, which dephosphorylates CFL1 to promote F-actin rearrangement and myofilament maturation.\",\n      \"evidence\": \"MS interactome of VCL in contracting vs. non-contracting cardiomyocytes; reciprocal Co-IP; zebrafish loss-of-function with myofilament phenotype; live imaging\",\n      \"pmids\": [\"31495694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this force-sensing mechanism operates in non-cardiac cell types was not tested\", \"Direct measurement of CFL1 activity (not just phosphorylation) at sites of VCL recruitment was lacking\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that SSH1 dephosphorylates SQSTM1/p62 independently of CFL1 activation clarified that the SSH1-CFL1 axis is one of multiple SSH1 outputs, refining the specificity of CFL1 regulation within the broader phosphatase network.\",\n      \"evidence\": \"SSH1 RNAi/overexpression with SQSTM1 Ser403 phospho-mutant epistasis and autophagy flux reporters in cell lines and primary neurons\",\n      \"pmids\": [\"33044112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether competition between CFL1 and SQSTM1 for SSH1 activity occurs in physiological settings was not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placing CFL1 downstream of RhoA/CDC42-LIMK signaling—modulated by Hhex via RhoGDIA—demonstrated that CFL1 phosphorylation status integrates multiple upstream GTPase inputs to control filopodium/lamellipodium formation and cancer cell motility.\",\n      \"evidence\": \"Co-IP of RhoGDIA with RhoA/CDC42; Western blot for p-CFL1; wound-healing assays in NSCLC cells with Hhex KD/OE\",\n      \"pmids\": [\"34321041\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct CFL1 activity assay was used; only phosphorylation was measured as a proxy\", \"Contribution of other actin regulators downstream of RhoA was not deconvolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Estradiol-mediated upregulation of total CFL1 and pCFL1 via LIMK placed CFL1 in a hormonal signaling axis that controls cortical actin remodeling and HIV-1 susceptibility in immune cells and mucosal tissue, expanding CFL1's role beyond cell migration to host-pathogen defense.\",\n      \"evidence\": \"Western blot after E2 treatment; LIMKi3 pharmacological inhibition and siRNA CFL1 KD with HIV-1 infection assays in PBMCs and endocervical explants\",\n      \"pmids\": [\"35418661\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CFL1 acts on HIV entry, post-entry trafficking, or both was not resolved\", \"In vivo hormonal regulation of CFL1 phosphorylation was not confirmed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Two independent studies consolidated the RhoA-LIMK-CFL1 cascade as a convergence point: HUNK kinase phosphorylates GEF-H1 to activate RhoA-LIMK-CFL1 signaling that stabilizes F-actin and inhibits EMT in CRC, while CFL1 was shown to promote PHGDH transcription and serine synthesis for ROS scavenging and sorafenib resistance in HCC, revealing a non-canonical transcriptional effector function.\",\n      \"evidence\": \"In vitro kinase assay for HUNK-GEF-H1; phospho-CFL1 Western blot and EMT readouts (CRC); transcriptome sequencing, siRNA CFL1 KD with metabolic readouts and in vivo nanoparticle delivery (HCC)\",\n      \"pmids\": [\"37193711\", \"37203277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CFL1 promotes PHGDH transcription is unknown—whether direct or via actin-dependent transcription factor regulation was not determined\", \"Whether CFL1's metabolic role extends beyond HCC was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CFL1 overexpression in bone marrow MSCs rescues migration via the CCL5/CCR1 axis, identifying CCR1 as a transcriptional target downstream of CFL1 and positioning CFL1 as a regulator of chemokine receptor expression beyond its canonical actin-severing role.\",\n      \"evidence\": \"Lentiviral CFL1 OE; RNA-seq identifying CCR1; Transwell migration with CCR1 inhibitor rescue; in vivo NOD mouse model\",\n      \"pmids\": [\"38183912\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CFL1 regulates CCR1 transcription directly or through actin-dependent nuclear signaling was not resolved\", \"Relevance to other autoimmune conditions was not explored\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Bidirectional manipulation of Cfl1 in hippocampal dentate gyrus established that CFL1 levels regulate synaptic strength at molecular layer synapses and reward-seeking behavior, providing the first causal link between CFL1-mediated actin dynamics and circuit-level neural function.\",\n      \"evidence\": \"AAV-mediated Cfl1 OE and KD in mouse DG; two-photon imaging; electrophysiology of ML-DG synapses; IntelliCage behavioral assays; RNA-seq\",\n      \"pmids\": [\"40931167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CFL1 acts presynaptically, postsynaptically, or both was not determined\", \"Phosphorylation-dependent regulation of CFL1 in this circuit was not examined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Two studies extended CFL1's roles to immune cell ontogeny and senescence: expression of non-functional CFL1 in CTMCs caused their complete developmental absence, while c-Myc was shown to transcriptionally activate CFL1 via E-box promoter elements, with CFL1 required for c-Myc-induced senescence through F-actin and nuclear G-actin remodeling.\",\n      \"evidence\": \"Conditional knock-in mouse (Mcpt5-Cre-nf-Cfl1) with mast cell quantification and anaphylaxis models; ChIP-qPCR for c-Myc at CFL1 E-boxes; CFL1 KD rescuing senescence; Co-IP of c-Myc and CFL1\",\n      \"pmids\": [\"41684538\", \"41888102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The developmental stage at which CFL1 activity is required for CTMC specification versus survival is unknown\", \"Functional significance of the physical c-Myc–CFL1 protein interaction beyond transcriptional regulation is not established\", \"Whether CFL1's senescence role is relevant in vivo was not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how CFL1 exerts its non-canonical transcriptional effects (promoting PHGDH and CCR1 expression)—whether through nuclear actin dynamics, chromatin remodeling, or indirect signaling—and whether its diverse tissue-specific roles (mast cell development, synaptic regulation, chemoresistance) share a unified actin-dependent mechanism or involve distinct effector pathways.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of CFL1 regulation at phospho-Ser3 in a cellular context\", \"Nuclear functions of CFL1 beyond actin-cofilin translocation are mechanistically undefined\", \"Whether CFL1 and ADF/destrin have truly non-redundant roles in each tissue context has not been systematically tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 3, 4, 7, 11, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 3, 4, 7, 11, 13]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3, 4, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"LIMK1\",\n      \"SSH1\",\n      \"VCL\",\n      \"MYC\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Cofilin-1 (CFL1) is an essential actin-severing and depolymerizing protein whose regulated cycling between active (dephosphorylated) and inactive (Ser3-phosphorylated) states drives actin cytoskeletal remodeling in virtually all non-muscle cell types. LIM-kinase 1 phosphorylates CFL1-Ser3 downstream of Rac and Rho/ROCK signaling to inactivate it, while the Slingshot phosphatase SSH1 dephosphorylates and reactivates it, and this toggle controls lamellipodium dynamics, cell migration, neural tube closure, connective-tissue mast cell development, cardiac myofilament maturation, and hippocampal synaptic function [PMID:9655398, PMID:10436159, PMID:11832213, PMID:31495694, PMID:41684538, PMID:40931167]. Beyond cytoskeletal regulation, CFL1 is transcriptionally induced by c-Myc at E-box elements in its promoter and is required for oncogene-induced senescence and the associated secretory phenotype, and it promotes PHGDH-dependent serine metabolism to confer sorafenib resistance in hepatocellular carcinoma [PMID:41888102, PMID:37203277]. Genetic variants in CFL1 are associated with increased risk of spina bifida and alcohol use disorder, consistent with its indispensable roles in neural tube closure and hippocampal reward circuitry [PMID:17352815, PMID:40931167].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Cloning and chromosomal mapping of human CFL1 to 11q13 established its identity as a non-muscle actin-binding protein distinct from muscle-type CFL2, providing the molecular entry point for functional studies.\",\n      \"evidence\": \"cDNA cloning from promyelocytic library, somatic cell hybrid PCR, and FISH mapping\",\n      \"pmids\": [\"8800436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional assay performed at this stage\", \"Regulation of CFL1 activity was unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of LIM-kinase 1 as the direct Ser3 kinase for CFL1 resolved how Rac signaling controls actin depolymerization, establishing the foundational LIMK1→p-CFL1 inactivation mechanism.\",\n      \"evidence\": \"In vitro kinase assay with site-directed Ser3 mutagenesis, actin depolymerization assay, dominant-negative LIMK1 in cultured cells\",\n      \"pmids\": [\"9655398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream activation of LIMK1 by Rho-family effectors not yet mapped\", \"No phosphatase for CFL1 reactivation had been identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Placing ROCK upstream of LIMK in a Rho→ROCK→LIMK→CFL1 cascade explained how Rho-induced stress fiber formation and neurite retraction converge on cofilin inactivation.\",\n      \"evidence\": \"Pharmacological ROCK inhibition (Y-27632), epistasis with dominant-negative constructs in HeLa and neuroblastoma cells\",\n      \"pmids\": [\"10436159\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of CFL1 reactivation still unknown\", \"Whether additional kinases target CFL1-Ser3 remained open\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery of the Slingshot (SSH) phosphatase family as the CFL1-Ser3 phosphatase completed the phospho-cycling circuit, showing that F-actin-bound SSH dephosphorylates p-CFL1 to restore severing activity.\",\n      \"evidence\": \"Cell-free phosphatase assay, Drosophila loss-of-function genetics showing elevated F-actin and p-cofilin, mammalian overexpression rescuing LIMK1-induced phenotypes\",\n      \"pmids\": [\"11832213\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial and temporal regulation of SSH1 in specific tissues was uncharacterized\", \"Whether SSH has CFL1-independent substrates was unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Association of CFL1 SNPs with spina bifida, combined with the neural tube closure failure in Cfl1-knockout mice, established CFL1 as a developmental disease gene and demonstrated its in vivo essentiality.\",\n      \"evidence\": \"Population-based case-control SNP association study referencing prior Cfl1 KO lethality at E10.5\",\n      \"pmids\": [\"17352815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific CFL1 variants were not functionally validated\", \"Contribution of CFL1 versus other actin regulators to neural tube closure was not dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of a VCL→SSH1→CFL axis in contracting zebrafish cardiomyocytes revealed that mechanical force-dependent cofilin activation drives myofilament maturation, extending CFL1 function beyond migration into cardiac development.\",\n      \"evidence\": \"Zebrafish VCL knockout, interactome mass spectrometry, co-immunoprecipitation, live imaging of myofilament assembly\",\n      \"pmids\": [\"31495694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this axis operates identically in mammalian cardiomyocytes was not tested\", \"Structural basis of VCL-SSH1 recruitment was not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that SSH1 dephosphorylates SQSTM1/p62 independently of CFL1 clarified that CFL1 is not the sole physiological substrate of its activating phosphatase, defining the boundary of CFL1 involvement in autophagy.\",\n      \"evidence\": \"RNAi knockdown, phospho-mutant constructs, fluorescent autophagy reporters in primary neurons and cell lines\",\n      \"pmids\": [\"33044112\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CFL1 itself has any direct role in autophagy remained unclear\", \"Other potential SSH1 substrates besides CFL1 and SQSTM1 were not surveyed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Two independent studies placed CFL1 phosphorylation status as a convergence node for upstream oncogenic signaling: the Hhex→RHOGDIA→RHOA/CDC42 axis suppresses CFL1 phosphorylation to restrict lung cancer migration, while the AGAP2-AS1/miR-182-5p ceRNA axis upregulates CFL1 expression to promote colorectal cancer EMT.\",\n      \"evidence\": \"Co-IP, wound-healing assays, luciferase miRNA-target reporters, xenograft rescue experiments\",\n      \"pmids\": [\"34321041\", \"34838479\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neither study demonstrated direct CFL1-actin severing in the cancer context biochemically\", \"Whether CFL1 expression level versus phosphorylation status is the more critical variable in vivo was not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of HUNK→GEF-H1(pSer645)→RhoA→LIMK-1→p-CFL1 as a metastasis-suppressive cascade in CRC extended the kinase hierarchy upstream of CFL1, while CFL1-driven PHGDH transcription and serine metabolism explained sorafenib resistance in HCC, revealing a non-cytoskeletal transcriptional function for CFL1.\",\n      \"evidence\": \"In vitro HUNK kinase assay with Ser645 mutagenesis, epistasis in CRC cells; CFL1 siRNA nanoparticle delivery with PHGDH promoter assays and ROS measurement in HCC xenografts\",\n      \"pmids\": [\"37193711\", \"37203277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CFL1 activates PHGDH transcription mechanistically (as a cytoskeletal protein) was not explained\", \"Whether CFL1 enters the nucleus to regulate transcription directly was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multiple 2024 studies broadened CFL1's physiological scope: conditional knockout showed CFL1 is absolutely required for connective-tissue mast cell development; c-Myc was identified as a direct transcriptional activator of CFL1 required for oncogene-induced senescence; and neuronal CFL1 was shown to mediate tumor-nerve crosstalk in HNSCC.\",\n      \"evidence\": \"Mcpt5-Cre conditional knock-in mouse with anaphylaxis models; ChIP-qPCR and Co-IP showing c-Myc–CFL1 promoter binding and physical interaction; conditional neuronal CFL1 KO in HNSCC model\",\n      \"pmids\": [\"41684538\", \"41888102\", \"38353363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CFL1 loss specifically prevents CTMC but not basophil development was not mechanistically resolved\", \"Whether c-Myc–CFL1 physical interaction has a function beyond transcriptional regulation is unknown\", \"Neuronal CFL1 contribution to perineural invasion needs replication\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Gain- and loss-of-function manipulation of Cfl1 in hippocampal dentate gyrus demonstrated that CFL1 regulates synaptic transmission and reward-seeking behavior, and human CFL1 SNPs were associated with alcohol use disorder risk, establishing CFL1 as a neuromodulatory factor in addiction circuitry.\",\n      \"evidence\": \"Viral vector Cfl overexpression/knockdown in DG, electrophysiology at ML-DG synapses, IntelliCage behavioral paradigm, human SNP association and mRNA correlation analysis\",\n      \"pmids\": [\"40931167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific CFL1-dependent actin substrates at hippocampal synapses were not identified\", \"Whether CFL1 phosphorylation or total expression drives the AUD-related phenotype is unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how CFL1 activates transcription of genes such as PHGDH (whether through nuclear translocation or indirect signaling), the structural basis of the CFL1-actin severing/depolymerization mechanism at atomic resolution in a mammalian context, and the cell-type-specific determinants that make certain lineages (CTMCs, neural tube, hippocampal neurons) uniquely dependent on CFL1 despite ubiquitous expression.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of human CFL1 bound to F-actin during severing\", \"Nuclear function of CFL1 is mechanistically undefined\", \"Redundancy with ADF/CFL2 across tissues is poorly mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 2, 6, 8, 18, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 2, 6, 8, 13, 18, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 8, 13]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [19, 20]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"LIMK1\",\n      \"SSH1\",\n      \"ROCK1\",\n      \"VCL\",\n      \"MYC\",\n      \"ACTA1\",\n      \"RHOA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}