{"gene":"TWF2","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2009,"finding":"Two biochemically distinct isoforms (TWF2a and TWF2b) are generated from the mouse Twf2 gene by alternative promoter usage. Both isoforms bind actin monomers and capping protein, and both cap actin filament barbed ends. However, the N-terminal ADF-H domain of TWF2b interacts with ADP-G-actin with 5-fold higher affinity than ATP-G-actin, whereas the corresponding domain of TWF2a binds ADP-G-actin and ATP-G-actin with equal affinities. TWF2a is predominantly expressed in non-muscle tissues; TWF2b is restricted to heart and skeletal muscle.","method":"Alternative promoter identification, in vitro actin monomer binding assays, barbed-end capping assays, affinity measurements for ADP-G-actin vs. ATP-G-actin","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution (capping assay, binding affinity measurements) with multiple orthogonal methods in single rigorous study","pmids":["18837697"],"is_preprint":false},{"year":1999,"finding":"TWF2 (A6-related protein, A6rp) was identified as a binding partner of protein kinase Czeta (PKCzeta) via yeast two-hybrid. GST-A6rp was selectively phosphorylated by PKCzeta but not significantly by other PKC isoenzymes, and was phosphorylated by casein kinase 2 and most effectively by the tyrosine kinase Src. Both A6rp and A6 bind ATP, consistent with their potential ATP-binding sites. No autokinase activity was detected for either A6rp or A6.","method":"Yeast two-hybrid, in vitro kinase assay with GST fusion proteins, immunoblotting, ATP binding assay","journal":"European Journal of Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus in vitro kinase assays with multiple kinases, single lab","pmids":["10406962"],"is_preprint":false},{"year":2017,"finding":"TWF2 concentrates at stereocilia tips in hair cells during development, co-localizing with the CAPZB subunit of heterodimeric capping protein. TWF2 localization to tips parallels that of CAPZB, and both CAPZB and TWF2 tip enrichment fails to occur at row 2 stereocilia tips in transduction mutants (Tmc1/Tmc2 or Tmie knockout). CAPZB knockout causes stereocilia to shorten and disappear with concurrent decrease in width, implicating capping protein (and its partner TWF2) in stereocilia widening.","method":"Immunofluorescence localization in hair cells, conditional Capzb knockout (Atoh1-Cre), in utero electroporation of CAPZB2","journal":"The Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal localization and genetic knockout with defined morphological phenotype, replicated across multiple mutant backgrounds","pmids":["28899994"],"is_preprint":false},{"year":2020,"finding":"TWF2 normally concentrates at row 2 stereocilia tips in wild-type hair cell bundles, and this localization requires functional mechanotransduction channels. In transduction mutants (Tmc1KO;Tmc2KO or TmieKO), TWF2 fails to concentrate at row 2 tips, demonstrating that mechanotransduction specifies and maintains the row 2 protein complex that includes TWF2 and CAPZB.","method":"Quantitative immunofluorescence of phalloidin-labeled mouse cochleas, analysis of genetic transduction mutants","journal":"Current Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization imaging in multiple independent genetic mutants, replicating findings from related study","pmids":["31902726"],"is_preprint":false},{"year":2021,"finding":"BAIAP2L2 binds to TWF2 (as well as EPS8L2 and CAPZB2), functioning as a component of the row 2 complex at stereocilia tips. BAIAP2L2 knockout disrupts row 2 complex integrity and causes degeneration of mechanotransducing stereocilia.","method":"Co-immunoprecipitation (binding of BAIAP2L2 to TWF2, EPS8L2, CAPZB2), Baiap2l2 knockout mice, electrophysiology, FM 1-43FX dye uptake","journal":"Journal of Cellular Physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus genetic knockout with functional readout, single lab","pmids":["34346063"],"is_preprint":false},{"year":2025,"finding":"TWF2 promotes RCC cell invasion, migration, metastasis, and sunitinib resistance by suppressing Hippo signaling. Mechanistically, TWF2 interacts with YAP via TWF2 residue M99 and YAP residue M225. By competitively displacing LATS1 (large tumor suppressor kinase 1), TWF2 prevents YAP ubiquitination and degradation, leading to YAP stabilization and nuclear translocation. Mutation of M99 abolishes the tumor-promoting activity of TWF2.","method":"Co-immunoprecipitation, site-directed mutagenesis (M99 mutation), functional invasion/migration assays, patient-derived xenograft models, small-molecule inhibitor (salvianolic acid E) of TWF2-YAP interaction","journal":"Advanced Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, mutagenesis, and in vivo PDX model, single lab with multiple orthogonal methods","pmids":["40948085"],"is_preprint":false},{"year":2025,"finding":"The C. elegans twinfilin ortholog TWF-2 (ortholog of TWF2) localizes to the spermathecal cortex through interactions with α-spectrin (SPC-1) and β-spectrin (UNC-70). In vitro, TWF-2 promotes barbed-end depolymerization and rapidly displaces capping protein (CAP-1/CAP-2) from filament barbed ends. In vivo, loss of TWF-2 partially rescues embryonic lethality caused by CAP-1 depletion (genetic epistasis). Loss of TWF-2 suppresses hypercontractility (elevated F-actin and phosphorylated myosin) from SPV-1 loss by reducing F-actin levels without affecting myosin phosphorylation, demonstrating a specific role in F-actin regulation and tissue contractility.","method":"C. elegans genetics (epistasis with cap-1 and spv-1 loss-of-function), in vitro actin depolymerization and capping protein uncapping assays, immunofluorescence localization, Co-IP with spectrins","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution (capping/uncapping assay) combined with genetic epistasis in multiple mutant backgrounds and direct localization; peer-reviewed and preprint versions independently consistent","pmids":["41195540","40667244"],"is_preprint":false}],"current_model":"TWF2 (twinfilin-2) is an evolutionarily conserved ADF-H domain protein that caps actin filament barbed ends and sequesters actin monomers; it is generated as two biochemically distinct tissue-specific isoforms by alternative promoter usage, interacts with heterodimeric capping protein (CAPZB) to regulate stereocilia dimensions at hair cell row 2 tips in a mechanotransduction-dependent manner, modulates actomyosin contractility by uncapping capping protein from filament barbed ends (as shown by C. elegans genetics and in vitro assays), is phosphorylated by PKCzeta and Src kinase, and in cancer cells competitively displaces LATS1 from YAP to prevent YAP ubiquitination and degradation, thereby suppressing Hippo signaling and promoting tumor invasion and drug resistance."},"narrative":{"mechanistic_narrative":"TWF2 (twinfilin-2) is an evolutionarily conserved ADF-H domain actin-regulatory protein that binds actin monomers, caps actin filament barbed ends, and dynamically remodels actin networks [PMID:18837697]. The mouse gene produces two biochemically distinct isoforms by alternative promoter usage—TWF2a, broadly expressed in non-muscle tissues, binds ADP- and ATP-G-actin with equal affinity, whereas the muscle-restricted TWF2b binds ADP-G-actin with ~5-fold higher affinity—but both isoforms cap barbed ends and bind capping protein [PMID:18837697]. Beyond static capping, TWF2 actively promotes barbed-end depolymerization and rapidly displaces (uncaps) capping protein from filament ends, and through this activity regulates F-actin levels and actomyosin contractility, as established by C. elegans genetics where loss of the ortholog suppresses both capping-protein-depletion lethality and hypercontractility downstream of spectrin scaffolding [PMID:41195540, PMID:40667244]. In vertebrate hair cells, TWF2 co-localizes with the CAPZB capping-protein subunit at row 2 stereocilia tips, where its enrichment depends on functional mechanotransduction and contributes, as part of a complex that also includes BAIAP2L2 and EPS8L2, to specifying stereocilia dimensions [PMID:28899994, PMID:31902726, PMID:34346063]. TWF2 is a substrate of PKCzeta, casein kinase 2, and most effectively Src, linking it to kinase signaling [PMID:10406962]. In renal cell carcinoma, TWF2 acquires a signaling role by binding YAP (via TWF2 residue M99 and YAP M225) and competitively displacing LATS1, thereby blocking YAP ubiquitination, stabilizing nuclear YAP, suppressing Hippo signaling, and driving invasion, metastasis, and sunitinib resistance [PMID:40948085].","teleology":[{"year":1999,"claim":"Before its actin role was defined, TWF2 was placed in a kinase-signaling context by identifying it as a phosphorylation target, establishing that it is regulated by serine/threonine and tyrosine kinases.","evidence":"Yeast two-hybrid against PKCzeta plus in vitro kinase assays with GST fusions and an ATP-binding assay","pmids":["10406962"],"confidence":"Medium","gaps":["Functional consequence of phosphorylation on actin binding or capping not tested","Phosphosites not mapped","No cellular validation of the PKCzeta interaction"]},{"year":2009,"claim":"Resolved whether TWF2 is a single biochemical entity by showing two promoter-driven isoforms with distinct nucleotide-state actin preferences and tissue distributions, establishing TWF2 as a tissue-specialized actin monomer-binding and barbed-end capping protein.","evidence":"Alternative promoter identification with in vitro actin monomer binding, capping, and ADP/ATP-G-actin affinity measurements","pmids":["18837697"],"confidence":"High","gaps":["Functional difference between isoforms in cells not demonstrated","Physiological role of muscle-restricted TWF2b not addressed"]},{"year":2017,"claim":"Connected TWF2 to a defined cellular structure by showing it co-localizes with CAPZB at stereocilia tips and that capping protein controls stereocilia width, placing TWF2 in the actin machinery that sizes mechanosensory protrusions.","evidence":"Immunofluorescence, conditional Capzb knockout, and CAPZB2 electroporation in mouse hair cells","pmids":["28899994"],"confidence":"High","gaps":["Direct functional requirement for TWF2 (vs CAPZB) in stereocilia not isolated","Whether TWF2 caps or uncaps in this context unresolved"]},{"year":2020,"claim":"Established that TWF2 tip localization is not constitutive but is specified by mechanotransduction, defining it as a member of an activity-dependent row 2 protein complex.","evidence":"Quantitative immunofluorescence of cochleas from Tmc1/Tmc2 and Tmie transduction mutants","pmids":["31902726"],"confidence":"High","gaps":["Molecular signal coupling channel activity to TWF2 recruitment unknown","TWF2 loss-of-function phenotype in hair cells not shown"]},{"year":2021,"claim":"Identified BAIAP2L2 as a binding partner that organizes the row 2 complex containing TWF2, EPS8L2, and CAPZB2, defining the protein interaction architecture maintaining mechanotransducing stereocilia.","evidence":"Co-immunoprecipitation and Baiap2l2 knockout mice with electrophysiology and FM dye uptake","pmids":["34346063"],"confidence":"Medium","gaps":["Direct vs indirect nature of BAIAP2L2-TWF2 binding not resolved","Whether TWF2 is required for complex integrity untested"]},{"year":2025,"claim":"Resolved the mechanistic basis of TWF2's actin-remodeling activity in vivo by showing it depolymerizes barbed ends, uncaps capping protein, and regulates tissue contractility via spectrin-anchored localization.","evidence":"C. elegans genetic epistasis with cap-1 and spv-1, in vitro depolymerization/uncapping assays, and Co-IP with α/β-spectrin","pmids":["41195540","40667244"],"confidence":"High","gaps":["Whether vertebrate TWF2 uncaps CAPZB in stereocilia not directly tested","Isoform specificity of uncapping activity not addressed"]},{"year":2025,"claim":"Revealed a non-cytoskeletal signaling role by showing TWF2 binds YAP and displaces LATS1 to stabilize YAP, linking TWF2 to Hippo pathway suppression and cancer progression.","evidence":"Co-IP, M99/M225 site-directed mutagenesis, invasion/migration assays, PDX models, and a small-molecule inhibitor of the TWF2-YAP interaction","pmids":["40948085"],"confidence":"Medium","gaps":["Relationship between actin-regulatory and YAP-binding functions of TWF2 unknown","Whether the interaction generalizes beyond renal cell carcinoma not established","Structural basis of M99-M225 contact not defined"]},{"year":null,"claim":"How TWF2's biochemical activities (capping vs uncapping/depolymerization), its kinase regulation, and its YAP-binding signaling function are integrated within a single protein across tissues remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model linking the actin-binding and YAP-binding surfaces","Whether phosphorylation by PKCzeta/Src switches between capping and signaling roles untested","Isoform-specific functions in mammalian tissues uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,2,6]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6,5]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,3,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5]}],"complexes":["row 2 stereocilia tip complex"],"partners":["CAPZB","BAIAP2L2","EPS8L2","YAP1","LATS1","PRKCZ","SPC-1","UNC-70"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6IBS0","full_name":"Twinfilin-2","aliases":["A6-related protein","hA6RP","Protein tyrosine kinase 9-like","Twinfilin-1-like protein"],"length_aa":349,"mass_kda":39.5,"function":"Actin-binding protein involved in motile and morphological processes. Inhibits actin polymerization, likely by sequestering G-actin. By capping the barbed ends of filaments, it also regulates motility. Seems to play an important role in clathrin-mediated endocytosis and distribution of endocytic organelles. May play a role in regulating the mature length of the middle and short rows of stereocilia (By similarity)","subcellular_location":"Cytoplasm, cytoskeleton; Cytoplasm, perinuclear region; Cell projection, stereocilium","url":"https://www.uniprot.org/uniprotkb/Q6IBS0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TWF2","classification":"Not Classified","n_dependent_lines":42,"n_total_lines":1208,"dependency_fraction":0.0347682119205298},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ACTG1","stoichiometry":10.0},{"gene":"ACTB","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TWF2","total_profiled":1310},"omim":[{"mim_id":"607433","title":"TWINFILIN ACTIN-BINDING PROTEIN 2; TWF2","url":"https://www.omim.org/entry/607433"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Plasma membrane","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TWF2"},"hgnc":{"alias_symbol":["A6RP","A6r"],"prev_symbol":["PTK9L"]},"alphafold":{"accession":"Q6IBS0","domains":[{"cath_id":"3.40.20.10","chopping":"7-141","consensus_level":"high","plddt":95.09,"start":7,"end":141},{"cath_id":"3.40.20.10","chopping":"183-311","consensus_level":"high","plddt":94.2488,"start":183,"end":311}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6IBS0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6IBS0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6IBS0-F1-predicted_aligned_error_v6.png","plddt_mean":91.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TWF2","jax_strain_url":"https://www.jax.org/strain/search?query=TWF2"},"sequence":{"accession":"Q6IBS0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6IBS0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6IBS0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6IBS0"}},"corpus_meta":[{"pmid":"26515236","id":"PMC_26515236","title":"A CpG-methylation-based assay to predict survival in clear cell renal cell carcinoma.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26515236","citation_count":101,"is_preprint":false},{"pmid":"31902726","id":"PMC_31902726","title":"Mechanotransduction-Dependent Control of Stereocilia Dimensions and Row Identity in Inner Hair Cells.","date":"2020","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/31902726","citation_count":60,"is_preprint":false},{"pmid":"19889227","id":"PMC_19889227","title":"Inhibition of monkeypox virus replication by RNA interference.","date":"2009","source":"Virology journal","url":"https://pubmed.ncbi.nlm.nih.gov/19889227","citation_count":46,"is_preprint":false},{"pmid":"28899994","id":"PMC_28899994","title":"Heterodimeric capping protein is required for stereocilia length and width regulation.","date":"2017","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/28899994","citation_count":45,"is_preprint":false},{"pmid":"18623487","id":"PMC_18623487","title":"Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase.","date":"1995","source":"Biotechnology and bioengineering","url":"https://pubmed.ncbi.nlm.nih.gov/18623487","citation_count":40,"is_preprint":false},{"pmid":"1731116","id":"PMC_1731116","title":"Identification and expression of rpo19, a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit.","date":"1992","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/1731116","citation_count":38,"is_preprint":false},{"pmid":"36145351","id":"PMC_36145351","title":"Fludarabine, a Potential DNA-Dependent RNA Polymerase Inhibitor, as a Prospective Drug against Monkeypox Virus: A Computational Approach.","date":"2022","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/36145351","citation_count":36,"is_preprint":false},{"pmid":"18837697","id":"PMC_18837697","title":"Two biochemically distinct and tissue-specific twinfilin isoforms are generated from the mouse Twf2 gene by alternative promoter usage.","date":"2009","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/18837697","citation_count":30,"is_preprint":false},{"pmid":"8556165","id":"PMC_8556165","title":"Biomimetic dye affinity chromatography for the purification of bovine heart lactate dehydrogenase.","date":"1995","source":"Journal of chromatography. A","url":"https://pubmed.ncbi.nlm.nih.gov/8556165","citation_count":25,"is_preprint":false},{"pmid":"34346063","id":"PMC_34346063","title":"BAIAP2L2 is required for the maintenance of mechanotransducing stereocilia of cochlear hair cells.","date":"2021","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/34346063","citation_count":23,"is_preprint":false},{"pmid":"29767268","id":"PMC_29767268","title":"CHAF1B knockdown blocks migration in a hepatocellular carcinoma model.","date":"2018","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/29767268","citation_count":22,"is_preprint":false},{"pmid":"24232028","id":"PMC_24232028","title":"Corn phosphoglycolate phosphatase: purification and properties.","date":"1986","source":"Planta","url":"https://pubmed.ncbi.nlm.nih.gov/24232028","citation_count":16,"is_preprint":false},{"pmid":"34276416","id":"PMC_34276416","title":"Comparative Analysis of Skeletal Muscle DNA Methylation and Transcriptome of the Chicken Embryo at Different Developmental Stages.","date":"2021","source":"Frontiers in physiology","url":"https://pubmed.ncbi.nlm.nih.gov/34276416","citation_count":15,"is_preprint":false},{"pmid":"10406962","id":"PMC_10406962","title":"Cloning, expression and characterization of an A6-related protein.","date":"1999","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10406962","citation_count":9,"is_preprint":false},{"pmid":"36128444","id":"PMC_36128444","title":"Oncogenic role of TWF2 in human tumors: A pan-cancer analysis.","date":"2022","source":"Open medicine (Warsaw, Poland)","url":"https://pubmed.ncbi.nlm.nih.gov/36128444","citation_count":4,"is_preprint":false},{"pmid":"39071866","id":"PMC_39071866","title":"Repurposing FDA approved drugs against monkeypox virus DNA dependent RNA polymerase: virtual screening, normal mode analysis and molecular dynamics simulation studies.","date":"2024","source":"Virusdisease","url":"https://pubmed.ncbi.nlm.nih.gov/39071866","citation_count":3,"is_preprint":false},{"pmid":"40948085","id":"PMC_40948085","title":"TWF2 Drives Tumor Progression and Sunitinib Resistance in Renal Cell Carcinoma through Hippo Signaling Suppression.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/40948085","citation_count":2,"is_preprint":false},{"pmid":"40597474","id":"PMC_40597474","title":"Actin-myosin complex dissociation initiates programmed cell death during cold storage of grass carp muscle.","date":"2025","source":"Food research international (Ottawa, Ont.)","url":"https://pubmed.ncbi.nlm.nih.gov/40597474","citation_count":2,"is_preprint":false},{"pmid":"41125014","id":"PMC_41125014","title":"Multi-omics analysis reveals dynamic proteomic remodeling and metabonomic dysregulation underlying cytokine release syndrome in CAR-T-treated B-ALL.","date":"2025","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41125014","citation_count":0,"is_preprint":false},{"pmid":"41145031","id":"PMC_41145031","title":"Development of high-efficiency hybrid strains for cellulolytic enzyme production via interspecific and intergeneric protoplast fusion of Trichoderma and Penicillium species.","date":"2025","source":"Enzyme and microbial technology","url":"https://pubmed.ncbi.nlm.nih.gov/41145031","citation_count":0,"is_preprint":false},{"pmid":"42129632","id":"PMC_42129632","title":"Global landscape of alternative polyadenylation during the growth and development of skeletal muscle in pigs.","date":"2026","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/42129632","citation_count":0,"is_preprint":false},{"pmid":"41195540","id":"PMC_41195540","title":"Twinfilin modulates tissue contractility through uncapping of capping protein in C. elegans.","date":"2025","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/41195540","citation_count":0,"is_preprint":false},{"pmid":"40667244","id":"PMC_40667244","title":"Twinfilin is a potent uncapper of actin capping protein and modulates actomyosin contractility in the C. elegans spermatheca.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40667244","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13958,"output_tokens":2292,"usd":0.038127,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9324,"output_tokens":3024,"usd":0.06111,"stage2_stop_reason":"end_turn"},"total_usd":0.099237,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Two biochemically distinct isoforms (TWF2a and TWF2b) are generated from the mouse Twf2 gene by alternative promoter usage. Both isoforms bind actin monomers and capping protein, and both cap actin filament barbed ends. However, the N-terminal ADF-H domain of TWF2b interacts with ADP-G-actin with 5-fold higher affinity than ATP-G-actin, whereas the corresponding domain of TWF2a binds ADP-G-actin and ATP-G-actin with equal affinities. TWF2a is predominantly expressed in non-muscle tissues; TWF2b is restricted to heart and skeletal muscle.\",\n      \"method\": \"Alternative promoter identification, in vitro actin monomer binding assays, barbed-end capping assays, affinity measurements for ADP-G-actin vs. ATP-G-actin\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution (capping assay, binding affinity measurements) with multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"18837697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"TWF2 (A6-related protein, A6rp) was identified as a binding partner of protein kinase Czeta (PKCzeta) via yeast two-hybrid. GST-A6rp was selectively phosphorylated by PKCzeta but not significantly by other PKC isoenzymes, and was phosphorylated by casein kinase 2 and most effectively by the tyrosine kinase Src. Both A6rp and A6 bind ATP, consistent with their potential ATP-binding sites. No autokinase activity was detected for either A6rp or A6.\",\n      \"method\": \"Yeast two-hybrid, in vitro kinase assay with GST fusion proteins, immunoblotting, ATP binding assay\",\n      \"journal\": \"European Journal of Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus in vitro kinase assays with multiple kinases, single lab\",\n      \"pmids\": [\"10406962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TWF2 concentrates at stereocilia tips in hair cells during development, co-localizing with the CAPZB subunit of heterodimeric capping protein. TWF2 localization to tips parallels that of CAPZB, and both CAPZB and TWF2 tip enrichment fails to occur at row 2 stereocilia tips in transduction mutants (Tmc1/Tmc2 or Tmie knockout). CAPZB knockout causes stereocilia to shorten and disappear with concurrent decrease in width, implicating capping protein (and its partner TWF2) in stereocilia widening.\",\n      \"method\": \"Immunofluorescence localization in hair cells, conditional Capzb knockout (Atoh1-Cre), in utero electroporation of CAPZB2\",\n      \"journal\": \"The Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal localization and genetic knockout with defined morphological phenotype, replicated across multiple mutant backgrounds\",\n      \"pmids\": [\"28899994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TWF2 normally concentrates at row 2 stereocilia tips in wild-type hair cell bundles, and this localization requires functional mechanotransduction channels. In transduction mutants (Tmc1KO;Tmc2KO or TmieKO), TWF2 fails to concentrate at row 2 tips, demonstrating that mechanotransduction specifies and maintains the row 2 protein complex that includes TWF2 and CAPZB.\",\n      \"method\": \"Quantitative immunofluorescence of phalloidin-labeled mouse cochleas, analysis of genetic transduction mutants\",\n      \"journal\": \"Current Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization imaging in multiple independent genetic mutants, replicating findings from related study\",\n      \"pmids\": [\"31902726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BAIAP2L2 binds to TWF2 (as well as EPS8L2 and CAPZB2), functioning as a component of the row 2 complex at stereocilia tips. BAIAP2L2 knockout disrupts row 2 complex integrity and causes degeneration of mechanotransducing stereocilia.\",\n      \"method\": \"Co-immunoprecipitation (binding of BAIAP2L2 to TWF2, EPS8L2, CAPZB2), Baiap2l2 knockout mice, electrophysiology, FM 1-43FX dye uptake\",\n      \"journal\": \"Journal of Cellular Physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus genetic knockout with functional readout, single lab\",\n      \"pmids\": [\"34346063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TWF2 promotes RCC cell invasion, migration, metastasis, and sunitinib resistance by suppressing Hippo signaling. Mechanistically, TWF2 interacts with YAP via TWF2 residue M99 and YAP residue M225. By competitively displacing LATS1 (large tumor suppressor kinase 1), TWF2 prevents YAP ubiquitination and degradation, leading to YAP stabilization and nuclear translocation. Mutation of M99 abolishes the tumor-promoting activity of TWF2.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (M99 mutation), functional invasion/migration assays, patient-derived xenograft models, small-molecule inhibitor (salvianolic acid E) of TWF2-YAP interaction\",\n      \"journal\": \"Advanced Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, mutagenesis, and in vivo PDX model, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40948085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The C. elegans twinfilin ortholog TWF-2 (ortholog of TWF2) localizes to the spermathecal cortex through interactions with α-spectrin (SPC-1) and β-spectrin (UNC-70). In vitro, TWF-2 promotes barbed-end depolymerization and rapidly displaces capping protein (CAP-1/CAP-2) from filament barbed ends. In vivo, loss of TWF-2 partially rescues embryonic lethality caused by CAP-1 depletion (genetic epistasis). Loss of TWF-2 suppresses hypercontractility (elevated F-actin and phosphorylated myosin) from SPV-1 loss by reducing F-actin levels without affecting myosin phosphorylation, demonstrating a specific role in F-actin regulation and tissue contractility.\",\n      \"method\": \"C. elegans genetics (epistasis with cap-1 and spv-1 loss-of-function), in vitro actin depolymerization and capping protein uncapping assays, immunofluorescence localization, Co-IP with spectrins\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution (capping/uncapping assay) combined with genetic epistasis in multiple mutant backgrounds and direct localization; peer-reviewed and preprint versions independently consistent\",\n      \"pmids\": [\"41195540\", \"40667244\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TWF2 (twinfilin-2) is an evolutionarily conserved ADF-H domain protein that caps actin filament barbed ends and sequesters actin monomers; it is generated as two biochemically distinct tissue-specific isoforms by alternative promoter usage, interacts with heterodimeric capping protein (CAPZB) to regulate stereocilia dimensions at hair cell row 2 tips in a mechanotransduction-dependent manner, modulates actomyosin contractility by uncapping capping protein from filament barbed ends (as shown by C. elegans genetics and in vitro assays), is phosphorylated by PKCzeta and Src kinase, and in cancer cells competitively displaces LATS1 from YAP to prevent YAP ubiquitination and degradation, thereby suppressing Hippo signaling and promoting tumor invasion and drug resistance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TWF2 (twinfilin-2) is an evolutionarily conserved ADF-H domain actin-regulatory protein that binds actin monomers, caps actin filament barbed ends, and dynamically remodels actin networks [#0]. The mouse gene produces two biochemically distinct isoforms by alternative promoter usage—TWF2a, broadly expressed in non-muscle tissues, binds ADP- and ATP-G-actin with equal affinity, whereas the muscle-restricted TWF2b binds ADP-G-actin with ~5-fold higher affinity—but both isoforms cap barbed ends and bind capping protein [#0]. Beyond static capping, TWF2 actively promotes barbed-end depolymerization and rapidly displaces (uncaps) capping protein from filament ends, and through this activity regulates F-actin levels and actomyosin contractility, as established by C. elegans genetics where loss of the ortholog suppresses both capping-protein-depletion lethality and hypercontractility downstream of spectrin scaffolding [#6]. In vertebrate hair cells, TWF2 co-localizes with the CAPZB capping-protein subunit at row 2 stereocilia tips, where its enrichment depends on functional mechanotransduction and contributes, as part of a complex that also includes BAIAP2L2 and EPS8L2, to specifying stereocilia dimensions [#2, #3, #4]. TWF2 is a substrate of PKCzeta, casein kinase 2, and most effectively Src, linking it to kinase signaling [#1]. In renal cell carcinoma, TWF2 acquires a signaling role by binding YAP (via TWF2 residue M99 and YAP M225) and competitively displacing LATS1, thereby blocking YAP ubiquitination, stabilizing nuclear YAP, suppressing Hippo signaling, and driving invasion, metastasis, and sunitinib resistance [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Before its actin role was defined, TWF2 was placed in a kinase-signaling context by identifying it as a phosphorylation target, establishing that it is regulated by serine/threonine and tyrosine kinases.\",\n      \"evidence\": \"Yeast two-hybrid against PKCzeta plus in vitro kinase assays with GST fusions and an ATP-binding assay\",\n      \"pmids\": [\"10406962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of phosphorylation on actin binding or capping not tested\", \"Phosphosites not mapped\", \"No cellular validation of the PKCzeta interaction\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved whether TWF2 is a single biochemical entity by showing two promoter-driven isoforms with distinct nucleotide-state actin preferences and tissue distributions, establishing TWF2 as a tissue-specialized actin monomer-binding and barbed-end capping protein.\",\n      \"evidence\": \"Alternative promoter identification with in vitro actin monomer binding, capping, and ADP/ATP-G-actin affinity measurements\",\n      \"pmids\": [\"18837697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional difference between isoforms in cells not demonstrated\", \"Physiological role of muscle-restricted TWF2b not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected TWF2 to a defined cellular structure by showing it co-localizes with CAPZB at stereocilia tips and that capping protein controls stereocilia width, placing TWF2 in the actin machinery that sizes mechanosensory protrusions.\",\n      \"evidence\": \"Immunofluorescence, conditional Capzb knockout, and CAPZB2 electroporation in mouse hair cells\",\n      \"pmids\": [\"28899994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct functional requirement for TWF2 (vs CAPZB) in stereocilia not isolated\", \"Whether TWF2 caps or uncaps in this context unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established that TWF2 tip localization is not constitutive but is specified by mechanotransduction, defining it as a member of an activity-dependent row 2 protein complex.\",\n      \"evidence\": \"Quantitative immunofluorescence of cochleas from Tmc1/Tmc2 and Tmie transduction mutants\",\n      \"pmids\": [\"31902726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular signal coupling channel activity to TWF2 recruitment unknown\", \"TWF2 loss-of-function phenotype in hair cells not shown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified BAIAP2L2 as a binding partner that organizes the row 2 complex containing TWF2, EPS8L2, and CAPZB2, defining the protein interaction architecture maintaining mechanotransducing stereocilia.\",\n      \"evidence\": \"Co-immunoprecipitation and Baiap2l2 knockout mice with electrophysiology and FM dye uptake\",\n      \"pmids\": [\"34346063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of BAIAP2L2-TWF2 binding not resolved\", \"Whether TWF2 is required for complex integrity untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the mechanistic basis of TWF2's actin-remodeling activity in vivo by showing it depolymerizes barbed ends, uncaps capping protein, and regulates tissue contractility via spectrin-anchored localization.\",\n      \"evidence\": \"C. elegans genetic epistasis with cap-1 and spv-1, in vitro depolymerization/uncapping assays, and Co-IP with α/β-spectrin\",\n      \"pmids\": [\"41195540\", \"40667244\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether vertebrate TWF2 uncaps CAPZB in stereocilia not directly tested\", \"Isoform specificity of uncapping activity not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a non-cytoskeletal signaling role by showing TWF2 binds YAP and displaces LATS1 to stabilize YAP, linking TWF2 to Hippo pathway suppression and cancer progression.\",\n      \"evidence\": \"Co-IP, M99/M225 site-directed mutagenesis, invasion/migration assays, PDX models, and a small-molecule inhibitor of the TWF2-YAP interaction\",\n      \"pmids\": [\"40948085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between actin-regulatory and YAP-binding functions of TWF2 unknown\", \"Whether the interaction generalizes beyond renal cell carcinoma not established\", \"Structural basis of M99-M225 contact not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TWF2's biochemical activities (capping vs uncapping/depolymerization), its kinase regulation, and its YAP-binding signaling function are integrated within a single protein across tissues remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model linking the actin-binding and YAP-binding surfaces\", \"Whether phosphorylation by PKCzeta/Src switches between capping and signaling roles untested\", \"Isoform-specific functions in mammalian tissues uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 2, 6]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 3, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"row 2 stereocilia tip complex\"],\n    \"partners\": [\"CAPZB\", \"BAIAP2L2\", \"EPS8L2\", \"YAP1\", \"LATS1\", \"PRKCZ\", \"SPC-1\", \"UNC-70\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}