{"gene":"INF2","run_date":"2026-04-28T18:06:54","timeline":{"discoveries":[{"year":2009,"finding":"INF2 is peripherally bound to the cytoplasmic face of the endoplasmic reticulum (ER) via a C-terminal farnesyl (CAAX) modification; farnesylation is required but not sufficient for ER association, with ionic interactions also important. The WH2 motif functions as a DAD that binds the DID with Kd ~1.1 µM, and this DID-DAD interaction inhibits INF2's depolymerization activity but not its nucleation activity. DAD/WH2 mutations that abolish DID-DAD binding cause ER collapse around the nucleus with actin accumulation.","method":"GFP-fusion live-cell imaging, membrane fractionation/extraction, farnesylation inhibitor treatment, in vitro binding assay (apparent Kd measurement), site-directed mutagenesis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (biochemical binding assay, mutagenesis, cell imaging, fractionation) in single study with rigorous controls","pmids":["19366733"],"is_preprint":false},{"year":2009,"finding":"Mutations in the diaphanous inhibitory domain (DID) of INF2 cause autosomal dominant FSGS; all disease-causing mutations reside in the DID, a region that interacts with the DAD and competes for actin monomer binding, implicating disrupted autoinhibitory DID-DAD interaction in disease.","method":"Linkage analysis, sequencing of familial FSGS cohort, conservation analysis of mutated residues","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis linking DID mutations to disease; functional mechanism inferred from domain knowledge, confirmed by biochemical studies in companion papers","pmids":["20023659"],"is_preprint":false},{"year":2010,"finding":"INF2 interacts with MAL2 and regulates basolateral-to-apical transcytosis and lumen formation in epithelial cells. Both actin polymerization and depolymerization activities of INF2 are required. INF2 binds Cdc42 in a GTP-loaded-dependent manner, and Cdc42 and INF2 together regulate MAL2 vesicle dynamics and transcytosis.","method":"Co-immunoprecipitation, knockdown (siRNA), live videomicroscopy, organotypic culture lumen formation assay, GTPase binding assay","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, functional knockdown with specific phenotypic readout, multiple orthogonal methods","pmids":["20493814"],"is_preprint":false},{"year":2010,"finding":"INF2 interacts with MAL in T lymphocytes and colocalizes at the cell periphery, pericentriolar endosomes, and along microtubules. Knockdown of INF2 reduces MAL+ transport vesicle formation and Lck levels at the plasma membrane, impairing immunological synapse formation. Both actin polymerization and depolymerization activities of INF2 are required for efficient Lck targeting. Cdc42 and Rac1, which bind INF2, regulate this transport.","method":"Co-immunoprecipitation, siRNA knockdown, videomicroscopy, flow cytometry (surface Lck), immunological synapse assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, KD with specific phenotypic readout, multiple orthogonal assays","pmids":["20881207"],"is_preprint":false},{"year":2011,"finding":"INF2 DID binds directly to the DADs of mDia1, mDia2, and mDia3 (diaphanous-related formins); this interaction inhibits mDia-mediated Rho-activated actin polymerization and SRF-responsive transcription. Disease-causing INF2 DID mutations (E184K, R218Q) show decreased capacity to inhibit SRF activation and gene transcription, suggesting that DID mutations impair inhibition of mDia signaling.","method":"Yeast two-hybrid screen, in vitro binding assay, co-immunoprecipitation, colocalization (immunofluorescence), SRF-luciferase reporter assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro binding, reciprocal Co-IP, functional reporter assay with mutagenesis","pmids":["21278336"],"is_preprint":false},{"year":2011,"finding":"INF2 exists as two C-terminal splice variants: CAAX (ER-localized via farnesyl group, actin-independent) and non-CAAX (localizes to an actin-dependent meshwork). Suppression of INF2-non-CAAX causes Golgi apparatus fragmentation, an effect reversed by latrunculin B treatment. Discrete actin patches in the peri-Golgi region are reduced upon INF2 suppression.","method":"siRNA knockdown, digitonin extraction (fractionation), latrunculin B treatment, fluorescence microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — KD with specific organelle phenotype, pharmacological rescue, isoform-specific localization","pmids":["21998196"],"is_preprint":false},{"year":2011,"finding":"INF2 (FH1-FH2-C construct) binds microtubules with high affinity (Kd < 100 nM) and bundles microtubules, reducing catastrophe rate. Microtubule bundling requires the C-terminus of INF2. Actin monomers inhibit microtubule binding/bundling by INF2 but microtubules do not inhibit INF2-mediated actin polymerization, distinguishing INF2 from mDia1/mDia2.","method":"In vitro microtubule co-sedimentation, TIRF microscopy, biochemical binding assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro assays with quantitative affinity measurements and domain mutagenesis","pmids":["21998204"],"is_preprint":false},{"year":2011,"finding":"INF2 mutations causing CMT with FSGS perturb the INF2-MAL-CDC42 pathway, resulting in actin cytoskeleton disorganization, enhanced INF2 binding to CDC42, and mislocalization of INF2, MAL, and CDC42 in Schwann cells. INF2 colocalizes and interacts with MAL in Schwann cells.","method":"Co-immunoprecipitation, immunohistochemistry, cell culture functional studies with patient-derived mutations","journal":"The New England journal of medicine","confidence":"High","confidence_rationale":"Tier 2 — Co-IP in relevant cell type, multiple disease mutations tested, colocalization validated","pmids":["22187985"],"is_preprint":false},{"year":2012,"finding":"INF2 FH2 domain mutations I643A and K792A have unexpected effects: I643A causes tight capping of a subset of filaments and nearly abolishes severing/depolymerization without abolishing barbed-end binding; K792A causes slow elongation with minor effects on severing. In cells, I643A cannot promote actin polymerization because it is unable to elongate in the presence of capping protein.","method":"Pyrene-actin assembly assay, TIRF microscopy, site-directed mutagenesis, cell expression assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis and multiple orthogonal assays","pmids":["22879592"],"is_preprint":false},{"year":2012,"finding":"INF2 is required for formation of detyrosinated (stable) microtubules after T cell receptor engagement, and this is essential for centrosome reorientation to the immunological synapse. The FH2 domain of INF2 mediates centrosome repositioning and can rescue DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells, placing INF2 downstream of or parallel to these factors.","method":"siRNA knockdown, genetic epistasis rescue experiments, immunofluorescence for detyrosinated tubulin, centrosome polarization assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — KD with specific phenotypic readout, epistasis rescue, domain mapping","pmids":["22986496"],"is_preprint":false},{"year":2013,"finding":"ER-localized INF2 mediates actin polymerization at ER-mitochondria contact sites to drive mitochondrial constriction and fission, functioning upstream of Drp1. Actin filaments accumulate between mitochondria and INF2-enriched ER at constriction sites. INF2 knockdown reduces mitochondrial fission.","method":"siRNA knockdown, live-cell imaging, electron microscopy, dominant-negative INF2 expression, epistasis with Drp1","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 — KD with specific organelle phenotype, epistasis placing INF2 upstream of Drp1, electron microscopy of constriction sites; widely replicated","pmids":["23349293"],"is_preprint":false},{"year":2014,"finding":"INF2 severs actin filaments through FH2 domain encirclement of the filament; a single INF2 dimer can sever filaments in a phosphate-release-dependent manner. The C-terminal DAD/WH2 motif increases severing potency 40-fold by binding adjacent actin protomers to further disrupt filament structure. Helical 3D reconstruction at 20 Å resolution shows FH2 encircling the filament.","method":"Two-color TIRF microscopy, pyrene-actin assay, ADP/ADP-Pi filament binding assay, electron microscopy (helical reconstruction), domain truncation/mutagenesis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 — structural EM plus in vitro reconstitution with domain mutagenesis, multiple orthogonal methods","pmids":["24412206"],"is_preprint":false},{"year":2014,"finding":"INF2 knockdown in zebrafish causes edema, podocyte dysfunction, and altered glomerular filtration barrier associated with mistrafficking of slit diaphragm proteins (nephrin/podocin) and disinhibited mDia (Dia2) activity. This phenotype is rescued by wild-type INF2 but not disease-causing INF2 mutants, and also rescued by loss of RhoA or Dia2, genetically placing INF2 as a negative regulator of Rho/Dia signaling in podocytes.","method":"Morpholino knockdown in zebrafish, rescue with wild-type vs. mutant INF2, double knockdown epistasis (RhoA, Dia2), immunofluorescence of slit diaphragm proteins","journal":"EBioMedicine","confidence":"High","confidence_rationale":"Tier 2 — in vivo epistasis with specific phenotype, mutant rescue experiment, multiple genetic perturbations","pmids":["26086034"],"is_preprint":false},{"year":2015,"finding":"INF2 generates short actin filaments (<60 nm) that continuously turn over through a cycle of barbed-end elongation, severing, and WH2-mediated depolymerization. The rate-limiting step is nucleotide exchange on actin monomers after release from INF2. Profilin accelerates turnover 6-fold and shifts the equilibrium toward polymerization.","method":"Pyrene-actin assay, TIRF microscopy, biochemical kinetics with domain truncations and profilin addition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mechanistic dissection of kinetics","pmids":["26124273"],"is_preprint":false},{"year":2015,"finding":"C. elegans EXC-6 (INF2 ortholog) regulates F-actin and microtubule cytoskeleton at excretory cell tips during tubulogenesis; disease-associated INF2 mutations cause constitutive activity in this system. Activated INF2 can substitute for EXC-6, and genetic analysis places EXC-6 function in organizing both F-actin and MT arrays for basolateral outgrowth and lumen formation.","method":"C. elegans genetics, live imaging, rescue with activated/mutant human INF2, genetic epistasis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — ortholog functional characterization with genetic epistasis and human INF2 rescue; disease mutations shown to cause constitutive activation","pmids":["25771894"],"is_preprint":false},{"year":2016,"finding":"INF2 acts downstream of mDia1 in a formin activation cascade to stabilize detyrosinated microtubules (Glu MTs) in fibroblasts after LPA stimulation. IQGAP1 scaffolds this cascade by directly binding the C-terminus of INF2 and facilitating LPA-induced mDia1-INF2 interaction and INF2 localization to MTs. Mutants disrupting mDia1-INF2 interaction fail to rescue MT stability in cells depleted of the respective formin.","method":"siRNA knockdown epistasis, direct N-IQGAP1/C-INF2 binding assay (pulldown), LPA-induced colocalization, formin interaction mutants","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — epistasis, direct binding assay, formin interaction mutants, multiple orthogonal approaches","pmids":["27030671"],"is_preprint":false},{"year":2016,"finding":"Mice with the FSGS-associated INF2 R218Q knockin mutation show impaired reversal of protamine sulfate-induced podocyte foot process effacement; mutant podocytes display persistent cytoplasmic nephrin/podocin aggregation, nephrin phosphorylation, and impaired recovery of mDia membrane localization, demonstrating that normal INF2 is required for actin-based recovery from podocyte injury.","method":"Knockin mouse model, protamine sulfate/heparin perfusion model, immunofluorescence of slit diaphragm proteins, podocyte primary culture","journal":"Kidney international","confidence":"High","confidence_rationale":"Tier 2 — disease knockin mouse with specific injury model and multiple molecular readouts","pmids":["27350175"],"is_preprint":false},{"year":2017,"finding":"SPOP (CUL3-RBX1 E3 ubiquitin ligase adaptor) recognizes a Ser/Thr-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2 that reduces INF2 ER localization and mitochondrially associated DRP1 puncta formation without causing INF2 degradation, thereby inhibiting mitochondrial fission. Cancer-associated SPOP mutants increase INF2 ER localization and promote fission through dominant-negative inhibition of endogenous SPOP.","method":"Co-immunoprecipitation, ubiquitination assay, subcellular fractionation, DRP1 puncta quantification, SPOP mutant expression","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, in vivo ubiquitination assay, localization with functional consequence, multiple disease-associated mutants tested","pmids":["28448495"],"is_preprint":false},{"year":2019,"finding":"INF2 is inhibited by a complex of lysine-acetylated actin (KAc-actin) bound to cyclase-associated protein (CAP), purified from mouse brain tissue. This inhibition requires the INF2 DID. Treatment with HDAC6 releases INF2 inhibition, and HDAC6 inhibitors block cellular INF2 activation. Disease-associated INF2 DID mutants are poorly inhibited by CAP-KAc-actin, suggesting FSGS/CMT mutations reduce CAP-KAc-actin binding.","method":"Biochemical purification of inhibitory complex from brain tissue, in vitro actin polymerization assay, HDAC6 treatment, HDAC6 inhibitor (tubastatin A) in cells, mutagenesis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 — purification of endogenous inhibitor, in vitro reconstitution, pharmacological validation in cells, disease mutants tested","pmids":["30962575"],"is_preprint":false},{"year":2019,"finding":"Lysine residues K50 and K61 on actin are the key sites for CAP/KAc-actin-mediated INF2 inhibition. K50Q- and K61Q-actin (acetylmimetic) bound to CAP2 inhibit full-length INF2 in a DID-dependent manner. The CAP WH2 domain binds INF2-DID with submicromolar affinity; INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin, supporting a bridging model between INF2 DID and DAD.","method":"Lysine-to-glutamine acetylmimetic mutations, in vitro INF2 inhibition assay, direct binding studies, U2OS cell overexpression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with acetylmimetic mutations, quantitative binding assays, cellular validation","pmids":["31871199"],"is_preprint":false},{"year":2020,"finding":"INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved by cathepsin proteases, liberating the N-terminal DID fragment. The N-terminal fragment localizes to podocyte foot processes (but not with FSGS mutations), binds mDIA1, and promotes cell spreading in a cleavage-dependent manner. FSGS-associated R218Q mutation impairs N-fragment localization and function but not cleavage itself.","method":"Western blot (isoform/cleavage analysis), cathepsin inhibitors, co-immunoprecipitation (N-fragment/mDIA1), podocyte spreading assay, immunofluorescence","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 — multiple methods including protease inhibition, Co-IP, and functional cell assay in disease-relevant cell type","pmids":["31924668"],"is_preprint":false},{"year":2020,"finding":"INF2 mutations cause constitutive activation of formin activity and a deregulated calcium-mediated actin reset (CaAR) stress response. FSGS-only mutations are distinguishable from CMT+FSGS mutations by degree of INF2 activation, enabling genotype-phenotype correlation. This was validated in primary patient cells and Drosophila nephrocytes.","method":"Quantitative live-cell imaging of CaAR, primary patient cells, Drosophila nephrocyte functional assay, panel of >50 disease mutants","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 — systematic functional characterization of large mutation panel in multiple model systems","pmids":["32444357"],"is_preprint":false},{"year":2020,"finding":"INF2 interacts with dynein light chain 1 (DYNLL1), and pathogenic INF2 mutations (R218Q) disrupt this interaction, diverting dynein-mediated post-endocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport rescues this defect.","method":"Yeast two-hybrid screen, live cell imaging, fluorescent/surface biotinylation trafficking assays, INF2 transgenic mouse model, dynein antagonism","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by trafficking assays, in vivo mouse model, pharmacological rescue","pmids":["33443052"],"is_preprint":false},{"year":2022,"finding":"The N-terminal extension of INF2 forms two α-helices; the first helix contains the sole calmodulin (CaM)-binding site (CaMBS) with key residues W11, L14, L18 (1-4-8 motif). CaM C-terminal lobe directly interacts with this helix. Ca2+/CaM binding activates INF2: INF2 KO cells do not form the perinuclear F-actin ring or respond to Ca2+ elevation with actin polymerization; expression of INF2 with inactivated CaMBS fails to rescue these defects.","method":"NMR spectroscopy, biochemical binding assays, INF2 KO cell complementation, Ca2+ stimulation assay, CaMBS mutagenesis (W11, L14, L18)","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with mutagenesis, KO rescue experiment, functional calcium response assay","pmids":["36306014"],"is_preprint":false},{"year":2022,"finding":"Ischemia and excess NMDA receptor activation cause INF2-dependent actin reorganization (actinification) within the somatodendritic compartment: F-actin depolymerizes from spines and polymerizes into stable filaments in dendritic shafts/soma. Ca2+ influx combined with spine F-actin depolymerization activates INF2. INF2 silencing renders neurons vulnerable to ischemic death; INF2 overexpression is protective.","method":"siRNA knockdown, INF2 overexpression, live-cell F-actin imaging, NMDA application, photothrombotic stroke model in mouse, ion flux measurements","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — KD and OE with specific phenotypic readout, in vitro and in vivo models, mechanistic Ca2+ requirement established","pmids":["36229429"],"is_preprint":false},{"year":2022,"finding":"INF2 forms an 8S complex with actin (4 actin monomers:2 INF2 dimers) in which actin monomers are arranged in parallel orientation. The 8S particles can seed actin assembly. INF2 accelerates the disassembly of oxidized (Mox) F-actin.","method":"Negative-stain electron microscopy (2D class averages), chemical crosslinking, pyrene-actin assembly seeding assay","journal":"Journal of biomolecular structure & dynamics","confidence":"Medium","confidence_rationale":"Tier 1-2 — EM structural characterization and biochemical assay, but single study with limited resolution","pmids":["35343388"],"is_preprint":false},{"year":2024,"finding":"AMPK phosphorylates INF2 at Ser1077 under energy stress, increasing INF2 localization to the ER and enhancing DRP1 recruitment to mitochondria, thereby promoting mitochondrial fission and endometrial cancer cell growth.","method":"In vitro kinase assay, phospho-specific immunoblot, ER localization imaging, DRP1 co-localization, siRNA knockdown, immunohistochemistry","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro kinase assay with specific phosphosite, localization with functional consequence, single lab","pmids":["38233384"],"is_preprint":false},{"year":2024,"finding":"FBXO7 E3 ubiquitin ligase ubiquitinates INF2 and targets it for degradation, thereby inhibiting INF2-DRP1 axis-mediated mitochondrial fission. ECa-associated FBXO7 mutants are defective in INF2 ubiquitination and degradation, promoting mitochondrial hyper-fission.","method":"Co-immunoprecipitation, ubiquitination assay, protein stability assay (cycloheximide chase), siRNA knockdown, DRP1 inhibitor (Mdivi-1)","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, in vivo ubiquitination assay, functional rescue, single lab","pmids":["37344480"],"is_preprint":false},{"year":2024,"finding":"INF2 R218Q knockin mice (but not INF2 knockout mice) are susceptible to glomerular disease upon puromycin aminonucleoside challenge, demonstrating a gain-of-function disease mechanism. R218Q INF2 alters actin cytoskeleton content and INF2 localization. Adhesion and mitochondria-related pathways are enriched in R218Q glomeruli. Patient-derived organoid podocytes with S186P INF2 recapitulate adhesion and mitochondrial phenotypes.","method":"Knockin vs. knockout mouse comparison, puromycin aminonucleoside injury model, colocalization, co-immunoprecipitation, actin measurements, RNA-seq, human kidney organoids","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — rigorous KI vs KO comparison with in vivo injury model, multiple molecular readouts, human organoid validation","pmids":["39536114"],"is_preprint":false},{"year":2024,"finding":"INF2 R218Q mutation disrupts sequestration of DYNLL1 by INF2, allowing DYNLL1-PI31 interaction that promotes dynein-mediated transport of nephrin to the proteasome for degradation. Proteasome inhibition with bortezomib stabilizes nephrin in R218Q podocytes and ameliorates FSGS in vivo.","method":"Co-immunoprecipitation (DYNLL1-PI31), siRNA knockdown of DYNLL1/PI31, proteasome inhibitor treatment, nephrin trafficking assay, R218Q KI mouse puromycin model","journal":"Kidney360","confidence":"High","confidence_rationale":"Tier 2 — mechanistic Co-IP, genetic and pharmacological interventions, in vivo validation","pmids":["39621430"],"is_preprint":false},{"year":2024,"finding":"Pathogenic INF2 variants cause ER dysmorphism (shift from tubular to sheet-like ER), mitochondrial fragmentation with peripheral misdistribution, and impaired lysosomal trafficking; CMT+FSGS variants cause more severe ER and mitochondrial disruption than FSGS-only variants, linked to greater cytoskeletal disorganization.","method":"High-resolution live imaging of HeLa cells expressing INF2 variants, ER/mitochondria morphology quantification, actin and MT inhibitor treatments","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — live imaging with functional correlates, systematic comparison of multiple variants, single lab","pmids":["39337270"],"is_preprint":false},{"year":2024,"finding":"Pathogenic INF2 promotes translocation of transcriptional cofactor MRTF to the nucleus, causing sustained MRTF-SRF transcriptional complex activation, profound transcriptome reprogramming, multipolar spindle assembly, multi-micronucleation, and cell death. Reducing MRTF-SRF activation mitigates nuclear abnormalities. Inactivation of the INF2 catalytic domain prevents aberrant nuclei.","method":"RNA sequencing, MRTF localization imaging, retroviral/plasmid expression of pathogenic INF2 in renal epithelial and primary podocyte cells, catalytic domain inactivation","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 — RNA-seq with pathway analysis, MRTF localization assay, catalytic mutant, single lab","pmids":["38916773"],"is_preprint":false},{"year":2024,"finding":"INF2-mediated actin polymerization at ER-organelle contact sites regulates the size and movement of mitochondria, endosomes, and lysosomes; ER-associated actin consistently marks fission sites for all three organelle types.","method":"Live imaging of organelle dynamics, ER-associated actin visualization, INF2 knockdown/overexpression, organelle fission site analysis","journal":"Research square (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 — live imaging with functional consequence, extends INF2-organelle fission concept; preprint, not peer-reviewed","pmids":["39184068"],"is_preprint":true},{"year":2024,"finding":"CAP1 and CAP2 (cyclase-associated proteins) inhibit INF2 in neurons to permit dendritic spine maturation. Overactivation of INF2 impairs spine maturation, and INF2 inactivation rescues spine defects in CAP-deficient hippocampal neurons, placing CAP-mediated INF2 inhibition as a molecular switch for filopodia-to-mature-spine transition.","method":"Genetic inactivation (KO) of CAP1/CAP2, INF2 overactivation/inactivation in hippocampal neurons, spine density/morphology imaging","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis in neurons with specific morphological readout, multiple perturbations","pmids":["39154297"],"is_preprint":false},{"year":2025,"finding":"Piezo1/Ca2+ signaling activates INF2 to induce widespread actin cytoskeletal remodeling, promoting de-adhesion and mesenchymal-to-amoeboid transition (MAT) in melanoma cells in confined environments. INF2 activation is required for cells to effectively migrate in mechanically challenging environments.","method":"Piezo1 inhibition/activation, Ca2+ imaging, INF2 KD, confinement microchannels, amoeboid migration quantification, de-adhesion assay","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — KD with specific migration phenotype, mechanistic Ca2+ requirement, confinement model; single lab","pmids":["40120583"],"is_preprint":false},{"year":2026,"finding":"INF2 autoinhibition does not require CAP proteins but does require actin monomer 'buffering' by profilin or thymosin; Ca2+-bound calmodulin (CALM) activates INF2 through binding to the N-terminus; the N-terminus has additional regulatory roles beyond CALM binding. This was established with a novel cell-free INF2 regulation assay.","method":"Cell-free in vitro assay for INF2 regulation, calmodulin binding assay, profilin/thymosin titration, N-terminus mutants","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — reconstituted cell-free assay with multiple perturbations, revises prior model with rigorous controls","pmids":["41498749"],"is_preprint":false},{"year":2020,"finding":"INF2 negatively regulates CFTR levels at the plasma membrane: reduction of INF2 levels promotes CFTR trafficking to the PM under EPAC1 activation, identified by protein interaction profiling and validated by knockdown.","method":"Protein interaction profiling (proteomics), siRNA knockdown, surface CFTR measurement","journal":"The Biochemical journal","confidence":"Low","confidence_rationale":"Tier 3 — proteomics pulldown followed by KD with single readout, single lab","pmids":["32573649"],"is_preprint":false},{"year":2021,"finding":"EV71 infection causes INF2 cleavage at Asp1051, mediated predominantly by activated caspase-2. Subcellular localization of INF2 and caspase-2 is altered in infected cells, suggesting caspase-2-mediated INF2 cleavage is involved in viral replication organelle formation.","method":"Caspase inhibitor treatment, site-directed mutagenesis of cleavage site, subcellular fractionation/immunofluorescence","journal":"Frontiers in microbiology","confidence":"Low","confidence_rationale":"Tier 3 — mechanistic mutagenesis of cleavage site with pharmacological validation, but single lab and limited functional follow-up","pmids":["34046026"],"is_preprint":false},{"year":2023,"finding":"Drebrin protects assembled actin filaments from INF2-mediated severing without inhibiting INF2's polymerization activity; truncated drebrin (DrbA1-300) is sufficient for this protection. This competitive regulation of actin dynamics is relevant to specialized cells such as neurons and podocytes.","method":"In vitro severing/polymerization assays, electron microscopy, TIRF microscopy, domain truncation","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro reconstitution with domain truncation and imaging, single lab","pmids":["38158176"],"is_preprint":false},{"year":2025,"finding":"The WH2/DAD of INF2 forms a single α-helix (by NMR); the hydrophobic N-terminal region of the WH2/DAD is essential for INF2-mediated actin polymerization and for inducing nuclear abnormalities. Deletion of the entire WH2/DAD or its hydrophobic region abolishes INF2 activity. The WH2/DAD undergoes a conformational change to facilitate actin binding.","method":"NMR spectroscopy, deletion/point mutants of WH2/DAD, cell expression of INF2 variants with F-actin and nuclear phenotype quantification","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with mutagenesis and cellular functional validation","pmids":["40993919"],"is_preprint":false},{"year":2025,"finding":"ERM proteins (Ezrin, Radixin, Moesin) localize to the nuclear envelope and contribute to perinuclear actin rim formation downstream of INF2/Ca2+ activation; ERM overexpression increases perinuclear actin rim levels while ERM knockdown reduces them. Emerin (a nuclear membrane actin-binding protein) is not required for the perinuclear actin rim.","method":"ERM knockdown/overexpression, emerin KO, Ca2+-induced actin rim imaging in melanoma cells","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 — KD/OE with single readout, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.02.22.639617"],"is_preprint":true}],"current_model":"INF2 is an ER-anchored formin (CAAX isoform) that both polymerizes and severs/depolymerizes actin filaments via its FH2 domain encirclement mechanism and C-terminal WH2/DAD motif; it is kept in a 'facilitated autoinhibited' state by a CAP/lysine-acetylated-actin complex requiring DID, and is activated by Ca2+/calmodulin binding to its N-terminal helix or by actin monomer buffering proteins (profilin, thymosin); active INF2 drives mitochondrial fission upstream of Drp1, regulates Golgi integrity, promotes detyrosinated microtubule stabilization, controls vesicular transcytosis through MAL2/Cdc42, negatively regulates mDia/Rho signaling through DID-DAD interactions, and sequesters DYNLL1 to prevent aberrant nephrin trafficking; all FSGS/CMT-causing DID mutations constitutively activate INF2 through a gain-of-function mechanism, leading to uncontrolled actin polymerization, MRTF-SRF transcriptional reprogramming, mitotic abnormalities, and dysregulated organelle dynamics in podocytes and Schwann cells."},"narrative":{"teleology":[{"year":2009,"claim":"Establishing that INF2 is an ER-anchored formin regulated by intramolecular DID–DAD autoinhibition resolved how its unique severing and nucleation activities are compartmentalized and restrained at the ER membrane.","evidence":"GFP-fusion imaging, membrane fractionation, farnesylation inhibitor treatment, and in vitro Kd measurement of DID–DAD interaction in mammalian cells","pmids":["19366733"],"confidence":"High","gaps":["How autoinhibition is relieved by upstream signals was unknown","Structural basis of ER targeting beyond farnesylation remained unclear"]},{"year":2009,"claim":"Identification of INF2 DID mutations as causative for autosomal dominant FSGS established the first disease link and implicated disrupted autoinhibition as the disease mechanism.","evidence":"Linkage analysis and sequencing of familial FSGS cohorts","pmids":["20023659"],"confidence":"Medium","gaps":["Functional proof that DID mutations cause gain-of-function (vs. loss-of-function) was not yet provided","Mechanism by which podocytes are specifically vulnerable was unknown"]},{"year":2010,"claim":"Discovery that INF2 partners with MAL/MAL2 and Cdc42 to regulate vesicular transcytosis and lumen formation revealed a trafficking function beyond simple actin remodeling.","evidence":"Co-immunoprecipitation, siRNA knockdown, and live videomicroscopy in epithelial cells and T lymphocytes","pmids":["20493814","20881207"],"confidence":"High","gaps":["Direct structural basis of INF2–MAL interaction was not defined","Whether this pathway operates in podocytes or Schwann cells was not tested"]},{"year":2011,"claim":"Demonstration that INF2 DID binds and inhibits mDia formins, with FSGS mutations impairing this inhibition, established INF2 as a negative regulator of Rho/mDia signaling and SRF transcription — a trans-regulatory function unprecedented for formins.","evidence":"Yeast two-hybrid, in vitro binding, Co-IP, SRF-luciferase reporter with disease mutants; extended in vivo by zebrafish epistasis showing Dia2/RhoA rescue of INF2 loss","pmids":["21278336","26086034"],"confidence":"High","gaps":["Whether INF2–mDia interaction occurs under physiological conditions in podocytes remained unresolved","Relative contribution of mDia inhibition vs. direct actin remodeling to FSGS pathogenesis was unclear"]},{"year":2011,"claim":"Identification of two splice isoforms (CAAX and non-CAAX) with distinct localizations (ER vs. cytosol/Golgi) and the finding that INF2 binds microtubules with high affinity broadened INF2's functional repertoire to include Golgi maintenance and microtubule regulation.","evidence":"siRNA isoform-specific knockdown, digitonin fractionation, latrunculin rescue of Golgi; in vitro MT cosedimentation and TIRF microscopy","pmids":["21998196","21998204"],"confidence":"High","gaps":["How INF2 chooses between actin and MT substrates in vivo was not determined","Physiological signals regulating the non-CAAX isoform were unknown"]},{"year":2013,"claim":"The landmark finding that ER-localized INF2 drives actin polymerization at ER–mitochondria contacts to promote Drp1-dependent mitochondrial fission established the INF2–Drp1 axis as a core organelle fission mechanism.","evidence":"siRNA knockdown, live-cell and electron microscopy of ER–mitochondria constriction sites, epistasis with Drp1","pmids":["23349293"],"confidence":"High","gaps":["How INF2 activation is spatially restricted to contact sites was unknown","Whether INF2 acts at fission sites of other organelles was untested"]},{"year":2014,"claim":"Structural and single-molecule analyses revealed that INF2 severs filaments by FH2 encirclement, with the WH2/DAD amplifying severing 40-fold, providing a mechanistic basis for INF2's unique dual polymerization/severing activity.","evidence":"Two-color TIRF, pyrene-actin assays, 20 Å EM helical reconstruction, domain mutagenesis","pmids":["24412206"],"confidence":"High","gaps":["High-resolution structure of FH2 encircling the filament was not achieved","How severing vs. elongation is regulated in the cell remained unclear"]},{"year":2016,"claim":"Placement of INF2 downstream of mDia1 in an IQGAP1-scaffolded cascade for detyrosinated microtubule stabilization integrated INF2 into the LPA/Rho signaling axis and explained its role in centrosome reorientation at the immunological synapse.","evidence":"siRNA epistasis, direct IQGAP1–INF2 binding assay, LPA stimulation, formin interaction mutants in fibroblasts and T cells","pmids":["27030671","22986496"],"confidence":"High","gaps":["How INF2 biochemically promotes microtubule detyrosination was not elucidated","Whether IQGAP1 scaffolding operates in podocytes was untested"]},{"year":2017,"claim":"Discovery that SPOP-mediated atypical polyubiquitination displaces INF2 from the ER without degradation revealed a non-degradative ubiquitin-based regulatory mechanism controlling mitochondrial fission, with relevance to cancer-associated SPOP mutations.","evidence":"Co-IP, ubiquitination assay, subcellular fractionation, DRP1 puncta quantification with SPOP cancer mutants","pmids":["28448495"],"confidence":"High","gaps":["The ubiquitin chain type and specific lysine sites on INF2 were not identified","Whether SPOP regulation occurs in podocytes or neurons was unknown"]},{"year":2019,"claim":"Identification of CAP/lysine-acetylated-actin as a 'facilitated autoinhibition' complex that locks INF2 via its DID — with disease mutations disrupting this — provided the first physiological inhibitor and unified the gain-of-function disease mechanism.","evidence":"Purification of inhibitory complex from brain, in vitro reconstitution with acetylmimetic actins, HDAC6 pharmacology, disease mutant testing","pmids":["30962575","31871199"],"confidence":"High","gaps":["Structural basis of the CAP–KAc-actin–DID ternary complex was not resolved","Relative contribution of CAP inhibition vs. DID–DAD autoinhibition in vivo was debated"]},{"year":2020,"claim":"Multiple advances solidified the gain-of-function disease model: INF2 R218Q knockin mice showed impaired podocyte recovery, FSGS mutations constitutively activated the calcium-mediated actin response (CaAR), and pathogenic INF2 disrupted DYNLL1 sequestration causing nephrin mistrafficking to lysosomes.","evidence":"R218Q knockin mouse with injury model, CaAR quantification across >50 mutants in patient cells and Drosophila, yeast two-hybrid and nephrin trafficking assays","pmids":["27350175","32444357","33443052"],"confidence":"High","gaps":["Whether CMT phenotype involves the same DYNLL1/nephrin pathway was unknown","Cell-type-specific activation thresholds explaining FSGS-only vs. CMT+FSGS genotype–phenotype correlation were not mechanistically resolved"]},{"year":2022,"claim":"Identification of the N-terminal calmodulin-binding site and demonstration that Ca²⁺/CaM is the physiological activator of INF2 resolved the long-standing question of how INF2 autoinhibition is relieved, and connected INF2 to calcium signaling in neurons (ischemia-protective actinification) and beyond.","evidence":"NMR of N-terminal helices, CaMBS mutagenesis with KO rescue, Ca²⁺-stimulated actin rim assays; NMDA/ischemia models with INF2 silencing/overexpression","pmids":["36306014","36229429"],"confidence":"High","gaps":["Whether CaM releases the CAP/KAc-actin complex or acts independently was not determined","Structural details of full-length INF2 conformational change upon CaM binding were lacking"]},{"year":2024,"claim":"Definitive proof that FSGS-causing INF2 mutations act via gain-of-function came from comparison of knockin vs. knockout mice: only R218Q knockin (not KO) developed glomerular disease, and pathogenic INF2 drove MRTF-SRF nuclear translocation, transcriptome reprogramming, and mitotic catastrophe.","evidence":"KI vs. KO mouse with puromycin injury, RNA-seq of R218Q glomeruli, MRTF imaging, human kidney organoids; catalytic-dead mutant rescue","pmids":["39536114","38916773"],"confidence":"High","gaps":["Whether MRTF-SRF inhibition is a viable therapeutic strategy in vivo was not tested","Contribution of mitotic abnormalities to slowly progressive podocyte loss in human disease was not established"]},{"year":2024,"claim":"Extension of the DYNLL1 mechanism showed that released DYNLL1 binds PI31 to redirect nephrin to proteasomal degradation, and proteasome inhibition rescued nephrin levels in vivo, providing a potential therapeutic angle for INF2-FSGS.","evidence":"Co-IP of DYNLL1–PI31, bortezomib treatment in R218Q KI mouse puromycin model, nephrin trafficking assays","pmids":["39621430"],"confidence":"High","gaps":["Long-term efficacy and safety of proteasome inhibition for FSGS is unknown","Whether other slit diaphragm proteins are similarly affected was not tested"]},{"year":2025,"claim":"A reconstituted cell-free system revised the autoinhibition model: CAP proteins are dispensable for INF2 autoinhibition, which instead requires actin monomer buffering by profilin or thymosin β4, and the N-terminus has regulatory functions beyond CaM binding.","evidence":"Cell-free INF2 regulation assay with profilin/thymosin titration, CaM binding, N-terminal mutants","pmids":["41498749"],"confidence":"High","gaps":["The precise role of the N-terminal region beyond CaM binding is not yet defined","How profilin-mediated autoinhibition integrates with CAP/KAc-actin inhibition in cells is unresolved"]},{"year":null,"claim":"A high-resolution structure of full-length INF2 in autoinhibited and active states, the spatial mechanisms governing INF2 activation selectively at ER–organelle contact sites, and whether therapeutic targeting of the gain-of-function mechanism can prevent or reverse FSGS/CMT remain major open questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of full-length INF2 or INF2–actin filament complex exists","Mechanism of spatial restriction of INF2 activation to specific ER subdomains is unknown","No therapeutic strategy targeting INF2 gain-of-function has been validated in clinical settings"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,6,8,11,13,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,12,15,18,19]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,5,10,17,26]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[6,9,15]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,20]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[10,17,26,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,15,23,34]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,3,22,29]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,7,28,31]}],"complexes":[],"partners":["MAL2","MAL","CDC42","DIAPH1","IQGAP1","DYNLL1","SPOP","CALM1"],"other_free_text":[]},"mechanistic_narrative":"INF2 is a multifunctional formin that uniquely combines actin nucleation/elongation with filament severing and depolymerization activities, enabling rapid actin turnover at endoplasmic reticulum–organelle contact sites and controlling organelle dynamics, vesicular trafficking, and microtubule stability. The ER-anchored CAAX isoform drives actin polymerization at ER–mitochondria contacts to promote Drp1-dependent mitochondrial fission, while the non-CAAX isoform maintains Golgi integrity; INF2 is activated by Ca²⁺/calmodulin binding to its N-terminal helix and kept autoinhibited by actin monomer buffering (profilin/thymosin) and a CAP/lysine-acetylated-actin complex engaging the DID [PMID:23349293, PMID:36306014, PMID:41498749, PMID:30962575]. INF2 also stabilizes detyrosinated microtubules downstream of mDia1 via IQGAP1 scaffolding, sequesters DYNLL1 to protect nephrin from degradation, and negatively regulates mDia/Rho-mediated SRF transcription through direct DID–DAD cross-interactions with diaphanous formins [PMID:27030671, PMID:33443052, PMID:21278336]. Autosomal dominant mutations in the INF2 DID cause focal segmental glomerulosclerosis (FSGS), alone or with Charcot-Marie-Tooth neuropathy, through a gain-of-function mechanism in which constitutive INF2 activation leads to uncontrolled actin polymerization, MRTF-SRF transcriptional reprogramming, nephrin mistrafficking, and dysregulated organelle dynamics [PMID:20023659, PMID:39536114, PMID:38916773]."},"prefetch_data":{"uniprot":{"accession":"Q27J81","full_name":"Inverted formin-2","aliases":["HBEBP2-binding protein C"],"length_aa":1249,"mass_kda":135.6,"function":"Severs actin filaments and accelerates their polymerization and depolymerization","subcellular_location":"Cytoplasm, perinuclear region","url":"https://www.uniprot.org/uniprotkb/Q27J81/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/INF2","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ACTB","stoichiometry":0.2},{"gene":"ACTG1","stoichiometry":0.2},{"gene":"CALD1","stoichiometry":0.2},{"gene":"CALM1","stoichiometry":0.2},{"gene":"CALM2","stoichiometry":0.2},{"gene":"CALM3","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"DYNLL1","stoichiometry":0.2},{"gene":"DYNLL2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/INF2","total_profiled":1310},"omim":[{"mim_id":"614455","title":"CHARCOT-MARIE-TOOTH DISEASE, DOMINANT INTERMEDIATE E; CMTDIE","url":"https://www.omim.org/entry/614455"},{"mim_id":"613237","title":"FOCAL SEGMENTAL GLOMERULOSCLEROSIS 5; FSGS5","url":"https://www.omim.org/entry/613237"},{"mim_id":"610982","title":"INVERTED FORMIN 2; INF2","url":"https://www.omim.org/entry/610982"},{"mim_id":"606627","title":"DISHEVELLED-ASSOCIATED ACTIVATOR OF MORPHOGENESIS 2; DAAM2","url":"https://www.omim.org/entry/606627"},{"mim_id":"606482","title":"CHARCOT-MARIE-TOOTH DISEASE, DOMINANT INTERMEDIATE B; CMTDIB","url":"https://www.omim.org/entry/606482"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/INF2"},"hgnc":{"alias_symbol":["MGC13251"],"prev_symbol":["C14orf151","C14orf173"]},"alphafold":{"accession":"Q27J81","domains":[{"cath_id":"1.20.58.630","chopping":"636-712","consensus_level":"high","plddt":88.5465,"start":636,"end":712},{"cath_id":"1.20.58.2220","chopping":"729-936","consensus_level":"high","plddt":92.9675,"start":729,"end":936}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q27J81","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q27J81-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q27J81-F1-predicted_aligned_error_v6.png","plddt_mean":66.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=INF2","jax_strain_url":"https://www.jax.org/strain/search?query=INF2"},"sequence":{"accession":"Q27J81","fasta_url":"https://rest.uniprot.org/uniprotkb/Q27J81.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q27J81/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q27J81"}},"corpus_meta":[{"pmid":"23349293","id":"PMC_23349293","title":"An 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The WH2 motif functions as a DAD that binds the DID with Kd ~1.1 µM, and this DID-DAD interaction inhibits INF2's depolymerization activity but not its nucleation activity. DAD/WH2 mutations that abolish DID-DAD binding cause ER collapse around the nucleus with actin accumulation.\",\n      \"method\": \"GFP-fusion live-cell imaging, membrane fractionation/extraction, farnesylation inhibitor treatment, in vitro binding assay (apparent Kd measurement), site-directed mutagenesis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (biochemical binding assay, mutagenesis, cell imaging, fractionation) in single study with rigorous controls\",\n      \"pmids\": [\"19366733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mutations in the diaphanous inhibitory domain (DID) of INF2 cause autosomal dominant FSGS; all disease-causing mutations reside in the DID, a region that interacts with the DAD and competes for actin monomer binding, implicating disrupted autoinhibitory DID-DAD interaction in disease.\",\n      \"method\": \"Linkage analysis, sequencing of familial FSGS cohort, conservation analysis of mutated residues\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis linking DID mutations to disease; functional mechanism inferred from domain knowledge, confirmed by biochemical studies in companion papers\",\n      \"pmids\": [\"20023659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"INF2 interacts with MAL2 and regulates basolateral-to-apical transcytosis and lumen formation in epithelial cells. Both actin polymerization and depolymerization activities of INF2 are required. INF2 binds Cdc42 in a GTP-loaded-dependent manner, and Cdc42 and INF2 together regulate MAL2 vesicle dynamics and transcytosis.\",\n      \"method\": \"Co-immunoprecipitation, knockdown (siRNA), live videomicroscopy, organotypic culture lumen formation assay, GTPase binding assay\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, functional knockdown with specific phenotypic readout, multiple orthogonal methods\",\n      \"pmids\": [\"20493814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"INF2 interacts with MAL in T lymphocytes and colocalizes at the cell periphery, pericentriolar endosomes, and along microtubules. Knockdown of INF2 reduces MAL+ transport vesicle formation and Lck levels at the plasma membrane, impairing immunological synapse formation. Both actin polymerization and depolymerization activities of INF2 are required for efficient Lck targeting. Cdc42 and Rac1, which bind INF2, regulate this transport.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, videomicroscopy, flow cytometry (surface Lck), immunological synapse assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, KD with specific phenotypic readout, multiple orthogonal assays\",\n      \"pmids\": [\"20881207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2 DID binds directly to the DADs of mDia1, mDia2, and mDia3 (diaphanous-related formins); this interaction inhibits mDia-mediated Rho-activated actin polymerization and SRF-responsive transcription. Disease-causing INF2 DID mutations (E184K, R218Q) show decreased capacity to inhibit SRF activation and gene transcription, suggesting that DID mutations impair inhibition of mDia signaling.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assay, co-immunoprecipitation, colocalization (immunofluorescence), SRF-luciferase reporter assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro binding, reciprocal Co-IP, functional reporter assay with mutagenesis\",\n      \"pmids\": [\"21278336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2 exists as two C-terminal splice variants: CAAX (ER-localized via farnesyl group, actin-independent) and non-CAAX (localizes to an actin-dependent meshwork). Suppression of INF2-non-CAAX causes Golgi apparatus fragmentation, an effect reversed by latrunculin B treatment. Discrete actin patches in the peri-Golgi region are reduced upon INF2 suppression.\",\n      \"method\": \"siRNA knockdown, digitonin extraction (fractionation), latrunculin B treatment, fluorescence microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KD with specific organelle phenotype, pharmacological rescue, isoform-specific localization\",\n      \"pmids\": [\"21998196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2 (FH1-FH2-C construct) binds microtubules with high affinity (Kd < 100 nM) and bundles microtubules, reducing catastrophe rate. Microtubule bundling requires the C-terminus of INF2. Actin monomers inhibit microtubule binding/bundling by INF2 but microtubules do not inhibit INF2-mediated actin polymerization, distinguishing INF2 from mDia1/mDia2.\",\n      \"method\": \"In vitro microtubule co-sedimentation, TIRF microscopy, biochemical binding assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro assays with quantitative affinity measurements and domain mutagenesis\",\n      \"pmids\": [\"21998204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2 mutations causing CMT with FSGS perturb the INF2-MAL-CDC42 pathway, resulting in actin cytoskeleton disorganization, enhanced INF2 binding to CDC42, and mislocalization of INF2, MAL, and CDC42 in Schwann cells. INF2 colocalizes and interacts with MAL in Schwann cells.\",\n      \"method\": \"Co-immunoprecipitation, immunohistochemistry, cell culture functional studies with patient-derived mutations\",\n      \"journal\": \"The New England journal of medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP in relevant cell type, multiple disease mutations tested, colocalization validated\",\n      \"pmids\": [\"22187985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"INF2 FH2 domain mutations I643A and K792A have unexpected effects: I643A causes tight capping of a subset of filaments and nearly abolishes severing/depolymerization without abolishing barbed-end binding; K792A causes slow elongation with minor effects on severing. In cells, I643A cannot promote actin polymerization because it is unable to elongate in the presence of capping protein.\",\n      \"method\": \"Pyrene-actin assembly assay, TIRF microscopy, site-directed mutagenesis, cell expression assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis and multiple orthogonal assays\",\n      \"pmids\": [\"22879592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"INF2 is required for formation of detyrosinated (stable) microtubules after T cell receptor engagement, and this is essential for centrosome reorientation to the immunological synapse. The FH2 domain of INF2 mediates centrosome repositioning and can rescue DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells, placing INF2 downstream of or parallel to these factors.\",\n      \"method\": \"siRNA knockdown, genetic epistasis rescue experiments, immunofluorescence for detyrosinated tubulin, centrosome polarization assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KD with specific phenotypic readout, epistasis rescue, domain mapping\",\n      \"pmids\": [\"22986496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ER-localized INF2 mediates actin polymerization at ER-mitochondria contact sites to drive mitochondrial constriction and fission, functioning upstream of Drp1. Actin filaments accumulate between mitochondria and INF2-enriched ER at constriction sites. INF2 knockdown reduces mitochondrial fission.\",\n      \"method\": \"siRNA knockdown, live-cell imaging, electron microscopy, dominant-negative INF2 expression, epistasis with Drp1\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KD with specific organelle phenotype, epistasis placing INF2 upstream of Drp1, electron microscopy of constriction sites; widely replicated\",\n      \"pmids\": [\"23349293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"INF2 severs actin filaments through FH2 domain encirclement of the filament; a single INF2 dimer can sever filaments in a phosphate-release-dependent manner. The C-terminal DAD/WH2 motif increases severing potency 40-fold by binding adjacent actin protomers to further disrupt filament structure. Helical 3D reconstruction at 20 Å resolution shows FH2 encircling the filament.\",\n      \"method\": \"Two-color TIRF microscopy, pyrene-actin assay, ADP/ADP-Pi filament binding assay, electron microscopy (helical reconstruction), domain truncation/mutagenesis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural EM plus in vitro reconstitution with domain mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"24412206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"INF2 knockdown in zebrafish causes edema, podocyte dysfunction, and altered glomerular filtration barrier associated with mistrafficking of slit diaphragm proteins (nephrin/podocin) and disinhibited mDia (Dia2) activity. This phenotype is rescued by wild-type INF2 but not disease-causing INF2 mutants, and also rescued by loss of RhoA or Dia2, genetically placing INF2 as a negative regulator of Rho/Dia signaling in podocytes.\",\n      \"method\": \"Morpholino knockdown in zebrafish, rescue with wild-type vs. mutant INF2, double knockdown epistasis (RhoA, Dia2), immunofluorescence of slit diaphragm proteins\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo epistasis with specific phenotype, mutant rescue experiment, multiple genetic perturbations\",\n      \"pmids\": [\"26086034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"INF2 generates short actin filaments (<60 nm) that continuously turn over through a cycle of barbed-end elongation, severing, and WH2-mediated depolymerization. The rate-limiting step is nucleotide exchange on actin monomers after release from INF2. Profilin accelerates turnover 6-fold and shifts the equilibrium toward polymerization.\",\n      \"method\": \"Pyrene-actin assay, TIRF microscopy, biochemical kinetics with domain truncations and profilin addition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mechanistic dissection of kinetics\",\n      \"pmids\": [\"26124273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"C. elegans EXC-6 (INF2 ortholog) regulates F-actin and microtubule cytoskeleton at excretory cell tips during tubulogenesis; disease-associated INF2 mutations cause constitutive activity in this system. Activated INF2 can substitute for EXC-6, and genetic analysis places EXC-6 function in organizing both F-actin and MT arrays for basolateral outgrowth and lumen formation.\",\n      \"method\": \"C. elegans genetics, live imaging, rescue with activated/mutant human INF2, genetic epistasis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ortholog functional characterization with genetic epistasis and human INF2 rescue; disease mutations shown to cause constitutive activation\",\n      \"pmids\": [\"25771894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"INF2 acts downstream of mDia1 in a formin activation cascade to stabilize detyrosinated microtubules (Glu MTs) in fibroblasts after LPA stimulation. IQGAP1 scaffolds this cascade by directly binding the C-terminus of INF2 and facilitating LPA-induced mDia1-INF2 interaction and INF2 localization to MTs. Mutants disrupting mDia1-INF2 interaction fail to rescue MT stability in cells depleted of the respective formin.\",\n      \"method\": \"siRNA knockdown epistasis, direct N-IQGAP1/C-INF2 binding assay (pulldown), LPA-induced colocalization, formin interaction mutants\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis, direct binding assay, formin interaction mutants, multiple orthogonal approaches\",\n      \"pmids\": [\"27030671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mice with the FSGS-associated INF2 R218Q knockin mutation show impaired reversal of protamine sulfate-induced podocyte foot process effacement; mutant podocytes display persistent cytoplasmic nephrin/podocin aggregation, nephrin phosphorylation, and impaired recovery of mDia membrane localization, demonstrating that normal INF2 is required for actin-based recovery from podocyte injury.\",\n      \"method\": \"Knockin mouse model, protamine sulfate/heparin perfusion model, immunofluorescence of slit diaphragm proteins, podocyte primary culture\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — disease knockin mouse with specific injury model and multiple molecular readouts\",\n      \"pmids\": [\"27350175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPOP (CUL3-RBX1 E3 ubiquitin ligase adaptor) recognizes a Ser/Thr-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2 that reduces INF2 ER localization and mitochondrially associated DRP1 puncta formation without causing INF2 degradation, thereby inhibiting mitochondrial fission. Cancer-associated SPOP mutants increase INF2 ER localization and promote fission through dominant-negative inhibition of endogenous SPOP.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, subcellular fractionation, DRP1 puncta quantification, SPOP mutant expression\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, in vivo ubiquitination assay, localization with functional consequence, multiple disease-associated mutants tested\",\n      \"pmids\": [\"28448495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"INF2 is inhibited by a complex of lysine-acetylated actin (KAc-actin) bound to cyclase-associated protein (CAP), purified from mouse brain tissue. This inhibition requires the INF2 DID. Treatment with HDAC6 releases INF2 inhibition, and HDAC6 inhibitors block cellular INF2 activation. Disease-associated INF2 DID mutants are poorly inhibited by CAP-KAc-actin, suggesting FSGS/CMT mutations reduce CAP-KAc-actin binding.\",\n      \"method\": \"Biochemical purification of inhibitory complex from brain tissue, in vitro actin polymerization assay, HDAC6 treatment, HDAC6 inhibitor (tubastatin A) in cells, mutagenesis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purification of endogenous inhibitor, in vitro reconstitution, pharmacological validation in cells, disease mutants tested\",\n      \"pmids\": [\"30962575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Lysine residues K50 and K61 on actin are the key sites for CAP/KAc-actin-mediated INF2 inhibition. K50Q- and K61Q-actin (acetylmimetic) bound to CAP2 inhibit full-length INF2 in a DID-dependent manner. The CAP WH2 domain binds INF2-DID with submicromolar affinity; INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin, supporting a bridging model between INF2 DID and DAD.\",\n      \"method\": \"Lysine-to-glutamine acetylmimetic mutations, in vitro INF2 inhibition assay, direct binding studies, U2OS cell overexpression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with acetylmimetic mutations, quantitative binding assays, cellular validation\",\n      \"pmids\": [\"31871199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved by cathepsin proteases, liberating the N-terminal DID fragment. The N-terminal fragment localizes to podocyte foot processes (but not with FSGS mutations), binds mDIA1, and promotes cell spreading in a cleavage-dependent manner. FSGS-associated R218Q mutation impairs N-fragment localization and function but not cleavage itself.\",\n      \"method\": \"Western blot (isoform/cleavage analysis), cathepsin inhibitors, co-immunoprecipitation (N-fragment/mDIA1), podocyte spreading assay, immunofluorescence\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including protease inhibition, Co-IP, and functional cell assay in disease-relevant cell type\",\n      \"pmids\": [\"31924668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2 mutations cause constitutive activation of formin activity and a deregulated calcium-mediated actin reset (CaAR) stress response. FSGS-only mutations are distinguishable from CMT+FSGS mutations by degree of INF2 activation, enabling genotype-phenotype correlation. This was validated in primary patient cells and Drosophila nephrocytes.\",\n      \"method\": \"Quantitative live-cell imaging of CaAR, primary patient cells, Drosophila nephrocyte functional assay, panel of >50 disease mutants\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic functional characterization of large mutation panel in multiple model systems\",\n      \"pmids\": [\"32444357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2 interacts with dynein light chain 1 (DYNLL1), and pathogenic INF2 mutations (R218Q) disrupt this interaction, diverting dynein-mediated post-endocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport rescues this defect.\",\n      \"method\": \"Yeast two-hybrid screen, live cell imaging, fluorescent/surface biotinylation trafficking assays, INF2 transgenic mouse model, dynein antagonism\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by trafficking assays, in vivo mouse model, pharmacological rescue\",\n      \"pmids\": [\"33443052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The N-terminal extension of INF2 forms two α-helices; the first helix contains the sole calmodulin (CaM)-binding site (CaMBS) with key residues W11, L14, L18 (1-4-8 motif). CaM C-terminal lobe directly interacts with this helix. Ca2+/CaM binding activates INF2: INF2 KO cells do not form the perinuclear F-actin ring or respond to Ca2+ elevation with actin polymerization; expression of INF2 with inactivated CaMBS fails to rescue these defects.\",\n      \"method\": \"NMR spectroscopy, biochemical binding assays, INF2 KO cell complementation, Ca2+ stimulation assay, CaMBS mutagenesis (W11, L14, L18)\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with mutagenesis, KO rescue experiment, functional calcium response assay\",\n      \"pmids\": [\"36306014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ischemia and excess NMDA receptor activation cause INF2-dependent actin reorganization (actinification) within the somatodendritic compartment: F-actin depolymerizes from spines and polymerizes into stable filaments in dendritic shafts/soma. Ca2+ influx combined with spine F-actin depolymerization activates INF2. INF2 silencing renders neurons vulnerable to ischemic death; INF2 overexpression is protective.\",\n      \"method\": \"siRNA knockdown, INF2 overexpression, live-cell F-actin imaging, NMDA application, photothrombotic stroke model in mouse, ion flux measurements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KD and OE with specific phenotypic readout, in vitro and in vivo models, mechanistic Ca2+ requirement established\",\n      \"pmids\": [\"36229429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"INF2 forms an 8S complex with actin (4 actin monomers:2 INF2 dimers) in which actin monomers are arranged in parallel orientation. The 8S particles can seed actin assembly. INF2 accelerates the disassembly of oxidized (Mox) F-actin.\",\n      \"method\": \"Negative-stain electron microscopy (2D class averages), chemical crosslinking, pyrene-actin assembly seeding assay\",\n      \"journal\": \"Journal of biomolecular structure & dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — EM structural characterization and biochemical assay, but single study with limited resolution\",\n      \"pmids\": [\"35343388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AMPK phosphorylates INF2 at Ser1077 under energy stress, increasing INF2 localization to the ER and enhancing DRP1 recruitment to mitochondria, thereby promoting mitochondrial fission and endometrial cancer cell growth.\",\n      \"method\": \"In vitro kinase assay, phospho-specific immunoblot, ER localization imaging, DRP1 co-localization, siRNA knockdown, immunohistochemistry\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro kinase assay with specific phosphosite, localization with functional consequence, single lab\",\n      \"pmids\": [\"38233384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO7 E3 ubiquitin ligase ubiquitinates INF2 and targets it for degradation, thereby inhibiting INF2-DRP1 axis-mediated mitochondrial fission. ECa-associated FBXO7 mutants are defective in INF2 ubiquitination and degradation, promoting mitochondrial hyper-fission.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, protein stability assay (cycloheximide chase), siRNA knockdown, DRP1 inhibitor (Mdivi-1)\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, in vivo ubiquitination assay, functional rescue, single lab\",\n      \"pmids\": [\"37344480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"INF2 R218Q knockin mice (but not INF2 knockout mice) are susceptible to glomerular disease upon puromycin aminonucleoside challenge, demonstrating a gain-of-function disease mechanism. R218Q INF2 alters actin cytoskeleton content and INF2 localization. Adhesion and mitochondria-related pathways are enriched in R218Q glomeruli. Patient-derived organoid podocytes with S186P INF2 recapitulate adhesion and mitochondrial phenotypes.\",\n      \"method\": \"Knockin vs. knockout mouse comparison, puromycin aminonucleoside injury model, colocalization, co-immunoprecipitation, actin measurements, RNA-seq, human kidney organoids\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rigorous KI vs KO comparison with in vivo injury model, multiple molecular readouts, human organoid validation\",\n      \"pmids\": [\"39536114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"INF2 R218Q mutation disrupts sequestration of DYNLL1 by INF2, allowing DYNLL1-PI31 interaction that promotes dynein-mediated transport of nephrin to the proteasome for degradation. Proteasome inhibition with bortezomib stabilizes nephrin in R218Q podocytes and ameliorates FSGS in vivo.\",\n      \"method\": \"Co-immunoprecipitation (DYNLL1-PI31), siRNA knockdown of DYNLL1/PI31, proteasome inhibitor treatment, nephrin trafficking assay, R218Q KI mouse puromycin model\",\n      \"journal\": \"Kidney360\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic Co-IP, genetic and pharmacological interventions, in vivo validation\",\n      \"pmids\": [\"39621430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Pathogenic INF2 variants cause ER dysmorphism (shift from tubular to sheet-like ER), mitochondrial fragmentation with peripheral misdistribution, and impaired lysosomal trafficking; CMT+FSGS variants cause more severe ER and mitochondrial disruption than FSGS-only variants, linked to greater cytoskeletal disorganization.\",\n      \"method\": \"High-resolution live imaging of HeLa cells expressing INF2 variants, ER/mitochondria morphology quantification, actin and MT inhibitor treatments\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with functional correlates, systematic comparison of multiple variants, single lab\",\n      \"pmids\": [\"39337270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Pathogenic INF2 promotes translocation of transcriptional cofactor MRTF to the nucleus, causing sustained MRTF-SRF transcriptional complex activation, profound transcriptome reprogramming, multipolar spindle assembly, multi-micronucleation, and cell death. Reducing MRTF-SRF activation mitigates nuclear abnormalities. Inactivation of the INF2 catalytic domain prevents aberrant nuclei.\",\n      \"method\": \"RNA sequencing, MRTF localization imaging, retroviral/plasmid expression of pathogenic INF2 in renal epithelial and primary podocyte cells, catalytic domain inactivation\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA-seq with pathway analysis, MRTF localization assay, catalytic mutant, single lab\",\n      \"pmids\": [\"38916773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"INF2-mediated actin polymerization at ER-organelle contact sites regulates the size and movement of mitochondria, endosomes, and lysosomes; ER-associated actin consistently marks fission sites for all three organelle types.\",\n      \"method\": \"Live imaging of organelle dynamics, ER-associated actin visualization, INF2 knockdown/overexpression, organelle fission site analysis\",\n      \"journal\": \"Research square (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with functional consequence, extends INF2-organelle fission concept; preprint, not peer-reviewed\",\n      \"pmids\": [\"39184068\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CAP1 and CAP2 (cyclase-associated proteins) inhibit INF2 in neurons to permit dendritic spine maturation. Overactivation of INF2 impairs spine maturation, and INF2 inactivation rescues spine defects in CAP-deficient hippocampal neurons, placing CAP-mediated INF2 inhibition as a molecular switch for filopodia-to-mature-spine transition.\",\n      \"method\": \"Genetic inactivation (KO) of CAP1/CAP2, INF2 overactivation/inactivation in hippocampal neurons, spine density/morphology imaging\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in neurons with specific morphological readout, multiple perturbations\",\n      \"pmids\": [\"39154297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Piezo1/Ca2+ signaling activates INF2 to induce widespread actin cytoskeletal remodeling, promoting de-adhesion and mesenchymal-to-amoeboid transition (MAT) in melanoma cells in confined environments. INF2 activation is required for cells to effectively migrate in mechanically challenging environments.\",\n      \"method\": \"Piezo1 inhibition/activation, Ca2+ imaging, INF2 KD, confinement microchannels, amoeboid migration quantification, de-adhesion assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with specific migration phenotype, mechanistic Ca2+ requirement, confinement model; single lab\",\n      \"pmids\": [\"40120583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"INF2 autoinhibition does not require CAP proteins but does require actin monomer 'buffering' by profilin or thymosin; Ca2+-bound calmodulin (CALM) activates INF2 through binding to the N-terminus; the N-terminus has additional regulatory roles beyond CALM binding. This was established with a novel cell-free INF2 regulation assay.\",\n      \"method\": \"Cell-free in vitro assay for INF2 regulation, calmodulin binding assay, profilin/thymosin titration, N-terminus mutants\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted cell-free assay with multiple perturbations, revises prior model with rigorous controls\",\n      \"pmids\": [\"41498749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2 negatively regulates CFTR levels at the plasma membrane: reduction of INF2 levels promotes CFTR trafficking to the PM under EPAC1 activation, identified by protein interaction profiling and validated by knockdown.\",\n      \"method\": \"Protein interaction profiling (proteomics), siRNA knockdown, surface CFTR measurement\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — proteomics pulldown followed by KD with single readout, single lab\",\n      \"pmids\": [\"32573649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EV71 infection causes INF2 cleavage at Asp1051, mediated predominantly by activated caspase-2. Subcellular localization of INF2 and caspase-2 is altered in infected cells, suggesting caspase-2-mediated INF2 cleavage is involved in viral replication organelle formation.\",\n      \"method\": \"Caspase inhibitor treatment, site-directed mutagenesis of cleavage site, subcellular fractionation/immunofluorescence\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — mechanistic mutagenesis of cleavage site with pharmacological validation, but single lab and limited functional follow-up\",\n      \"pmids\": [\"34046026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Drebrin protects assembled actin filaments from INF2-mediated severing without inhibiting INF2's polymerization activity; truncated drebrin (DrbA1-300) is sufficient for this protection. This competitive regulation of actin dynamics is relevant to specialized cells such as neurons and podocytes.\",\n      \"method\": \"In vitro severing/polymerization assays, electron microscopy, TIRF microscopy, domain truncation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with domain truncation and imaging, single lab\",\n      \"pmids\": [\"38158176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The WH2/DAD of INF2 forms a single α-helix (by NMR); the hydrophobic N-terminal region of the WH2/DAD is essential for INF2-mediated actin polymerization and for inducing nuclear abnormalities. Deletion of the entire WH2/DAD or its hydrophobic region abolishes INF2 activity. The WH2/DAD undergoes a conformational change to facilitate actin binding.\",\n      \"method\": \"NMR spectroscopy, deletion/point mutants of WH2/DAD, cell expression of INF2 variants with F-actin and nuclear phenotype quantification\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with mutagenesis and cellular functional validation\",\n      \"pmids\": [\"40993919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ERM proteins (Ezrin, Radixin, Moesin) localize to the nuclear envelope and contribute to perinuclear actin rim formation downstream of INF2/Ca2+ activation; ERM overexpression increases perinuclear actin rim levels while ERM knockdown reduces them. Emerin (a nuclear membrane actin-binding protein) is not required for the perinuclear actin rim.\",\n      \"method\": \"ERM knockdown/overexpression, emerin KO, Ca2+-induced actin rim imaging in melanoma cells\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — KD/OE with single readout, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.02.22.639617\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"INF2 is an ER-anchored formin (CAAX isoform) that both polymerizes and severs/depolymerizes actin filaments via its FH2 domain encirclement mechanism and C-terminal WH2/DAD motif; it is kept in a 'facilitated autoinhibited' state by a CAP/lysine-acetylated-actin complex requiring DID, and is activated by Ca2+/calmodulin binding to its N-terminal helix or by actin monomer buffering proteins (profilin, thymosin); active INF2 drives mitochondrial fission upstream of Drp1, regulates Golgi integrity, promotes detyrosinated microtubule stabilization, controls vesicular transcytosis through MAL2/Cdc42, negatively regulates mDia/Rho signaling through DID-DAD interactions, and sequesters DYNLL1 to prevent aberrant nephrin trafficking; all FSGS/CMT-causing DID mutations constitutively activate INF2 through a gain-of-function mechanism, leading to uncontrolled actin polymerization, MRTF-SRF transcriptional reprogramming, mitotic abnormalities, and dysregulated organelle dynamics in podocytes and Schwann cells.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"INF2 is a multifunctional formin that uniquely combines actin nucleation/elongation with filament severing and depolymerization activities, enabling rapid actin turnover at endoplasmic reticulum–organelle contact sites and controlling organelle dynamics, vesicular trafficking, and microtubule stability. The ER-anchored CAAX isoform drives actin polymerization at ER–mitochondria contacts to promote Drp1-dependent mitochondrial fission, while the non-CAAX isoform maintains Golgi integrity; INF2 is activated by Ca²⁺/calmodulin binding to its N-terminal helix and kept autoinhibited by actin monomer buffering (profilin/thymosin) and a CAP/lysine-acetylated-actin complex engaging the DID [PMID:23349293, PMID:36306014, PMID:41498749, PMID:30962575]. INF2 also stabilizes detyrosinated microtubules downstream of mDia1 via IQGAP1 scaffolding, sequesters DYNLL1 to protect nephrin from degradation, and negatively regulates mDia/Rho-mediated SRF transcription through direct DID–DAD cross-interactions with diaphanous formins [PMID:27030671, PMID:33443052, PMID:21278336]. Autosomal dominant mutations in the INF2 DID cause focal segmental glomerulosclerosis (FSGS), alone or with Charcot-Marie-Tooth neuropathy, through a gain-of-function mechanism in which constitutive INF2 activation leads to uncontrolled actin polymerization, MRTF-SRF transcriptional reprogramming, nephrin mistrafficking, and dysregulated organelle dynamics [PMID:20023659, PMID:39536114, PMID:38916773].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing that INF2 is an ER-anchored formin regulated by intramolecular DID–DAD autoinhibition resolved how its unique severing and nucleation activities are compartmentalized and restrained at the ER membrane.\",\n      \"evidence\": \"GFP-fusion imaging, membrane fractionation, farnesylation inhibitor treatment, and in vitro Kd measurement of DID–DAD interaction in mammalian cells\",\n      \"pmids\": [\"19366733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How autoinhibition is relieved by upstream signals was unknown\", \"Structural basis of ER targeting beyond farnesylation remained unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of INF2 DID mutations as causative for autosomal dominant FSGS established the first disease link and implicated disrupted autoinhibition as the disease mechanism.\",\n      \"evidence\": \"Linkage analysis and sequencing of familial FSGS cohorts\",\n      \"pmids\": [\"20023659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional proof that DID mutations cause gain-of-function (vs. loss-of-function) was not yet provided\", \"Mechanism by which podocytes are specifically vulnerable was unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery that INF2 partners with MAL/MAL2 and Cdc42 to regulate vesicular transcytosis and lumen formation revealed a trafficking function beyond simple actin remodeling.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, and live videomicroscopy in epithelial cells and T lymphocytes\",\n      \"pmids\": [\"20493814\", \"20881207\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of INF2–MAL interaction was not defined\", \"Whether this pathway operates in podocytes or Schwann cells was not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that INF2 DID binds and inhibits mDia formins, with FSGS mutations impairing this inhibition, established INF2 as a negative regulator of Rho/mDia signaling and SRF transcription — a trans-regulatory function unprecedented for formins.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, Co-IP, SRF-luciferase reporter with disease mutants; extended in vivo by zebrafish epistasis showing Dia2/RhoA rescue of INF2 loss\",\n      \"pmids\": [\"21278336\", \"26086034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether INF2–mDia interaction occurs under physiological conditions in podocytes remained unresolved\", \"Relative contribution of mDia inhibition vs. direct actin remodeling to FSGS pathogenesis was unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of two splice isoforms (CAAX and non-CAAX) with distinct localizations (ER vs. cytosol/Golgi) and the finding that INF2 binds microtubules with high affinity broadened INF2's functional repertoire to include Golgi maintenance and microtubule regulation.\",\n      \"evidence\": \"siRNA isoform-specific knockdown, digitonin fractionation, latrunculin rescue of Golgi; in vitro MT cosedimentation and TIRF microscopy\",\n      \"pmids\": [\"21998196\", \"21998204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How INF2 chooses between actin and MT substrates in vivo was not determined\", \"Physiological signals regulating the non-CAAX isoform were unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The landmark finding that ER-localized INF2 drives actin polymerization at ER–mitochondria contacts to promote Drp1-dependent mitochondrial fission established the INF2–Drp1 axis as a core organelle fission mechanism.\",\n      \"evidence\": \"siRNA knockdown, live-cell and electron microscopy of ER–mitochondria constriction sites, epistasis with Drp1\",\n      \"pmids\": [\"23349293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How INF2 activation is spatially restricted to contact sites was unknown\", \"Whether INF2 acts at fission sites of other organelles was untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Structural and single-molecule analyses revealed that INF2 severs filaments by FH2 encirclement, with the WH2/DAD amplifying severing 40-fold, providing a mechanistic basis for INF2's unique dual polymerization/severing activity.\",\n      \"evidence\": \"Two-color TIRF, pyrene-actin assays, 20 Å EM helical reconstruction, domain mutagenesis\",\n      \"pmids\": [\"24412206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of FH2 encircling the filament was not achieved\", \"How severing vs. elongation is regulated in the cell remained unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placement of INF2 downstream of mDia1 in an IQGAP1-scaffolded cascade for detyrosinated microtubule stabilization integrated INF2 into the LPA/Rho signaling axis and explained its role in centrosome reorientation at the immunological synapse.\",\n      \"evidence\": \"siRNA epistasis, direct IQGAP1–INF2 binding assay, LPA stimulation, formin interaction mutants in fibroblasts and T cells\",\n      \"pmids\": [\"27030671\", \"22986496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How INF2 biochemically promotes microtubule detyrosination was not elucidated\", \"Whether IQGAP1 scaffolding operates in podocytes was untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery that SPOP-mediated atypical polyubiquitination displaces INF2 from the ER without degradation revealed a non-degradative ubiquitin-based regulatory mechanism controlling mitochondrial fission, with relevance to cancer-associated SPOP mutations.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, subcellular fractionation, DRP1 puncta quantification with SPOP cancer mutants\",\n      \"pmids\": [\"28448495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The ubiquitin chain type and specific lysine sites on INF2 were not identified\", \"Whether SPOP regulation occurs in podocytes or neurons was unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of CAP/lysine-acetylated-actin as a 'facilitated autoinhibition' complex that locks INF2 via its DID — with disease mutations disrupting this — provided the first physiological inhibitor and unified the gain-of-function disease mechanism.\",\n      \"evidence\": \"Purification of inhibitory complex from brain, in vitro reconstitution with acetylmimetic actins, HDAC6 pharmacology, disease mutant testing\",\n      \"pmids\": [\"30962575\", \"31871199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the CAP–KAc-actin–DID ternary complex was not resolved\", \"Relative contribution of CAP inhibition vs. DID–DAD autoinhibition in vivo was debated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Multiple advances solidified the gain-of-function disease model: INF2 R218Q knockin mice showed impaired podocyte recovery, FSGS mutations constitutively activated the calcium-mediated actin response (CaAR), and pathogenic INF2 disrupted DYNLL1 sequestration causing nephrin mistrafficking to lysosomes.\",\n      \"evidence\": \"R218Q knockin mouse with injury model, CaAR quantification across >50 mutants in patient cells and Drosophila, yeast two-hybrid and nephrin trafficking assays\",\n      \"pmids\": [\"27350175\", \"32444357\", \"33443052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CMT phenotype involves the same DYNLL1/nephrin pathway was unknown\", \"Cell-type-specific activation thresholds explaining FSGS-only vs. CMT+FSGS genotype–phenotype correlation were not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of the N-terminal calmodulin-binding site and demonstration that Ca²⁺/CaM is the physiological activator of INF2 resolved the long-standing question of how INF2 autoinhibition is relieved, and connected INF2 to calcium signaling in neurons (ischemia-protective actinification) and beyond.\",\n      \"evidence\": \"NMR of N-terminal helices, CaMBS mutagenesis with KO rescue, Ca²⁺-stimulated actin rim assays; NMDA/ischemia models with INF2 silencing/overexpression\",\n      \"pmids\": [\"36306014\", \"36229429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CaM releases the CAP/KAc-actin complex or acts independently was not determined\", \"Structural details of full-length INF2 conformational change upon CaM binding were lacking\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Definitive proof that FSGS-causing INF2 mutations act via gain-of-function came from comparison of knockin vs. knockout mice: only R218Q knockin (not KO) developed glomerular disease, and pathogenic INF2 drove MRTF-SRF nuclear translocation, transcriptome reprogramming, and mitotic catastrophe.\",\n      \"evidence\": \"KI vs. KO mouse with puromycin injury, RNA-seq of R218Q glomeruli, MRTF imaging, human kidney organoids; catalytic-dead mutant rescue\",\n      \"pmids\": [\"39536114\", \"38916773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MRTF-SRF inhibition is a viable therapeutic strategy in vivo was not tested\", \"Contribution of mitotic abnormalities to slowly progressive podocyte loss in human disease was not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extension of the DYNLL1 mechanism showed that released DYNLL1 binds PI31 to redirect nephrin to proteasomal degradation, and proteasome inhibition rescued nephrin levels in vivo, providing a potential therapeutic angle for INF2-FSGS.\",\n      \"evidence\": \"Co-IP of DYNLL1–PI31, bortezomib treatment in R218Q KI mouse puromycin model, nephrin trafficking assays\",\n      \"pmids\": [\"39621430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Long-term efficacy and safety of proteasome inhibition for FSGS is unknown\", \"Whether other slit diaphragm proteins are similarly affected was not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A reconstituted cell-free system revised the autoinhibition model: CAP proteins are dispensable for INF2 autoinhibition, which instead requires actin monomer buffering by profilin or thymosin β4, and the N-terminus has regulatory functions beyond CaM binding.\",\n      \"evidence\": \"Cell-free INF2 regulation assay with profilin/thymosin titration, CaM binding, N-terminal mutants\",\n      \"pmids\": [\"41498749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The precise role of the N-terminal region beyond CaM binding is not yet defined\", \"How profilin-mediated autoinhibition integrates with CAP/KAc-actin inhibition in cells is unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of full-length INF2 in autoinhibited and active states, the spatial mechanisms governing INF2 activation selectively at ER–organelle contact sites, and whether therapeutic targeting of the gain-of-function mechanism can prevent or reverse FSGS/CMT remain major open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of full-length INF2 or INF2–actin filament complex exists\", \"Mechanism of spatial restriction of INF2 activation to specific ER subdomains is unknown\", \"No therapeutic strategy targeting INF2 gain-of-function has been validated in clinical settings\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 6, 8, 11, 13, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 12, 15, 18, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 5, 10, 17, 26]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [6, 9, 15]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [10, 17, 26, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 15, 23, 34]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 3, 22, 29]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 7, 28, 31]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MAL2\",\n      \"MAL\",\n      \"CDC42\",\n      \"DIAPH1\",\n      \"IQGAP1\",\n      \"DYNLL1\",\n      \"SPOP\",\n      \"CALM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}