{"gene":"FMNL3","run_date":"2026-06-09T23:54:44","timeline":{"discoveries":[{"year":2011,"finding":"The C-terminal 76 amino acids of FMNL3 contain a WH2-like sequence that binds actin monomers competitively with other WH2 domains and with profilin, and also binds filament barbed ends. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high-affinity inhibition of barbed end elongation. Mutation of two aliphatic residues blocking high-affinity actin binding does not affect polymerization enhancement, but mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement.","method":"In vitro actin polymerization assays, barbed-end elongation assays, mutagenesis of FMNL3 C-terminal domain","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple orthogonal biochemical assays in a single rigorous study","pmids":["22094460"],"is_preprint":false},{"year":2010,"finding":"The FH2 domain of FRL2/FMNL3 is required for filopodia assembly; the FH1 domain is interchangeable with that of FRL1, but the FH2 domain cannot be substituted by FRL1's FH2. A mutation compromising FRL2's barbed-end binding ability abolishes filopodia assembly. Full-length FMNL3 shows reduced filopodia-stimulating activity compared to FH1-FH2 constructs, suggesting autoinhibitory regulation by additional domains (GBD, DID, DAD).","method":"Overexpression of FH1-FH2 constructs and chimeric constructs in multiple cell types, barbed-end binding mutant analysis, fluorescence microscopy","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-swap chimeras, point mutagenesis, and multiple cell-type validation in a single focused mechanistic study","pmids":["20862687"],"is_preprint":false},{"year":2015,"finding":"In zebrafish, Cdc42 binds to and activates FMNL3 downstream of BMP/Arhgef9b signaling to promote assembly of endothelial filopodia during angiogenic sprouting of the caudal vein plexus. Active Cdc42 stimulates FMNL3 to drive filopodial extension.","method":"Genetic epistasis in zebrafish (morpholino knockdown, dominant-active/dominant-negative constructs), Co-IP/binding assays for Cdc42–FMNL3 interaction, in vivo live imaging","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis in vivo combined with direct binding evidence, replicated across multiple genetic conditions","pmids":["25584797"],"is_preprint":false},{"year":2012,"finding":"Activated FMNL3 triggers alignment of stabilized microtubules into parallel arrays directed toward the growing tip during endothelial cell elongation in angiogenesis. FMNL3 knockdown in zebrafish causes profound developmental angiogenesis defects rescued by human FMNL3 expression.","method":"siRNA knockdown in endothelial cells, co-culture angiogenesis assay, immunofluorescence of microtubules, morpholino knockdown and rescue in zebrafish","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function in two systems (mammalian cells and zebrafish) with defined morphological readouts and rescue experiment","pmids":["22275430"],"is_preprint":false},{"year":2012,"finding":"N-myristoylation of FMNL3 (at Gly2) is required for its plasma membrane localization and for induction of cellular morphological changes; replacing Gly2 with Ala or using an N-myristoylation inhibitor abolishes membrane association and morphological effects.","method":"Site-directed mutagenesis (Gly2Ala), pharmacological inhibition of N-myristoylation, immunofluorescence in HEK293T cells","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus pharmacological inhibition in a single lab, two orthogonal methods","pmids":["22790947"],"is_preprint":false},{"year":2014,"finding":"Endogenous FMNL3 localizes predominantly in a punctate pattern at the plasma membrane (>95% of puncta), enriches in filopodia and membrane ruffles and at nascent cell-cell adhesions. A small population of cytoplasmic puncta of endocytic origin enriches near cell-cell contacts and fuses with the plasma membrane there. FMNL3 siRNA suppression decreases filopodia number and compromises cell-cell adhesion in migrating cell sheets, indicating FMNL3 assembles actin-based protrusions specialized for cell-cell adhesion.","method":"Immunofluorescence of endogenous FMNL3, live GFP-FMNL3 imaging, FRAP, siRNA knockdown with filopodia counting and cell-cell adhesion assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous protein localization combined with live imaging, FRAP, and siRNA phenotypic rescue in a single focused study with multiple orthogonal methods","pmids":["25428984"],"is_preprint":false},{"year":2015,"finding":"FMNL3 interacts with Cdc42 and RhoJ (Rho family GTPases). FMNL3 and RhoJ are concentrated at the early apical membrane initiation site (AMIS) and regulate formation of radiating actin cables from this site. FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface during vascular lumenogenesis.","method":"Co-immunoprecipitation (FMNL3 with Cdc42 and RhoJ), immunofluorescence localization, siRNA/shRNA knockdown with podocalyxin trafficking assay in endothelial cells","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP binding plus functional knockdown with specific trafficking readout, single lab","pmids":["26299518"],"is_preprint":false},{"year":2018,"finding":"FMNL3 is enriched at >95% of filopodial tips in U2OS cells and its suppression reduces filopodial assembly by 90%. Ena/VASP proteins (VASP, Mena) contribute to filopodial assembly but are not consistently tip-localized; >85% of FMNL3-containing filopodia are associated with focal adhesions, placing FMNL3 as the dominant tip-localizing actin elongation factor for filopodia in this context.","method":"siRNA knockdown of FMNL3, VASP, and Mena; quantitative fluorescence microscopy of filopodial tips; co-localization with focal adhesions","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic siRNA knockdowns with quantitative imaging, single lab, multiple proteins tested","pmids":["30373894"],"is_preprint":false},{"year":2020,"finding":"FMNL3 concentrates at the oocyte cortex and spindle periphery. Depletion of FMNL3 causes failure of polar body extrusion and symmetric meiotic division due to spindle migration defects at late metaphase I, attributed to decreased cytoplasmic actin. FMNL3 interacts with the actin-bundling protein FASCIN, and this interaction regulates actin filaments required for spindle migration. Rescue by Fmnl3-EGFP mRNA injection confirmed specificity.","method":"Morpholino/siRNA depletion in mouse oocytes, co-immunoprecipitation (FMNL3–FASCIN), mRNA rescue injection, immunofluorescence","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction plus loss-of-function with mRNA rescue, single lab","pmids":["32167535"],"is_preprint":false},{"year":2024,"finding":"FMNL3 specifically interacts with the EMT transcription factor Twist1 (but not other EMT-TFs) and suppresses ubiquitin-mediated degradation of Twist1 by blocking the interaction between Twist1 and its ubiquitin transfer protein Rad23B. This stabilization of Twist1 enhances repression of CDH1 (E-cadherin) transcription and promotes breast cancer cell migration and invasion. Reciprocally, Twist1 directly binds the FMNL3 promoter to upregulate FMNL3 transcription.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, promoter binding/ChIP assay, in vitro migration/invasion assays in breast cancer cells","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, and functional readouts, single lab","pmids":["39582466"],"is_preprint":false},{"year":2017,"finding":"TGF-β1 treatment of NPC cells enhances FMNL3 expression concurrent with EMT induction. FMNL3 knockdown partially attenuates TGF-β1-promoted cell migration and associated EMT marker changes, and reduces EMT in xenograft tumors, placing FMNL3 downstream of TGF-β1 signaling in this context.","method":"siRNA knockdown of FMNL3, TGF-β1 stimulation, cell migration assays, EMT marker western blotting, xenograft assay","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, siRNA knockdown with phenotypic readout, no direct biochemical mechanism established beyond expression correlation","pmids":["28198387"],"is_preprint":false}],"current_model":"FMNL3 is a formin-family actin assembly factor whose FH2 domain drives filopodial assembly by maintaining barbed-end attachment during elongation; its C-terminal WH2-like sequence modulates actin polymerization; N-myristoylation at Gly2 anchors it to the plasma membrane; it is activated by GTP-bound Cdc42 (and RhoJ) downstream of BMP/Arhgef9b signaling to nucleate endothelial filopodia and organize radiating actin cables at the apical membrane initiation site for podocalyxin trafficking during lumenogenesis; it also aligns stabilized microtubules during endothelial elongation, interacts with FASCIN to regulate cytoplasmic actin for oocyte meiotic spindle migration, and in cancer contexts stabilizes Twist1 by blocking Rad23B-mediated ubiquitination to suppress E-cadherin and promote invasion."},"narrative":{"mechanistic_narrative":"FMNL3 is a formin-family actin assembly factor that builds filopodia and other actin-based protrusions and integrates them with cytoskeletal organization during cell migration, adhesion, and tissue morphogenesis [PMID:20862687, PMID:25428984]. Its FH2 domain maintains barbed-end attachment to drive filopodial elongation, and a barbed-end-binding mutation abolishes filopodia assembly; full-length protein shows reduced activity relative to FH1-FH2 constructs, consistent with autoinhibition by accessory domains [PMID:20862687]. A C-terminal WH2-like sequence binds actin monomers and barbed ends and, as a dimer, accelerates polymerization while inhibiting barbed-end elongation, with a cluster of C-terminal basic residues required for polymerization enhancement [PMID:22094460]. N-myristoylation at Gly2 anchors FMNL3 to the plasma membrane, where the endogenous protein concentrates as puncta at filopodial tips, membrane ruffles, and nascent cell-cell adhesions, and is the dominant tip-localizing elongation factor for filopodia [PMID:22790947, PMID:25428984, PMID:30373894]. FMNL3 is activated by GTP-bound Cdc42 and the related GTPase RhoJ: downstream of BMP/Arhgef9b signaling Cdc42 stimulates FMNL3 to nucleate endothelial filopodia during angiogenic sprouting, FMNL3 aligns stabilized microtubules into parallel arrays during endothelial elongation, and FMNL3 with RhoJ organizes radiating actin cables at the apical membrane initiation site to direct podocalyxin trafficking during vascular lumenogenesis [PMID:25584797, PMID:22275430, PMID:26299518]. Beyond protrusion assembly, FMNL3 interacts with the actin-bundling protein FASCIN to control cytoplasmic actin required for meiotic spindle migration and polar body extrusion in oocytes [PMID:32167535]. In cancer, FMNL3 stabilizes the EMT transcription factor Twist1 by blocking Rad23B-mediated ubiquitination, thereby reinforcing CDH1 (E-cadherin) repression and promoting migration and invasion, in a reciprocal loop in which Twist1 transactivates the FMNL3 promoter [PMID:39582466].","teleology":[{"year":2010,"claim":"Established that FMNL3 drives filopodia through its FH2 domain via barbed-end binding, defining its core actin-assembly mechanism and hinting at autoinhibitory regulation.","evidence":"Domain-swap chimeras and barbed-end-binding point mutants overexpressed in multiple cell types with fluorescence microscopy","pmids":["20862687"],"confidence":"High","gaps":["Did not resolve how the GBD/DID/DAD domains mediate autoinhibition or relief","No reconstitution of full-length protein activity"]},{"year":2011,"claim":"Defined a biochemically distinct C-terminal WH2-like module that binds actin monomers and barbed ends and tunes polymerization, separating actin-binding from polymerization-enhancing activities.","evidence":"In vitro actin polymerization and barbed-end elongation assays with targeted mutagenesis of the C-terminal domain","pmids":["22094460"],"confidence":"High","gaps":["How the WH2-like C terminus cooperates with the FH2 domain in the intact protein is unresolved","Physiological relevance of barbed-end inhibition not tested in cells"]},{"year":2012,"claim":"Linked FMNL3 to angiogenesis and showed it couples actin assembly to microtubule organization, extending its role beyond filopodia to directional cell elongation.","evidence":"siRNA knockdown in endothelial cells and morpholino knockdown with human FMNL3 rescue in zebrafish, plus microtubule immunofluorescence","pmids":["22275430"],"confidence":"High","gaps":["Direct molecular basis for microtubule alignment not identified","Whether FMNL3 binds microtubules directly is unknown"]},{"year":2012,"claim":"Identified N-myristoylation at Gly2 as the membrane-targeting requirement that licenses FMNL3's morphogenic activity.","evidence":"Gly2Ala mutagenesis and pharmacological N-myristoylation inhibition with immunofluorescence in HEK293T cells","pmids":["22790947"],"confidence":"Medium","gaps":["Single-lab evidence not independently replicated","Interplay between myristoylation and GTPase activation not addressed"]},{"year":2014,"claim":"Localized endogenous FMNL3 to plasma-membrane puncta at filopodia and nascent cell-cell adhesions, establishing a role in adhesion-specialized protrusions rather than only migration.","evidence":"Endogenous immunofluorescence, live GFP imaging, FRAP, and siRNA knockdown with filopodia counting and adhesion assays","pmids":["25428984"],"confidence":"High","gaps":["Molecular partners at cell-cell adhesions not identified","Mechanism of endocytic FMNL3 puncta recycling unclear"]},{"year":2015,"claim":"Placed FMNL3 in a signaling pathway by showing Cdc42 activates it downstream of BMP/Arhgef9b to build endothelial filopodia in vivo.","evidence":"Genetic epistasis in zebrafish with binding assays and live imaging","pmids":["25584797"],"confidence":"High","gaps":["Structural basis of Cdc42-mediated activation not defined","How autoinhibition is relieved by GTPase binding not shown"]},{"year":2015,"claim":"Extended GTPase regulation to RhoJ and connected FMNL3 to apical actin organization and podocalyxin trafficking during lumen formation.","evidence":"Co-IP of FMNL3 with Cdc42 and RhoJ, localization, and siRNA/shRNA knockdown with podocalyxin trafficking readout in endothelial cells","pmids":["26299518"],"confidence":"Medium","gaps":["Whether RhoJ activates FMNL3 like Cdc42 not directly tested","Mechanism linking actin cables to vesicular trafficking unresolved"]},{"year":2018,"claim":"Quantitatively established FMNL3 as the dominant tip-localizing filopodial elongation factor, distinguishing it from Ena/VASP proteins.","evidence":"Comparative siRNA knockdown of FMNL3, VASP, and Mena with quantitative tip imaging in U2OS cells","pmids":["30373894"],"confidence":"Medium","gaps":["Context-dependence beyond U2OS cells not established","Functional cooperation with focal adhesions mechanistically undefined"]},{"year":2020,"claim":"Revealed a FASCIN-dependent function for FMNL3 in regulating cytoplasmic actin for meiotic spindle migration, broadening its roles beyond protrusion assembly.","evidence":"Depletion in mouse oocytes, FMNL3-FASCIN Co-IP, and mRNA rescue with immunofluorescence","pmids":["32167535"],"confidence":"Medium","gaps":["Direct FMNL3-FASCIN binding interface not mapped","Single Co-IP without reciprocal structural validation"]},{"year":2024,"claim":"Identified a non-cytoskeletal, transcription-stabilizing role in which FMNL3 protects Twist1 from Rad23B-mediated ubiquitination to drive EMT and invasion.","evidence":"Reciprocal Co-IP, ubiquitination assay, ChIP/promoter binding, and migration/invasion assays in breast cancer cells","pmids":["39582466"],"confidence":"Medium","gaps":["How FMNL3 physically blocks the Twist1-Rad23B interaction is undefined","Whether this function requires actin-assembly activity not tested"]},{"year":null,"claim":"How FMNL3's autoinhibition is relieved by GTPase binding and membrane anchoring, and whether its cytoskeletal and Twist1-stabilizing functions are mechanistically linked, remain open.","evidence":"No timeline study reconstitutes full-length regulated FMNL3 or connects its actin-assembly and transcription-factor-stabilizing activities","pmids":[],"confidence":"Low","gaps":["No structure of regulated full-length FMNL3","No unified model linking protrusion assembly to Twist1 stabilization"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,5,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,5,7]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,8]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9]}],"complexes":[],"partners":["CDC42","RHOJ","FSCN1","TWIST1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IVF7","full_name":"Formin-like protein 3","aliases":["Formin homology 2 domain-containing protein 3","WW domain-binding protein 3","WBP-3"],"length_aa":1028,"mass_kda":117.2,"function":"Plays a role in the regulation of cell morphology and cytoskeletal organization. Required in the control of cell shape and migration. Required for developmental angiogenesis (By similarity). In this process, required for microtubule reorganization and for efficient endothelial cell elongation. In quiescent endothelial cells, triggers rearrangement of the actin cytoskeleton, but does not alter microtubule alignement","subcellular_location":"Cytoplasm; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8IVF7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FMNL3","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FMNL3","total_profiled":1310},"omim":[{"mim_id":"616288","title":"FORMIN-LIKE 3; FMNL3","url":"https://www.omim.org/entry/616288"},{"mim_id":"616285","title":"FORMIN-LIKE 2; FMNL2","url":"https://www.omim.org/entry/616285"},{"mim_id":"609381","title":"SYNTAXIN-BINDING PROTEIN 5-LIKE; STXBP5L","url":"https://www.omim.org/entry/609381"},{"mim_id":"606627","title":"DISHEVELLED-ASSOCIATED ACTIVATOR OF MORPHOGENESIS 2; DAAM2","url":"https://www.omim.org/entry/606627"},{"mim_id":"604656","title":"FORMIN-LIKE 1; FMNL1","url":"https://www.omim.org/entry/604656"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FMNL3"},"hgnc":{"alias_symbol":["DKFZp762B245","MGC45819","WBP3"],"prev_symbol":[]},"alphafold":{"accession":"Q8IVF7","domains":[{"cath_id":"-","chopping":"32-42_50-73","consensus_level":"high","plddt":85.3294,"start":32,"end":73},{"cath_id":"-","chopping":"75-154_198-262","consensus_level":"medium","plddt":85.8808,"start":75,"end":262},{"cath_id":"1.20.58","chopping":"642-721","consensus_level":"high","plddt":89.446,"start":642,"end":721},{"cath_id":"-","chopping":"786-828_918-953","consensus_level":"medium","plddt":90.4825,"start":786,"end":953}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IVF7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IVF7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IVF7-F1-predicted_aligned_error_v6.png","plddt_mean":76.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FMNL3","jax_strain_url":"https://www.jax.org/strain/search?query=FMNL3"},"sequence":{"accession":"Q8IVF7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IVF7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IVF7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IVF7"}},"corpus_meta":[{"pmid":"25584797","id":"PMC_25584797","title":"Cdc42 mediates Bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial filopodia in zebrafish.","date":"2015","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/25584797","citation_count":142,"is_preprint":false},{"pmid":"12684686","id":"PMC_12684686","title":"Identification and characterization of human FMNL1, FMNL2 and FMNL3 genes in silico.","date":"2003","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/12684686","citation_count":128,"is_preprint":false},{"pmid":"22094460","id":"PMC_22094460","title":"The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22094460","citation_count":62,"is_preprint":false},{"pmid":"20862687","id":"PMC_20862687","title":"Assembly of filopodia by the formin FRL2 (FMNL3).","date":"2010","source":"Cytoskeleton (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/20862687","citation_count":62,"is_preprint":false},{"pmid":"22275430","id":"PMC_22275430","title":"The formin FMNL3 is a cytoskeletal regulator of angiogenesis.","date":"2012","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/22275430","citation_count":37,"is_preprint":false},{"pmid":"25428984","id":"PMC_25428984","title":"The formin FMNL3 assembles plasma membrane protrusions that participate in cell-cell adhesion.","date":"2014","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/25428984","citation_count":35,"is_preprint":false},{"pmid":"28198387","id":"PMC_28198387","title":"High FMNL3 expression promotes nasopharyngeal carcinoma cell metastasis: role in TGF-β1-induced epithelia-to-mesenchymal transition.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28198387","citation_count":33,"is_preprint":false},{"pmid":"22790947","id":"PMC_22790947","title":"Protein N-myristoylation is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3.","date":"2012","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22790947","citation_count":32,"is_preprint":false},{"pmid":"26299518","id":"PMC_26299518","title":"The Formin FMNL3 Controls Early Apical Specification in Endothelial Cells by Regulating the Polarized Trafficking of Podocalyxin.","date":"2015","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/26299518","citation_count":28,"is_preprint":false},{"pmid":"27499915","id":"PMC_27499915","title":"FMNL2/FMNL3 formins are linked with oncogenic pathways and predict melanoma outcome.","date":"2016","source":"The journal of pathology. Clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/27499915","citation_count":28,"is_preprint":false},{"pmid":"30373894","id":"PMC_30373894","title":"Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly.","date":"2018","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/30373894","citation_count":25,"is_preprint":false},{"pmid":"27645894","id":"PMC_27645894","title":"MicroRNA-127 is a tumor suppressor in human esophageal squamous cell carcinoma through the regulation of oncogene FMNL3.","date":"2016","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/27645894","citation_count":24,"is_preprint":false},{"pmid":"32167535","id":"PMC_32167535","title":"FMNL3 regulates FASCIN for actin-mediated spindle migration and cytokinesis in mouse oocytes†.","date":"2020","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/32167535","citation_count":11,"is_preprint":false},{"pmid":"33847733","id":"PMC_33847733","title":"Novel role of LINC01013/miR-6795-5p/FMNL3 axis in the regulation of hepatocellular carcinoma stem cell features.","date":"2021","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/33847733","citation_count":10,"is_preprint":false},{"pmid":"39582466","id":"PMC_39582466","title":"FMNL3 Promotes Migration and Invasion of Breast Cancer Cells via Inhibiting Rad23B-Induced Ubiquitination of Twist1.","date":"2024","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/39582466","citation_count":3,"is_preprint":false},{"pmid":"36444244","id":"PMC_36444244","title":"FMNL3 is Overexpressed in Tumor Tissues and Predicts an Immuno-Hot Phenotype in Pancreatic Cancer.","date":"2022","source":"International journal of general medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36444244","citation_count":3,"is_preprint":false},{"pmid":"35428519","id":"PMC_35428519","title":"Hsa_circ_0081534 facilitates malignant phenotypes by sequestering miR-874-3p and upregulating FMNL3 in nasopharyngeal carcinoma.","date":"2022","source":"Auris, nasus, larynx","url":"https://pubmed.ncbi.nlm.nih.gov/35428519","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10702,"output_tokens":3152,"usd":0.039693,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10519,"output_tokens":3607,"usd":0.071385,"stage2_stop_reason":"end_turn"},"total_usd":0.111078,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"The C-terminal 76 amino acids of FMNL3 contain a WH2-like sequence that binds actin monomers competitively with other WH2 domains and with profilin, and also binds filament barbed ends. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high-affinity inhibition of barbed end elongation. Mutation of two aliphatic residues blocking high-affinity actin binding does not affect polymerization enhancement, but mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement.\",\n      \"method\": \"In vitro actin polymerization assays, barbed-end elongation assays, mutagenesis of FMNL3 C-terminal domain\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple orthogonal biochemical assays in a single rigorous study\",\n      \"pmids\": [\"22094460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The FH2 domain of FRL2/FMNL3 is required for filopodia assembly; the FH1 domain is interchangeable with that of FRL1, but the FH2 domain cannot be substituted by FRL1's FH2. A mutation compromising FRL2's barbed-end binding ability abolishes filopodia assembly. Full-length FMNL3 shows reduced filopodia-stimulating activity compared to FH1-FH2 constructs, suggesting autoinhibitory regulation by additional domains (GBD, DID, DAD).\",\n      \"method\": \"Overexpression of FH1-FH2 constructs and chimeric constructs in multiple cell types, barbed-end binding mutant analysis, fluorescence microscopy\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-swap chimeras, point mutagenesis, and multiple cell-type validation in a single focused mechanistic study\",\n      \"pmids\": [\"20862687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In zebrafish, Cdc42 binds to and activates FMNL3 downstream of BMP/Arhgef9b signaling to promote assembly of endothelial filopodia during angiogenic sprouting of the caudal vein plexus. Active Cdc42 stimulates FMNL3 to drive filopodial extension.\",\n      \"method\": \"Genetic epistasis in zebrafish (morpholino knockdown, dominant-active/dominant-negative constructs), Co-IP/binding assays for Cdc42–FMNL3 interaction, in vivo live imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis in vivo combined with direct binding evidence, replicated across multiple genetic conditions\",\n      \"pmids\": [\"25584797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Activated FMNL3 triggers alignment of stabilized microtubules into parallel arrays directed toward the growing tip during endothelial cell elongation in angiogenesis. FMNL3 knockdown in zebrafish causes profound developmental angiogenesis defects rescued by human FMNL3 expression.\",\n      \"method\": \"siRNA knockdown in endothelial cells, co-culture angiogenesis assay, immunofluorescence of microtubules, morpholino knockdown and rescue in zebrafish\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function in two systems (mammalian cells and zebrafish) with defined morphological readouts and rescue experiment\",\n      \"pmids\": [\"22275430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"N-myristoylation of FMNL3 (at Gly2) is required for its plasma membrane localization and for induction of cellular morphological changes; replacing Gly2 with Ala or using an N-myristoylation inhibitor abolishes membrane association and morphological effects.\",\n      \"method\": \"Site-directed mutagenesis (Gly2Ala), pharmacological inhibition of N-myristoylation, immunofluorescence in HEK293T cells\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus pharmacological inhibition in a single lab, two orthogonal methods\",\n      \"pmids\": [\"22790947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Endogenous FMNL3 localizes predominantly in a punctate pattern at the plasma membrane (>95% of puncta), enriches in filopodia and membrane ruffles and at nascent cell-cell adhesions. A small population of cytoplasmic puncta of endocytic origin enriches near cell-cell contacts and fuses with the plasma membrane there. FMNL3 siRNA suppression decreases filopodia number and compromises cell-cell adhesion in migrating cell sheets, indicating FMNL3 assembles actin-based protrusions specialized for cell-cell adhesion.\",\n      \"method\": \"Immunofluorescence of endogenous FMNL3, live GFP-FMNL3 imaging, FRAP, siRNA knockdown with filopodia counting and cell-cell adhesion assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous protein localization combined with live imaging, FRAP, and siRNA phenotypic rescue in a single focused study with multiple orthogonal methods\",\n      \"pmids\": [\"25428984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FMNL3 interacts with Cdc42 and RhoJ (Rho family GTPases). FMNL3 and RhoJ are concentrated at the early apical membrane initiation site (AMIS) and regulate formation of radiating actin cables from this site. FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface during vascular lumenogenesis.\",\n      \"method\": \"Co-immunoprecipitation (FMNL3 with Cdc42 and RhoJ), immunofluorescence localization, siRNA/shRNA knockdown with podocalyxin trafficking assay in endothelial cells\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP binding plus functional knockdown with specific trafficking readout, single lab\",\n      \"pmids\": [\"26299518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FMNL3 is enriched at >95% of filopodial tips in U2OS cells and its suppression reduces filopodial assembly by 90%. Ena/VASP proteins (VASP, Mena) contribute to filopodial assembly but are not consistently tip-localized; >85% of FMNL3-containing filopodia are associated with focal adhesions, placing FMNL3 as the dominant tip-localizing actin elongation factor for filopodia in this context.\",\n      \"method\": \"siRNA knockdown of FMNL3, VASP, and Mena; quantitative fluorescence microscopy of filopodial tips; co-localization with focal adhesions\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic siRNA knockdowns with quantitative imaging, single lab, multiple proteins tested\",\n      \"pmids\": [\"30373894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FMNL3 concentrates at the oocyte cortex and spindle periphery. Depletion of FMNL3 causes failure of polar body extrusion and symmetric meiotic division due to spindle migration defects at late metaphase I, attributed to decreased cytoplasmic actin. FMNL3 interacts with the actin-bundling protein FASCIN, and this interaction regulates actin filaments required for spindle migration. Rescue by Fmnl3-EGFP mRNA injection confirmed specificity.\",\n      \"method\": \"Morpholino/siRNA depletion in mouse oocytes, co-immunoprecipitation (FMNL3–FASCIN), mRNA rescue injection, immunofluorescence\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction plus loss-of-function with mRNA rescue, single lab\",\n      \"pmids\": [\"32167535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FMNL3 specifically interacts with the EMT transcription factor Twist1 (but not other EMT-TFs) and suppresses ubiquitin-mediated degradation of Twist1 by blocking the interaction between Twist1 and its ubiquitin transfer protein Rad23B. This stabilization of Twist1 enhances repression of CDH1 (E-cadherin) transcription and promotes breast cancer cell migration and invasion. Reciprocally, Twist1 directly binds the FMNL3 promoter to upregulate FMNL3 transcription.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, promoter binding/ChIP assay, in vitro migration/invasion assays in breast cancer cells\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, and functional readouts, single lab\",\n      \"pmids\": [\"39582466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TGF-β1 treatment of NPC cells enhances FMNL3 expression concurrent with EMT induction. FMNL3 knockdown partially attenuates TGF-β1-promoted cell migration and associated EMT marker changes, and reduces EMT in xenograft tumors, placing FMNL3 downstream of TGF-β1 signaling in this context.\",\n      \"method\": \"siRNA knockdown of FMNL3, TGF-β1 stimulation, cell migration assays, EMT marker western blotting, xenograft assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, siRNA knockdown with phenotypic readout, no direct biochemical mechanism established beyond expression correlation\",\n      \"pmids\": [\"28198387\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FMNL3 is a formin-family actin assembly factor whose FH2 domain drives filopodial assembly by maintaining barbed-end attachment during elongation; its C-terminal WH2-like sequence modulates actin polymerization; N-myristoylation at Gly2 anchors it to the plasma membrane; it is activated by GTP-bound Cdc42 (and RhoJ) downstream of BMP/Arhgef9b signaling to nucleate endothelial filopodia and organize radiating actin cables at the apical membrane initiation site for podocalyxin trafficking during lumenogenesis; it also aligns stabilized microtubules during endothelial elongation, interacts with FASCIN to regulate cytoplasmic actin for oocyte meiotic spindle migration, and in cancer contexts stabilizes Twist1 by blocking Rad23B-mediated ubiquitination to suppress E-cadherin and promote invasion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FMNL3 is a formin-family actin assembly factor that builds filopodia and other actin-based protrusions and integrates them with cytoskeletal organization during cell migration, adhesion, and tissue morphogenesis [#1, #5]. Its FH2 domain maintains barbed-end attachment to drive filopodial elongation, and a barbed-end-binding mutation abolishes filopodia assembly; full-length protein shows reduced activity relative to FH1-FH2 constructs, consistent with autoinhibition by accessory domains [#1]. A C-terminal WH2-like sequence binds actin monomers and barbed ends and, as a dimer, accelerates polymerization while inhibiting barbed-end elongation, with a cluster of C-terminal basic residues required for polymerization enhancement [#0]. N-myristoylation at Gly2 anchors FMNL3 to the plasma membrane, where the endogenous protein concentrates as puncta at filopodial tips, membrane ruffles, and nascent cell-cell adhesions, and is the dominant tip-localizing elongation factor for filopodia [#4, #5, #7]. FMNL3 is activated by GTP-bound Cdc42 and the related GTPase RhoJ: downstream of BMP/Arhgef9b signaling Cdc42 stimulates FMNL3 to nucleate endothelial filopodia during angiogenic sprouting, FMNL3 aligns stabilized microtubules into parallel arrays during endothelial elongation, and FMNL3 with RhoJ organizes radiating actin cables at the apical membrane initiation site to direct podocalyxin trafficking during vascular lumenogenesis [#2, #3, #6]. Beyond protrusion assembly, FMNL3 interacts with the actin-bundling protein FASCIN to control cytoplasmic actin required for meiotic spindle migration and polar body extrusion in oocytes [#8]. In cancer, FMNL3 stabilizes the EMT transcription factor Twist1 by blocking Rad23B-mediated ubiquitination, thereby reinforcing CDH1 (E-cadherin) repression and promoting migration and invasion, in a reciprocal loop in which Twist1 transactivates the FMNL3 promoter [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established that FMNL3 drives filopodia through its FH2 domain via barbed-end binding, defining its core actin-assembly mechanism and hinting at autoinhibitory regulation.\",\n      \"evidence\": \"Domain-swap chimeras and barbed-end-binding point mutants overexpressed in multiple cell types with fluorescence microscopy\",\n      \"pmids\": [\"20862687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how the GBD/DID/DAD domains mediate autoinhibition or relief\", \"No reconstitution of full-length protein activity\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined a biochemically distinct C-terminal WH2-like module that binds actin monomers and barbed ends and tunes polymerization, separating actin-binding from polymerization-enhancing activities.\",\n      \"evidence\": \"In vitro actin polymerization and barbed-end elongation assays with targeted mutagenesis of the C-terminal domain\",\n      \"pmids\": [\"22094460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the WH2-like C terminus cooperates with the FH2 domain in the intact protein is unresolved\", \"Physiological relevance of barbed-end inhibition not tested in cells\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked FMNL3 to angiogenesis and showed it couples actin assembly to microtubule organization, extending its role beyond filopodia to directional cell elongation.\",\n      \"evidence\": \"siRNA knockdown in endothelial cells and morpholino knockdown with human FMNL3 rescue in zebrafish, plus microtubule immunofluorescence\",\n      \"pmids\": [\"22275430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular basis for microtubule alignment not identified\", \"Whether FMNL3 binds microtubules directly is unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified N-myristoylation at Gly2 as the membrane-targeting requirement that licenses FMNL3's morphogenic activity.\",\n      \"evidence\": \"Gly2Ala mutagenesis and pharmacological N-myristoylation inhibition with immunofluorescence in HEK293T cells\",\n      \"pmids\": [\"22790947\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab evidence not independently replicated\", \"Interplay between myristoylation and GTPase activation not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Localized endogenous FMNL3 to plasma-membrane puncta at filopodia and nascent cell-cell adhesions, establishing a role in adhesion-specialized protrusions rather than only migration.\",\n      \"evidence\": \"Endogenous immunofluorescence, live GFP imaging, FRAP, and siRNA knockdown with filopodia counting and adhesion assays\",\n      \"pmids\": [\"25428984\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners at cell-cell adhesions not identified\", \"Mechanism of endocytic FMNL3 puncta recycling unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed FMNL3 in a signaling pathway by showing Cdc42 activates it downstream of BMP/Arhgef9b to build endothelial filopodia in vivo.\",\n      \"evidence\": \"Genetic epistasis in zebrafish with binding assays and live imaging\",\n      \"pmids\": [\"25584797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Cdc42-mediated activation not defined\", \"How autoinhibition is relieved by GTPase binding not shown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended GTPase regulation to RhoJ and connected FMNL3 to apical actin organization and podocalyxin trafficking during lumen formation.\",\n      \"evidence\": \"Co-IP of FMNL3 with Cdc42 and RhoJ, localization, and siRNA/shRNA knockdown with podocalyxin trafficking readout in endothelial cells\",\n      \"pmids\": [\"26299518\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RhoJ activates FMNL3 like Cdc42 not directly tested\", \"Mechanism linking actin cables to vesicular trafficking unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Quantitatively established FMNL3 as the dominant tip-localizing filopodial elongation factor, distinguishing it from Ena/VASP proteins.\",\n      \"evidence\": \"Comparative siRNA knockdown of FMNL3, VASP, and Mena with quantitative tip imaging in U2OS cells\",\n      \"pmids\": [\"30373894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Context-dependence beyond U2OS cells not established\", \"Functional cooperation with focal adhesions mechanistically undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a FASCIN-dependent function for FMNL3 in regulating cytoplasmic actin for meiotic spindle migration, broadening its roles beyond protrusion assembly.\",\n      \"evidence\": \"Depletion in mouse oocytes, FMNL3-FASCIN Co-IP, and mRNA rescue with immunofluorescence\",\n      \"pmids\": [\"32167535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct FMNL3-FASCIN binding interface not mapped\", \"Single Co-IP without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a non-cytoskeletal, transcription-stabilizing role in which FMNL3 protects Twist1 from Rad23B-mediated ubiquitination to drive EMT and invasion.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, ChIP/promoter binding, and migration/invasion assays in breast cancer cells\",\n      \"pmids\": [\"39582466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How FMNL3 physically blocks the Twist1-Rad23B interaction is undefined\", \"Whether this function requires actin-assembly activity not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FMNL3's autoinhibition is relieved by GTPase binding and membrane anchoring, and whether its cytoskeletal and Twist1-stabilizing functions are mechanistically linked, remain open.\",\n      \"evidence\": \"No timeline study reconstitutes full-length regulated FMNL3 or connects its actin-assembly and transcription-factor-stabilizing activities\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of regulated full-length FMNL3\", \"No unified model linking protrusion assembly to Twist1 stabilization\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 5, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 5, 7]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDC42\", \"RHOJ\", \"FSCN1\", \"TWIST1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}