{"gene":"INF2","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2009,"finding":"INF2 is peripherally bound to the cytoplasmic face of the endoplasmic reticulum (ER) in Swiss 3T3 cells. ER localization requires a C-terminal farnesyl (prenyl) group on INF2, though farnesylation alone is insufficient and ionic interactions also contribute. The WH2 motif of INF2 functions as a diaphanous autoregulatory domain (DAD) that binds to the N-terminal diaphanous inhibitory domain (DID) with an apparent Kd of 1.1 µM. The DID-DAD interaction does not inhibit actin nucleation but does inhibit INF2's depolymerization activity. DAD/WH2 point mutations that disrupt DID-DAD interaction also abolish depolymerization activity, and expression of these mutants causes ER collapse around the nucleus with actin filament accumulation around the collapsed ER.","method":"Subcellular fractionation, membrane extraction, immunofluorescence of endogenous and GFP-fusion INF2, farnesylation inhibitor experiments, in vitro binding assays (Kd measurement), pyrene-actin polymerization and depolymerization assays, site-directed mutagenesis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal in vitro biochemical assays plus mutagenesis plus cell imaging, single rigorous study with comprehensive mechanistic dissection","pmids":["19366733"],"is_preprint":false},{"year":2009,"finding":"Mutations in the diaphanous inhibitory domain (DID) of INF2 cause autosomal-dominant focal segmental glomerulosclerosis (FSGS). All nine independent disease-causing missense mutations are located within the DID, altering highly conserved residues, implicating this domain in podocyte actin regulation.","method":"Linkage analysis, candidate gene sequencing in 11 unrelated FSGS families, segregation analysis","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic evidence across 11 families but mechanism inferred from domain mapping without in vitro functional validation in this paper","pmids":["20023659"],"is_preprint":false},{"year":2010,"finding":"INF2 is a binding partner of MAL2 in epithelial cells. Cdc42 binds INF2 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are both necessary for basolateral-to-apical transcytosis and lumen formation in HepG2 and MDCK cells. Both the actin polymerization and depolymerization activities of INF2 are required for efficient transcytosis.","method":"Co-immunoprecipitation, siRNA knockdown, videomicroscopy, GTP-loading assays, organotypic culture lumen-formation assay","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple cell systems, functional rescue experiments with activity-dead INF2 mutants","pmids":["20493814"],"is_preprint":false},{"year":2010,"finding":"INF2 interacts with MAL in Schwann cells and T lymphocytes. In Jurkat T cells, INF2 colocalizes with MAL at the cell periphery and pericentriolar endosomes and along microtubules. Knockdown of INF2 reduces MAL+ transport vesicle formation and Lck levels at the plasma membrane, impairing immunological synapse formation. Cdc42 and Rac1 bind INF2 and regulate Lck transport. Both actin polymerization and depolymerization activities of INF2 are required for Lck targeting.","method":"Co-immunoprecipitation, siRNA knockdown, videomicroscopy, flow cytometry (surface Lck), immunological synapse assay, dominant-negative GTPase constructs","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, functional knockdown with defined phenotypic readouts, activity-mutant rescue experiments, multiple orthogonal methods","pmids":["20881207"],"is_preprint":false},{"year":2011,"finding":"INF2 mutations cause Charcot-Marie-Tooth neuropathy with FSGS. INF2 colocalizes and physically interacts with MAL in Schwann cells. Disease-causing INF2 DID mutants perturb the INF2-MAL-CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42, and mislocalization of INF2, MAL, and CDC42.","method":"Direct INF2 sequencing in CMT+FSGS patients, immunohistochemistry, co-immunoprecipitation, co-localization imaging, functional cell studies with mutant constructs","journal":"The New England journal of medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, colocalization, patient mutation analysis, functional perturbation experiments across multiple labs; replicated interaction with MAL","pmids":["22187985"],"is_preprint":false},{"year":2011,"finding":"INF2's DID domain interacts with the DADs of mDia1, mDia2, and mDia3. This interaction was confirmed by yeast two-hybrid, in vitro biochemical assays, and co-immunoprecipitation in cells. INF2_DID/mDia_DAD binding inhibits mDia-mediated, Rho-activated actin polymerization and SRF-responsive gene transcription. Disease-causing INF2 DID mutations (E184K, R218Q) decrease this inhibitory effect, suggesting DID-mDia DAD interaction is disrupted by FSGS mutations.","method":"Yeast two-hybrid screen, in vitro binding assays, co-immunoprecipitation, actin polymerization assays, SRF luciferase reporter assays, site-directed mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (Y2H, in vitro biochemistry, Co-IP, functional reporter assays) with disease mutant validation","pmids":["21278336"],"is_preprint":false},{"year":2011,"finding":"INF2 is expressed as two C-terminal splice variants: CAAX (farnesylated, ER-localized) and non-CAAX. The non-CAAX isoform localizes to an actin-dependent meshwork distinct from ER. Suppression of INF2-non-CAAX causes Golgi fragmentation, an effect counteracted by latrunculin B treatment. Discrete actin filament patches are present in the peri-Golgi region and are reduced upon INF2 suppression.","method":"RT-PCR (splice variant expression), siRNA knockdown, fluorescence microscopy (ER and Golgi markers), digitonin extraction (localization), actin drug treatment","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KD with defined Golgi phenotype, pharmacological epistasis, localization experiments, multiple orthogonal approaches in single study","pmids":["21998196"],"is_preprint":false},{"year":2011,"finding":"INF2, mDia1, and mDia2 all bind microtubules through constructs containing FH1, FH2, and C-terminal domains with Kd < 100 nM. INF2-FH1FH2C is a potent microtubule-bundling protein that reduces catastrophe rate; this bundling activity requires the C-terminus. Actin monomers inhibit microtubule binding/bundling by INF2 but not by mDia1 or mDia2. Microtubules do not inhibit actin polymerization by INF2, unlike mDia2.","method":"In vitro microtubule co-sedimentation assays, microtubule bundling assays, stoichiometry measurements, TIRF microscopy, actin competition assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple reconstitution-based in vitro assays with quantitative measurements; direct biochemical characterization","pmids":["21998204"],"is_preprint":false},{"year":2012,"finding":"INF2 FH2 domain mutations I643A and K792A have unexpected effects: neither abolishes barbed-end binding, but I643A causes tight capping of a subset of filaments and almost completely abolishes severing and depolymerization activity. K792A has small effects on polymerization and severing. I643A cannot induce actin polymerization in cells because it cannot promote assembly in the presence of capping protein.","method":"Pyrene-actin polymerization assays, TIRF microscopy, barbed-end capping assays, cell imaging with GFP-INF2 mutants, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis plus cellular validation, multiple orthogonal methods","pmids":["22879592"],"is_preprint":false},{"year":2012,"finding":"INF2 is required for formation of stabilized, detyrosinated (Glu) microtubules following T cell receptor (TCR) engagement, and is essential for centrosome reorientation to the immunological synapse. The FH2 domain of INF2 mediates centrosome repositioning and can restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Microtubule stabilization is required for centrosome polarization.","method":"siRNA knockdown, immunofluorescence microscopy (Glu-tubulin staining), dominant-negative and constitutively active GTPase constructs, centrosome reorientation assay, FH2 domain rescue experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KD with defined phenotypic readout, domain rescue experiments, epistasis with multiple GTPases and formins","pmids":["22986496"],"is_preprint":false},{"year":2013,"finding":"ER-localized INF2 drives actin polymerization at ER-mitochondria contact sites, which is required for efficient mitochondrial fission. INF2 functions upstream of Drp1 in fission. Actin filaments accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. INF2-induced actin polymerization drives initial mitochondrial constriction, enabling Drp1-driven secondary constriction.","method":"siRNA knockdown, live-cell imaging, super-resolution microscopy (SIM), phalloidin staining, dominant-negative Drp1 epistasis, rescue with INF2 constructs","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KD with defined mitochondrial phenotype, epistasis experiment placing INF2 upstream of Drp1, live imaging; widely replicated by subsequent studies","pmids":["23349293"],"is_preprint":false},{"year":2014,"finding":"INF2 severs actin filaments via a mechanism in which the FH2 domain encircles the filament (as revealed by 20 Å helical 3D electron microscopy reconstruction). INF2 binds stoichiometrically to filament sides and requires phosphate release. Unlike cofilin, INF2 binds ADP and ADP-Pi filaments equally well. As few as a single INF2 dimer can sever filaments. The C-terminal WH2/DAD motif increases severing potency 40-fold over FH1-FH2 alone.","method":"TIRF microscopy (two-color), pyrene-actin assays, electron microscopy (helical 3D reconstruction at 20 Å), phosphate release assays, truncation/domain analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural determination combined with multiple in vitro reconstitution assays and domain mutagenesis in single study","pmids":["24412206"],"is_preprint":false},{"year":2014,"finding":"INF2 knockdown in zebrafish causes glomerular edema, podocyte dysfunction, and altered glomerular filtration barrier with mistrafficking of slit diaphragm proteins and disinhibited mDia (Dia2) activity. This phenotype is rescued by wild-type human INF2 but not by FSGS-causing DID mutants. The INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2, placing INF2 functionally upstream of RhoA/Dia signaling in the glomerulus.","method":"Morpholino knockdown in zebrafish, rescue with human INF2 WT and mutants, genetic epistasis with RhoA and Dia2, podocyte trafficking assays","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic epistasis in zebrafish model, rescue experiments; single study","pmids":["26086034"],"is_preprint":false},{"year":2015,"finding":"INF2 creates short actin filaments (<60 nm) that continuously turn over through combined barbed-end elongation, severing, and WH2-mediated depolymerization. The rate-limiting step is nucleotide exchange (ADP→ATP) on actin monomers after release from the INF2/actin complex. Profilin addition accelerates filament turnover 6-fold (by accelerating nucleotide exchange) and shifts equilibrium toward longer filaments.","method":"Pyrene-actin assays, TIRF microscopy, nucleotide exchange assays, truncation analysis, profilin competition experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with multiple assays identifying rate-limiting step and profilin mechanism","pmids":["26124273"],"is_preprint":false},{"year":2015,"finding":"C. elegans EXC-6 (ortholog of human INF2) is required for tubulogenesis of the excretory cell; it regulates microtubule and F-actin accumulation at cell tips and dynamics of basolateral MTs. Activated human INF2 can substitute for EXC-6 in C. elegans, and FSGS/CMT disease-associated INF2 mutations cause constitutive activity in this in vivo assay.","method":"C. elegans genetics (exc-6 mutants), live imaging, rescue with human INF2 variants (WT and disease mutants), genetic analysis of cytoskeletal markers","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic analysis with functional rescue by human protein; ortholog study in C. elegans","pmids":["25771894"],"is_preprint":false},{"year":2016,"finding":"INF2 is necessary for mDia1-mediated induction of stable detyrosinated microtubules (Glu MTs) in fibroblasts after LPA treatment. mDia1 regulates INF2 localization to MTs. IQGAP1 associates directly with the C-terminus of INF2 and mediates the interaction between mDia1 and INF2, acting as a scaffold in an mDia1→INF2 formin activation cascade. Mutants that disrupt mDia1-INF2 interaction fail to rescue MT stability in cells depleted of the respective formin.","method":"siRNA knockdown, immunofluorescence (Glu-tubulin), co-immunoprecipitation, direct binding assays (IQGAP1-INF2 N-terminus), MT dynamics measurements, domain interaction mutants","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding assay plus Co-IP plus epistasis with multiple formin mutants plus MT dynamics measurements; multiple orthogonal methods","pmids":["27030671"],"is_preprint":false},{"year":2017,"finding":"SPOP (acting as adaptor of the CUL3-RBX1 E3 ubiquitin ligase) recognizes a Ser/Thr-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. This ubiquitination does not cause INF2 degradation but reduces INF2 ER localization and mitochondrially associated DRP1 puncta formation, thereby inhibiting INF2-mediated mitochondrial fission. INF2 mutants that escape SPOP-mediated ubiquitination promote more mitochondrial fission. Cancer-associated SPOP mutants increase INF2 ER localization and promote fission through dominant-negative inhibition of endogenous SPOP.","method":"Co-immunoprecipitation, ubiquitination assays, confocal microscopy (ER localization, DRP1 puncta), site-directed mutagenesis of SPOP-binding motif, SPOP knockout/knockdown","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, in-cell ubiquitination assays, functional readout (fission/DRP1 puncta), mutagenesis of binding motif, multiple orthogonal methods","pmids":["28448495"],"is_preprint":false},{"year":2019,"finding":"INF2 is inhibited by a complex of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin). This inhibition is dependent on the INF2 DID domain. Treatment with HDAC6 releases INF2 inhibition by deacetylating the CAP-KAc-actin complex; HDAC6 inhibitors block cellular INF2 activation. FSGS/CMT disease-associated INF2 DID mutants are poorly inhibited by CAP-KAc-actin, suggesting these diseases result from reduced CAP-KAc-actin binding.","method":"Biochemical purification from mouse brain tissue, in vitro actin polymerization assays, HDAC6 enzymatic treatment, HDAC6 inhibitor treatment, disease mutant analysis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — purification of endogenous inhibitor, reconstitution of inhibition in vitro, pharmacological manipulation, disease mutant validation; rigorous single study with multiple orthogonal methods","pmids":["30962575"],"is_preprint":false},{"year":2019,"finding":"Lysine residues K50 and K61 on actin are the relevant acetylation sites for CAP/KAc-actin-mediated INF2 inhibition. K50Q- and K61Q-actin (acetylmimetics), when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. The CAP WH2 domain binds INF2-DID with submicromolar affinity. INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. A model is proposed whereby CAP/KAc-actin bridges INF2 DID and DAD to enforce 'facilitated autoinhibition'.","method":"K→Q acetylmimetic mutations in actin, in vitro polymerization assays, direct-binding assays (fluorescence polarization), cell expression of mutant actins, TIRF microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific mutagenesis, in vitro reconstitution, quantitative binding assays, cellular validation; rigorous mechanistic dissection","pmids":["31871199"],"is_preprint":false},{"year":2020,"finding":"INF2-CAAX is the predominant podocyte isoform and is proteolytically cleaved by cathepsin proteases, liberating the N-terminal DID to function independently. The N-terminal fragment localizes to podocyte foot processes (dependent on cleavage), binds mDIA1, and promotes cell spreading; these functions are impaired by the FSGS-associated R218Q mutation without affecting cleavage itself.","method":"Immunoblot (cleavage detection), cathepsin inhibitor treatment, subcellular fractionation, co-immunoprecipitation (mDIA1), cell spreading assay, INF2 disease mutant analysis, human kidney disease tissue immunostaining","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, protease inhibition, localization, functional assay), disease mutant mechanistic validation","pmids":["31924668"],"is_preprint":false},{"year":2020,"finding":"INF2 mutations cause a deregulated calcium-mediated actin reset (CaAR) stress response in cells, resulting in constitutive formin activation. Mutations linked exclusively to FSGS can be distinguished from those causing FSGS+CMT based on degree of INF2 activation. This was validated in primary patient cells and Drosophila nephrocytes.","method":"Quantitative live-cell imaging of CaAR, expression of >50 INF2 mutant variants, validation in primary patient cells and Drosophila nephrocyte model","journal":"Journal of the American Society of Nephrology : JASN","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative functional assay across large panel of mutants, multiple systems; but single study with cell imaging as primary readout","pmids":["32444357"],"is_preprint":false},{"year":2020,"finding":"INF2 interacts with CFTR and regulates CFTR plasma membrane levels. Reduction of INF2 levels promotes CFTR trafficking to the plasma membrane under EPAC1 activation, indicating INF2 acts as a negative regulator of CFTR PM stability after cAMP/EPAC1 signaling.","method":"Protein interaction profiling (Co-IP/pulldown), siRNA knockdown of INF2, surface biotinylation assay for CFTR PM levels, EPAC1 activation with cAMP analog","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP interaction plus functional KD with defined readout; single study, limited mechanistic depth","pmids":["32573649"],"is_preprint":false},{"year":2020,"finding":"FSGS-causing INF2 mutation R218Q disrupts an interaction of INF2 with dynein light chain 1 (DYNLL1). This mutation diverts dynein-mediated post-endocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport rescues this effect.","method":"Yeast two-hybrid (INF2-DYNLL1 interaction), live cell imaging, fluorescent and surface biotinylation trafficking assays in cultured podocytes, dynein inhibitor treatment, R218Q knockin mouse model with puromycin nephropathy","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — Y2H interaction, trafficking assays, in vivo mouse model, pharmacological rescue; multiple orthogonal methods","pmids":["33443052"],"is_preprint":false},{"year":2022,"finding":"The N-terminal extension of INF2 contains a calmodulin-binding site (CaMBS) organized into two α-helices; the first helix binds Ca2+/calmodulin (CaM) directly through its C-terminal lobe, with residues W11, L14, and L18 (1-4-8 motif) being critical. This CaM interaction is required for INF2 activation: INF2 KO cells lack perinuclear F-actin ring, have low cytosolic F-actin, and do not respond to Ca2+ by making F-actin. Expression of INF2 with inactivated CaMBS fails to restore these defects and fails to translocate MRTF to the nucleus.","method":"NMR structure determination, biochemical binding assays, site-directed mutagenesis (W11, L14, L18), INF2 KO cell complementation, F-actin quantification, MRTF localization assay, Ca2+ stimulation experiments","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure combined with mutagenesis, biochemical binding, KO complementation, and multiple functional readouts in single study","pmids":["36306014"],"is_preprint":false},{"year":2022,"finding":"INF2 activation mediates actin reorganization (actinification) in neurons in response to NMDA receptor hyperactivation and ischemia: F-actin depolymerizes from dendritic spines and polymerizes into stable filaments in dendrite shafts and soma within <5 min. INF2 silencing renders neurons vulnerable to cell death; INF2 overexpression is protective against ischemia-induced death. Na+, Cl-, water, and Ca2+ influx combined with spine F-actin depolymerization are required together to activate INF2.","method":"siRNA knockdown of INF2 in cultured neurons, INF2 overexpression, bath-applied NMDA, photothrombotic stroke in mice, live F-actin imaging, cell viability assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — KD and OE with defined phenotype, in vitro and in vivo models, ionic requirements dissected systematically","pmids":["36229429"],"is_preprint":false},{"year":2024,"finding":"INF2 R218Q is a gain-of-function mutation: R218Q knockin mice develop FSGS upon PAN injury while INF2 knockout mice show minimal phenotype. Co-immunoprecipitation and cellular actin measurements show R218Q INF2 confers increased actin cytoskeleton activity. RNA expression analysis shows adhesion and mitochondria-related pathways are enriched in R218Q PAN-treated mice. Both R218Q mouse podocytes and human INF2-S186P organoid podocytes recapitulate adhesion and mitochondrial phenotypes.","method":"Knockin vs knockout mouse comparison with PAN-induced injury model, co-immunoprecipitation, actin content measurement, RNA-seq, human kidney organoids with INF2 patient mutation","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct knockin vs KO comparison in vivo, molecular mechanism (Co-IP, actin measurement), transcriptomics, human organoid validation; multiple orthogonal approaches","pmids":["39536114"],"is_preprint":false},{"year":2024,"finding":"AMPK phosphorylates INF2 at Ser1077 under energy stress conditions, leading to increased INF2 localization to the ER and enhanced DRP1 recruitment to mitochondria, thereby promoting mitochondrial fission and cancer cell growth.","method":"In vitro kinase assay, phosphorylation site mutagenesis (S1077A), confocal microscopy (ER localization, DRP1 puncta), immunohistochemistry of patient specimens correlating AMPK activity with phospho-INF2","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus mutagenesis plus cell imaging; single study with multiple methods but limited independent validation","pmids":["38233384"],"is_preprint":false},{"year":2024,"finding":"FBXO7 E3 ubiquitin ligase mediates ubiquitination and degradation of INF2, thereby restraining INF2-mediated DRP1 recruitment and mitochondrial division. Cancer-associated FBXO7 mutants are defective in INF2 ubiquitination and degradation, promoting mitochondrial hyper-division. The FBXO7-INF2-DRP1 axis controls endometrial cancer cell proliferation and apoptosis.","method":"Co-immunoprecipitation, in-cell ubiquitination assays, INF2 and DRP1 knockdown, DRP1 inhibitor Mdivi-1, immunohistochemistry of patient specimens","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, functional KD with phenotypic readouts; single lab study","pmids":["37344480"],"is_preprint":false},{"year":2024,"finding":"Pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton (both F-actin and microtubules). FSGS variants induce sheet-like ER, while CMT-FSGS variants cause severe ER dysmorphism with fragmented ER and INF2 aggregates. Pathogenic variants also induce mitochondrial fragmentation and dysregulate mitochondrial distribution, more severely in CMT-FSGS than FSGS-only variants.","method":"High-resolution live imaging in HeLa cells expressing WT and pathogenic INF2 variants, actin and microtubule inhibitor treatment, quantification of ER tubule/sheet ratio, mitochondrial morphology analysis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live cell imaging with pharmacological probes, comparative analysis of multiple variants; single study","pmids":["39337270"],"is_preprint":false},{"year":2024,"finding":"Pathogenic INF2 DID variants trigger translocation of the transcriptional cofactor MRTF into the nucleus, causing profound transcriptome reprogramming primarily through sustained MRTF-SRF complex activation. This leads to surplus microtubule-organizing centers, multipolar spindle assembly, multi-micronucleation, mitotic catastrophe, and cell death. Inactivation of INF2's catalytic domain prevents aberrant nuclei formation; reducing MRTF-SRF activation mitigates multi-micronucleation.","method":"Retrovirus and plasmid expression of WT and pathogenic INF2 variants, immunofluorescence microscopy, RNA sequencing, MRTF localization assay, INF2 catalytic domain inactivation, MRTF-SRF pathway inhibition","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-seq plus imaging with multiple controls including catalytic mutant and pathway inhibitor; single lab study","pmids":["38916773"],"is_preprint":false},{"year":2024,"finding":"The R218Q INF2 mutation disrupts sequestration of DYNLL1 by INF2, allowing DYNLL1 to interact with PI31, which promotes dynein-mediated transport of nephrin to the proteasome for degradation. Knockdown of DYNLL1 or PI31, dynein inactivation, or proteasome inhibition (bortezomib) each restore nephrin proteostasis in R218Q knockin podocytes. Bortezomib ameliorates R218Q podocytopathy and FSGS in vivo.","method":"Co-immunoprecipitation (DYNLL1-INF2, DYNLL1-PI31), siRNA knockdown (DYNLL1, PI31), dynein inhibitor, bortezomib treatment, R218Q knockin mouse PAN model, nephrin trafficking assays","journal":"Kidney360","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, multiple genetic and pharmacological interventions with defined molecular readout, in vivo validation; multiple orthogonal methods","pmids":["39621430"],"is_preprint":false},{"year":2024,"finding":"INF2-mediated actin polymerization at ER-organelle contacts regulates morphology and mobility of mitochondria, endosomes, and lysosomes. ER-associated actin consistently marks mitochondrial, endosomal, and lysosomal fission sites. INF2 (ER-anchored isoform) is a key regulator of morphology and mobility of these organelles.","method":"Live-cell imaging, INF2 knockdown/knockout, ER and organelle co-localization analysis, organelle morphology and motility quantification","journal":"Research square (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — live imaging with KD/KO and defined organelle phenotypes; preprint, single study","pmids":["39184068"],"is_preprint":true},{"year":2025,"finding":"INF2 is activated by calcium-bound calmodulin (CALM) through binding to the N-terminus. INF2 inhibition does not require CAP proteins (contradicting prior reports) but does require actin 'buffering' by monomer-binding proteins such as profilin or thymosin. The N-terminus plays roles in INF2 regulation beyond CALM binding.","method":"Novel cell-free reconstitution assay, calmodulin binding assays, actin polymerization assays with profilin/thymosin, CAP protein titration, N-terminal deletion mutants","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution assay with multiple conditions; single study that contradicts prior CAP requirement finding, awaiting independent replication","pmids":["41498749"],"is_preprint":false},{"year":2025,"finding":"Piezo1-mediated Ca2+ influx activates INF2, causing widespread actin cytoskeletal remodeling that promotes de-adhesion and drives mesenchymal-to-amoeboid transition (MAT) in confined environments in melanoma cells.","method":"Piezo1 knockdown/inhibition, INF2 knockdown, intracellular Ca2+ measurement, confinement assays (microchannels), cell migration quantification, actin staining","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD of both Piezo1 and INF2 with defined migration phenotype, multiple confinement conditions; single study","pmids":["40120583"],"is_preprint":false},{"year":2025,"finding":"INF2 co-localizes with HDAC6 and interacts with it, affecting tubulin acetylation and microtubule stability for mitochondrial transport during oocyte meiosis. INF2 co-localizes with cytoplasmic actin and its depletion reduces actin polymerization, causing spindle migration failure in mouse and porcine oocytes. INF2 associates with DRP1 and depletion disturbs mitochondrial distribution and fission. Exogenous INF2 mRNA rescues meiotic maturation defects in obese mouse oocytes.","method":"siRNA/morpholino knockdown in mouse and porcine oocytes, immunofluorescence microscopy, co-immunoprecipitation (INF2-HDAC6, INF2-DRP1), mRNA rescue experiment, JC-1 mitochondrial potential assay","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for two interactions, functional KD in two species with defined phenotypes, mRNA rescue; single study","pmids":["40180021"],"is_preprint":false},{"year":2026,"finding":"INF2-mediated actin filaments facilitate agonist-induced IP3R activity and regulate the formation and/or stability of IP3R clusters on the ER. INF2 physically interacts with IP3R isoforms, often at IP3R clusters. INF2-IP3R interaction is independent of INF2 actin polymerization activity, but the ability of INF2 to mediate IP3R clusters requires its actin polymerization activity. INF2 on the ER regulates IP3R cluster positioning to mediate ER-mitochondrial contacts and facilitate ER-to-mitochondrial calcium transfer.","method":"Co-immunoprecipitation (INF2-IP3R), INF2 knockdown, live-cell calcium imaging, IP3R cluster imaging, ER-mitochondria contact site analysis, INF2 activity mutants","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, KD with functional readout, activity mutant dissection; single study with multiple orthogonal methods","pmids":["42259291"],"is_preprint":false},{"year":2022,"finding":"INF2 and actin form an 8S complex (4 actin monomers : 2 INF2 dimer molecules) in which actin monomers are in a parallel orientation, as shown by chemical crosslinking and negative-staining electron microscopy 2D class averages. 8S particles can seed rapid actin assembly. INF2 also accelerates disassembly of oxidized Mox-F-actin.","method":"Negative-staining electron microscopy (2D class averages), chemical crosslinking, pyrene-actin assembly/disassembly assays, stoichiometry determination","journal":"Journal of biomolecular structure & dynamics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural EM plus biochemical assays; single study with limited resolution (no atomic structure)","pmids":["35343388"],"is_preprint":false},{"year":2024,"finding":"CAP1 and CAP2 inhibit INF2 to induce dendritic spine maturation in hippocampal neurons. INF2 overactivation causes an impaired spine maturation phenotype similar to CAP1/CAP2 double KO. INF2 inactivation largely rescues spine maturation defects in CAP-deficient neurons. CAPs act as a molecular switch controlling the transition from filopodia-like to mature spines partly by inhibiting INF2.","method":"Genetic inactivation of CAP1 and/or CAP2 in hippocampal neurons, INF2 overexpression and inactivation constructs, dendritic spine morphology analysis, epistasis experiments","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with CAP and INF2 manipulations plus defined synaptic phenotype; single study","pmids":["39154297"],"is_preprint":false},{"year":2025,"finding":"The WH2/DAD motif of INF2 forms a single α-helix (determined by NMR in H2O and TFE). The hydrophobic N-terminal region of the WH2/DAD is essential for INF2-mediated actin polymerization; cells expressing INF2 with only the hydrophobic region of WH2/DAD show higher F-actin and nuclear abnormalities phenocopying pathogenic DID variants. Deletion of the entire WH2/DAD or hydrophobic region alone abolishes INF2 activity.","method":"NMR structure determination, site-directed deletion/truncation of WH2/DAD subregions, cell F-actin quantification, nuclear abnormality scoring","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structure plus cell functional mutagenesis; single study","pmids":["40993919"],"is_preprint":false}],"current_model":"INF2 is an ER-anchored formin (CAAX isoform, farnesylated) that both polymerizes and severs/depolymerizes actin filaments: it nucleates actin through its FH2 domain, processively elongates barbed ends, and severs filaments by encircling them with FH2 while the C-terminal WH2/DAD motif disrupts filament structure after phosphate release; its regulation involves facilitated autoinhibition whereby a complex of CAP protein bound to lysine-acetylated actin (KAc-actin at K50/K61) bridges the DID and DAD domains, and is relieved by HDAC6-mediated actin deacetylation or by Ca2+/calmodulin binding to an N-terminal CaM-binding site; INF2-driven actin polymerization at ER–mitochondria contacts drives initial mitochondrial constriction upstream of Drp1-mediated fission, and INF2 also stabilizes microtubules, organizes IP3R clusters to facilitate ER-to-mitochondria calcium transfer, and regulates vesicular trafficking (transcytosis, Lck targeting via MAL, nephrin recycling via dynein light chain DYNLL1); all FSGS- and CMT-causing mutations cluster in the DID, act as gain-of-function alleles that constitutively activate actin polymerization, disrupt interactions with mDia DADs, CAP/KAc-actin, DYNLL1, and mDIA1, and ultimately drive MRTF-SRF transcriptional reprogramming, mitotic catastrophe, and progressive podocyte/Schwann cell loss."},"narrative":{"mechanistic_narrative":"INF2 is an ER-anchored formin that uniquely combines actin filament nucleation, processive barbed-end elongation, and filament severing/depolymerization to drive rapid actin turnover at organelle membranes [PMID:19366733, PMID:24412206, PMID:26124273]. Its catalytic core nucleates and elongates actin, while the FH2 domain encircles filament sides and, together with a C-terminal WH2/DAD motif, severs filaments after phosphate release [PMID:24412206, PMID:40993919]; this generates short, fast-turning-over filaments whose disassembly is rate-limited by nucleotide exchange and accelerated by profilin [PMID:26124273]. INF2 is held inactive by intramolecular DID-DAD autoinhibition that selectively suppresses depolymerization [PMID:19366733], and is activated by Ca2+/calmodulin binding to an N-terminal CaM-binding site required for stress-induced actin assembly and MRTF nuclear translocation [PMID:36306014, PMID:41498749]. The CAAX/farnesylated isoform anchors to the ER cytoplasmic face, where INF2-driven actin polymerization at ER-mitochondria contacts mediates initial mitochondrial constriction upstream of Drp1 fission [PMID:19366733, PMID:23349293], a function tuned by SPOP/CUL3 and FBXO7 ubiquitination and by AMPK phosphorylation [PMID:28448495, PMID:38233384, PMID:37344480], and extended to endosomal and lysosomal fission and to IP3R cluster organization that supports ER-to-mitochondria calcium transfer [PMID:42259291]. INF2 also bundles and stabilizes microtubules through its C-terminus, acting downstream of an mDia1→IQGAP1→INF2 cascade to build detyrosinated microtubules and reorient the centrosome [PMID:21998204, PMID:22986496, PMID:27030671], and regulates polarized membrane trafficking through MAL/MAL2 and Cdc42 [PMID:20493814, PMID:20881207]. INF2 functions as a node in Rho/mDia and MRTF-SRF transcriptional signaling, where it inhibits mDia-driven actin polymerization via DID-DAD contacts [PMID:21278336, PMID:26086034]. Mutations in the DID cause autosomal-dominant focal segmental glomerulosclerosis and Charcot-Marie-Tooth neuropathy [PMID:20023659, PMID:22187985]; these are gain-of-function alleles that escape CAP/acetylated-actin- and mDia-mediated inhibition, constitutively activate actin polymerization, and disrupt DYNLL1-dependent nephrin trafficking, ultimately driving MRTF-SRF reprogramming and mitotic catastrophe [PMID:30962575, PMID:33443052, PMID:39536114, PMID:38916773].","teleology":[{"year":2009,"claim":"Established INF2 as an ER-localized formin whose distinctive depolymerization activity is held in check by an intramolecular DID-DAD interaction, defining its core autoregulatory architecture.","evidence":"Subcellular fractionation, farnesylation inhibition, in vitro binding/Kd measurement, and pyrene-actin assays with DAD/WH2 mutants","pmids":["19366733"],"confidence":"High","gaps":["Did not establish the physiological trigger that relieves autoinhibition","Structural basis of FH2-mediated severing not yet defined"]},{"year":2009,"claim":"Linked INF2 to human disease by showing DID missense mutations cause autosomal-dominant FSGS, focusing attention on the autoregulatory domain in podocyte actin control.","evidence":"Linkage analysis and candidate gene sequencing across 11 FSGS families","pmids":["20023659"],"confidence":"Medium","gaps":["No in vitro functional consequence of the mutations demonstrated in this study","Cell type-specific mechanism not addressed"]},{"year":2010,"claim":"Placed INF2 in polarized membrane trafficking by identifying MAL/MAL2 and GTP-Cdc42 as partners required for transcytosis, lumen formation, and Lck-dependent immunological synapse assembly.","evidence":"Reciprocal Co-IP, siRNA knockdown, GTP-loading assays, and activity-dead mutant rescue in epithelial and T cells","pmids":["20493814","20881207"],"confidence":"High","gaps":["Whether trafficking roles depend on ER versus non-CAAX isoform unresolved","Direct substrate filaments for transcytosis not visualized"]},{"year":2011,"claim":"Defined INF2's signaling and cytoskeletal cross-talk: its DID binds mDia DADs to restrain Rho/SRF-driven actin polymerization, and its C-terminus bundles and stabilizes microtubules.","evidence":"Yeast two-hybrid, in vitro binding, Co-IP, SRF reporter assays, and microtubule co-sedimentation/bundling assays","pmids":["21278336","21998204","21998196"],"confidence":"High","gaps":["How DID-mDia inhibition is integrated with INF2's own actin activity unclear","Non-CAAX isoform role at Golgi mechanistically incomplete"]},{"year":2012,"claim":"Refined the catalytic mechanism and a microtubule-stabilization function: specific FH2 residues separate capping/severing from elongation, and INF2 is required for detyrosinated microtubule formation and centrosome reorientation.","evidence":"Pyrene-actin and TIRF assays with FH2 point mutants plus knockdown and FH2 rescue in T cells","pmids":["22879592","22986496"],"confidence":"High","gaps":["Coupling between actin and microtubule activities at the molecular level unresolved"]},{"year":2013,"claim":"Identified INF2's signature cellular role: ER-anchored actin polymerization at ER-mitochondria contacts drives initial mitochondrial constriction upstream of Drp1, establishing actin's role in fission.","evidence":"siRNA knockdown, super-resolution and live imaging, and Drp1 epistasis","pmids":["23349293"],"confidence":"High","gaps":["Signal initiating contact-site actin polymerization not defined","Quantitative contribution relative to Drp1 not resolved"]},{"year":2014,"claim":"Resolved the severing mechanism structurally, showing FH2 encircles filament sides and requires phosphate release, with the WH2/DAD boosting severing potency.","evidence":"20 Å helical 3D EM reconstruction, two-color TIRF, and phosphate-release/truncation assays","pmids":["24412206"],"confidence":"High","gaps":["Atomic-resolution structure of the severing complex not obtained"]},{"year":2014,"claim":"Provided in vivo validation that INF2 acts upstream of RhoA/Dia in the glomerulus and that FSGS DID mutants are loss-of-rescue alleles in this context.","evidence":"Zebrafish morpholino knockdown with human INF2 WT/mutant rescue and RhoA/Dia2 genetic epistasis","pmids":["26086034"],"confidence":"Medium","gaps":["Single model system","Mechanistic reconciliation with gain-of-function findings unresolved"]},{"year":2015,"claim":"Characterized INF2's rapid filament turnover kinetics, identifying nucleotide exchange as rate-limiting and profilin as an accelerator, and confirmed conserved in vivo function through the C. elegans ortholog.","evidence":"Pyrene/TIRF kinetics with profilin titration and C. elegans exc-6 rescue with human disease variants","pmids":["26124273","25771894"],"confidence":"High","gaps":["In vivo relevance of short-filament steady state not directly tested"]},{"year":2016,"claim":"Defined a formin activation cascade in which IQGAP1 scaffolds mDia1 to INF2 to build stable detyrosinated microtubules, linking actin and microtubule regulation hierarchically.","evidence":"Direct binding assays, Co-IP, knockdown, and MT dynamics measurements with interaction-disrupting mutants","pmids":["27030671"],"confidence":"High","gaps":["Whether INF2 microtubule and actin pools are spatially distinct unclear"]},{"year":2017,"claim":"Showed INF2 mitochondrial fission activity is restrained post-translationally by SPOP/CUL3 atypical ubiquitination that controls its ER localization rather than its stability.","evidence":"Co-IP, in-cell ubiquitination assays, ER/DRP1 imaging, and SPOP-binding motif mutagenesis","pmids":["28448495"],"confidence":"High","gaps":["Upstream signals controlling SPOP-INF2 engagement not defined"]},{"year":2019,"claim":"Discovered facilitated autoinhibition: a CAP·lysine-acetylated-actin complex bridges DID and DAD to inhibit INF2, relieved by HDAC6 deacetylation, and impaired by disease DID mutants.","evidence":"Endogenous inhibitor purification, in vitro reconstitution with K50/K61 acetylmimetics, HDAC6 treatment, and disease-mutant binding assays","pmids":["30962575","31871199"],"confidence":"High","gaps":["Cellular dynamics of acetylation-controlled switching not fully mapped","Later contradicted regarding CAP requirement for inhibition"]},{"year":2020,"claim":"Demonstrated that diverse FSGS/CMT mutations are gain-of-function alleles producing graded constitutive formin activation, and uncovered additional disease-relevant interactions with DYNLL1, CFTR, mDIA1, and a cathepsin-cleaved DID fragment.","evidence":"Quantitative CaAR live imaging across >50 mutants, Y2H, surface trafficking assays, protease inhibition, and a R218Q knockin mouse","pmids":["32444357","31924668","32573649","33443052"],"confidence":"High","gaps":["How a single DID determines both mitochondrial and trafficking phenotypes unresolved","Quantitative activation thresholds distinguishing FSGS from CMT not mechanistically explained"]},{"year":2022,"claim":"Identified Ca2+/calmodulin binding at an N-terminal site as a direct activating input required for stress-induced actin assembly and MRTF nuclear translocation, and extended INF2 activation to neuronal injury (actinification).","evidence":"NMR structure with 1-4-8 motif mutagenesis, KO complementation, MRTF assays, and neuronal NMDA/ischemia models","pmids":["36306014","36229429"],"confidence":"High","gaps":["Integration of CaM activation with DID-DAD/CAP autoinhibition not resolved","8S INF2:actin complex stoichiometry resolved only at low EM resolution (#36)"]},{"year":2024,"claim":"Established R218Q as a gain-of-function allele driving FSGS in vivo and dissected its downstream effects on actin, adhesion, mitochondria, nephrin proteostasis, MRTF-SRF reprogramming, and mitotic catastrophe.","evidence":"Knockin-versus-knockout mouse comparisons, organoids, RNA-seq, Co-IP, DYNLL1-PI31 trafficking dissection with bortezomib rescue, and pathogenic-variant ER/mitochondria imaging","pmids":["39536114","39621430","38916773","39337270"],"confidence":"Medium","gaps":["Tissue specificity of podocyte/Schwann vulnerability not fully explained","Causal ordering of mitochondrial versus adhesion phenotypes unresolved"]},{"year":2024,"claim":"Expanded regulatory and physiological scope: AMPK phosphorylation and FBXO7 ubiquitination tune INF2-DRP1 fission in cancer, CAP1/CAP2 inhibit INF2 during dendritic spine maturation, and ER-actin marks endosome/lysosome fission.","evidence":"Kinase and ubiquitination assays, neuronal genetic epistasis, and live organelle imaging (one preprint)","pmids":["38233384","37344480","39154297","39184068"],"confidence":"Medium","gaps":["Whether multiple regulatory inputs converge on the same INF2 pool unknown","Organelle-fission generality not yet peer-reviewed (#31)"]},{"year":2025,"claim":"Connected mechanosensory and meiotic contexts and revised the autoinhibition model, proposing actin buffering by profilin/thymosin rather than CAP is required, alongside refined structural definition of the WH2/DAD helix.","evidence":"Cell-free reconstitution, Piezo1/INF2 confinement migration assays, oocyte knockdown with Co-IP, and NMR of the WH2/DAD","pmids":["41498749","40120583","40180021","40993919"],"confidence":"Medium","gaps":["CAP-requirement contradiction (#32 vs #17/#18) awaits independent reconciliation","Single-study mechanistic claims for migration and meiosis"]},{"year":2026,"claim":"Defined an INF2-IP3R axis in which INF2 actin activity organizes IP3R clusters to position ER-mitochondria contacts and facilitate calcium transfer, integrating its fission and calcium-handling roles.","evidence":"Co-IP, knockdown, calcium and IP3R cluster imaging, and activity-mutant dissection","pmids":["42259291"],"confidence":"Medium","gaps":["Single study","Whether IP3R clustering precedes or follows fission machinery recruitment unclear"]},{"year":null,"claim":"How the multiple activating (Ca2+/CaM, HDAC6, mechanical/ionic) and inhibitory (CAP/acetyl-actin, profilin buffering, ubiquitination, phosphorylation) inputs are integrated in space and time to select between mitochondrial, microtubule, trafficking, and transcriptional outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling CAP-dependent versus actin-buffering-dependent autoinhibition","No atomic structure of activated full-length INF2 on a filament","Mechanism dictating cell-type-specific disease vulnerability undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,11,13]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[7,11,36]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,12]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[22,30]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,10,16,28]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,23]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7,15]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[9,29]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[10,16,27,31]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[2,3,22,30]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,12,23,29]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,4,25,29]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,23,29]}],"complexes":[],"partners":["MAL","MAL2","CDC42","DIAPH1","IQGAP1","DYNLL1","DNM1L","ITPR1"],"other_free_text":[]}},"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 actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2.","date":"2013","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/23349293","citation_count":674,"is_preprint":false},{"pmid":"20023659","id":"PMC_20023659","title":"Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis.","date":"2009","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20023659","citation_count":361,"is_preprint":false},{"pmid":"22187985","id":"PMC_22187985","title":"INF2 mutations in Charcot-Marie-Tooth disease with glomerulopathy.","date":"2011","source":"The New England journal of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/22187985","citation_count":219,"is_preprint":false},{"pmid":"21258034","id":"PMC_21258034","title":"Mutations in INF2 are a major cause of autosomal dominant focal segmental glomerulosclerosis.","date":"2011","source":"Journal of the American Society of Nephrology : 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formin INF2 regulates basolateral-to-apical transcytosis and lumen formation in association with Cdc42 and MAL2.","date":"2010","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/20493814","citation_count":83,"is_preprint":false},{"pmid":"30579254","id":"PMC_30579254","title":"INF2 regulates oxidative stress-induced apoptosis in epidermal HaCaT cells by modulating the HIF1 signaling pathway.","date":"2018","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/30579254","citation_count":80,"is_preprint":false},{"pmid":"21998196","id":"PMC_21998196","title":"Splice variant-specific cellular function of the formin INF2 in maintenance of Golgi architecture.","date":"2011","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21998196","citation_count":73,"is_preprint":false},{"pmid":"22986496","id":"PMC_22986496","title":"INF2 promotes the formation of detyrosinated microtubules necessary 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ER localization requires a C-terminal farnesyl (prenyl) group on INF2, though farnesylation alone is insufficient and ionic interactions also contribute. The WH2 motif of INF2 functions as a diaphanous autoregulatory domain (DAD) that binds to the N-terminal diaphanous inhibitory domain (DID) with an apparent Kd of 1.1 µM. The DID-DAD interaction does not inhibit actin nucleation but does inhibit INF2's depolymerization activity. DAD/WH2 point mutations that disrupt DID-DAD interaction also abolish depolymerization activity, and expression of these mutants causes ER collapse around the nucleus with actin filament accumulation around the collapsed ER.\",\n      \"method\": \"Subcellular fractionation, membrane extraction, immunofluorescence of endogenous and GFP-fusion INF2, farnesylation inhibitor experiments, in vitro binding assays (Kd measurement), pyrene-actin polymerization and depolymerization assays, site-directed mutagenesis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal in vitro biochemical assays plus mutagenesis plus cell imaging, single rigorous study with comprehensive mechanistic dissection\",\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 focal segmental glomerulosclerosis (FSGS). All nine independent disease-causing missense mutations are located within the DID, altering highly conserved residues, implicating this domain in podocyte actin regulation.\",\n      \"method\": \"Linkage analysis, candidate gene sequencing in 11 unrelated FSGS families, segregation analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic evidence across 11 families but mechanism inferred from domain mapping without in vitro functional validation in this paper\",\n      \"pmids\": [\"20023659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"INF2 is a binding partner of MAL2 in epithelial cells. Cdc42 binds INF2 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are both necessary for basolateral-to-apical transcytosis and lumen formation in HepG2 and MDCK cells. Both the actin polymerization and depolymerization activities of INF2 are required for efficient transcytosis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, videomicroscopy, GTP-loading assays, organotypic culture lumen-formation assay\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple cell systems, functional rescue experiments with activity-dead INF2 mutants\",\n      \"pmids\": [\"20493814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"INF2 interacts with MAL in Schwann cells and T lymphocytes. In Jurkat T cells, INF2 colocalizes with MAL at the cell periphery and pericentriolar endosomes and along microtubules. Knockdown of INF2 reduces MAL+ transport vesicle formation and Lck levels at the plasma membrane, impairing immunological synapse formation. Cdc42 and Rac1 bind INF2 and regulate Lck transport. Both actin polymerization and depolymerization activities of INF2 are required for Lck targeting.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, videomicroscopy, flow cytometry (surface Lck), immunological synapse assay, dominant-negative GTPase constructs\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, functional knockdown with defined phenotypic readouts, activity-mutant rescue experiments, multiple orthogonal methods\",\n      \"pmids\": [\"20881207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2 mutations cause Charcot-Marie-Tooth neuropathy with FSGS. INF2 colocalizes and physically interacts with MAL in Schwann cells. Disease-causing INF2 DID mutants perturb the INF2-MAL-CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42, and mislocalization of INF2, MAL, and CDC42.\",\n      \"method\": \"Direct INF2 sequencing in CMT+FSGS patients, immunohistochemistry, co-immunoprecipitation, co-localization imaging, functional cell studies with mutant constructs\",\n      \"journal\": \"The New England journal of medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, colocalization, patient mutation analysis, functional perturbation experiments across multiple labs; replicated interaction with MAL\",\n      \"pmids\": [\"22187985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2's DID domain interacts with the DADs of mDia1, mDia2, and mDia3. This interaction was confirmed by yeast two-hybrid, in vitro biochemical assays, and co-immunoprecipitation in cells. INF2_DID/mDia_DAD binding inhibits mDia-mediated, Rho-activated actin polymerization and SRF-responsive gene transcription. Disease-causing INF2 DID mutations (E184K, R218Q) decrease this inhibitory effect, suggesting DID-mDia DAD interaction is disrupted by FSGS mutations.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assays, co-immunoprecipitation, actin polymerization assays, SRF luciferase reporter assays, site-directed mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (Y2H, in vitro biochemistry, Co-IP, functional reporter assays) with disease mutant validation\",\n      \"pmids\": [\"21278336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2 is expressed as two C-terminal splice variants: CAAX (farnesylated, ER-localized) and non-CAAX. The non-CAAX isoform localizes to an actin-dependent meshwork distinct from ER. Suppression of INF2-non-CAAX causes Golgi fragmentation, an effect counteracted by latrunculin B treatment. Discrete actin filament patches are present in the peri-Golgi region and are reduced upon INF2 suppression.\",\n      \"method\": \"RT-PCR (splice variant expression), siRNA knockdown, fluorescence microscopy (ER and Golgi markers), digitonin extraction (localization), actin drug treatment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined Golgi phenotype, pharmacological epistasis, localization experiments, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"21998196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"INF2, mDia1, and mDia2 all bind microtubules through constructs containing FH1, FH2, and C-terminal domains with Kd < 100 nM. INF2-FH1FH2C is a potent microtubule-bundling protein that reduces catastrophe rate; this bundling activity requires the C-terminus. Actin monomers inhibit microtubule binding/bundling by INF2 but not by mDia1 or mDia2. Microtubules do not inhibit actin polymerization by INF2, unlike mDia2.\",\n      \"method\": \"In vitro microtubule co-sedimentation assays, microtubule bundling assays, stoichiometry measurements, TIRF microscopy, actin competition assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple reconstitution-based in vitro assays with quantitative measurements; direct biochemical characterization\",\n      \"pmids\": [\"21998204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"INF2 FH2 domain mutations I643A and K792A have unexpected effects: neither abolishes barbed-end binding, but I643A causes tight capping of a subset of filaments and almost completely abolishes severing and depolymerization activity. K792A has small effects on polymerization and severing. I643A cannot induce actin polymerization in cells because it cannot promote assembly in the presence of capping protein.\",\n      \"method\": \"Pyrene-actin polymerization assays, TIRF microscopy, barbed-end capping assays, cell imaging with GFP-INF2 mutants, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis plus cellular validation, multiple orthogonal methods\",\n      \"pmids\": [\"22879592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"INF2 is required for formation of stabilized, detyrosinated (Glu) microtubules following T cell receptor (TCR) engagement, and is essential for centrosome reorientation to the immunological synapse. The FH2 domain of INF2 mediates centrosome repositioning and can restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Microtubule stabilization is required for centrosome polarization.\",\n      \"method\": \"siRNA knockdown, immunofluorescence microscopy (Glu-tubulin staining), dominant-negative and constitutively active GTPase constructs, centrosome reorientation assay, FH2 domain rescue experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KD with defined phenotypic readout, domain rescue experiments, epistasis with multiple GTPases and formins\",\n      \"pmids\": [\"22986496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ER-localized INF2 drives actin polymerization at ER-mitochondria contact sites, which is required for efficient mitochondrial fission. INF2 functions upstream of Drp1 in fission. Actin filaments accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. INF2-induced actin polymerization drives initial mitochondrial constriction, enabling Drp1-driven secondary constriction.\",\n      \"method\": \"siRNA knockdown, live-cell imaging, super-resolution microscopy (SIM), phalloidin staining, dominant-negative Drp1 epistasis, rescue with INF2 constructs\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KD with defined mitochondrial phenotype, epistasis experiment placing INF2 upstream of Drp1, live imaging; widely replicated by subsequent studies\",\n      \"pmids\": [\"23349293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"INF2 severs actin filaments via a mechanism in which the FH2 domain encircles the filament (as revealed by 20 Å helical 3D electron microscopy reconstruction). INF2 binds stoichiometrically to filament sides and requires phosphate release. Unlike cofilin, INF2 binds ADP and ADP-Pi filaments equally well. As few as a single INF2 dimer can sever filaments. The C-terminal WH2/DAD motif increases severing potency 40-fold over FH1-FH2 alone.\",\n      \"method\": \"TIRF microscopy (two-color), pyrene-actin assays, electron microscopy (helical 3D reconstruction at 20 Å), phosphate release assays, truncation/domain analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural determination combined with multiple in vitro reconstitution assays and domain mutagenesis in single study\",\n      \"pmids\": [\"24412206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"INF2 knockdown in zebrafish causes glomerular edema, podocyte dysfunction, and altered glomerular filtration barrier with mistrafficking of slit diaphragm proteins and disinhibited mDia (Dia2) activity. This phenotype is rescued by wild-type human INF2 but not by FSGS-causing DID mutants. The INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2, placing INF2 functionally upstream of RhoA/Dia signaling in the glomerulus.\",\n      \"method\": \"Morpholino knockdown in zebrafish, rescue with human INF2 WT and mutants, genetic epistasis with RhoA and Dia2, podocyte trafficking assays\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic epistasis in zebrafish model, rescue experiments; single study\",\n      \"pmids\": [\"26086034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"INF2 creates short actin filaments (<60 nm) that continuously turn over through combined barbed-end elongation, severing, and WH2-mediated depolymerization. The rate-limiting step is nucleotide exchange (ADP→ATP) on actin monomers after release from the INF2/actin complex. Profilin addition accelerates filament turnover 6-fold (by accelerating nucleotide exchange) and shifts equilibrium toward longer filaments.\",\n      \"method\": \"Pyrene-actin assays, TIRF microscopy, nucleotide exchange assays, truncation analysis, profilin competition experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with multiple assays identifying rate-limiting step and profilin mechanism\",\n      \"pmids\": [\"26124273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"C. elegans EXC-6 (ortholog of human INF2) is required for tubulogenesis of the excretory cell; it regulates microtubule and F-actin accumulation at cell tips and dynamics of basolateral MTs. Activated human INF2 can substitute for EXC-6 in C. elegans, and FSGS/CMT disease-associated INF2 mutations cause constitutive activity in this in vivo assay.\",\n      \"method\": \"C. elegans genetics (exc-6 mutants), live imaging, rescue with human INF2 variants (WT and disease mutants), genetic analysis of cytoskeletal markers\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic analysis with functional rescue by human protein; ortholog study in C. elegans\",\n      \"pmids\": [\"25771894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"INF2 is necessary for mDia1-mediated induction of stable detyrosinated microtubules (Glu MTs) in fibroblasts after LPA treatment. mDia1 regulates INF2 localization to MTs. IQGAP1 associates directly with the C-terminus of INF2 and mediates the interaction between mDia1 and INF2, acting as a scaffold in an mDia1→INF2 formin activation cascade. Mutants that disrupt mDia1-INF2 interaction fail to rescue MT stability in cells depleted of the respective formin.\",\n      \"method\": \"siRNA knockdown, immunofluorescence (Glu-tubulin), co-immunoprecipitation, direct binding assays (IQGAP1-INF2 N-terminus), MT dynamics measurements, domain interaction mutants\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding assay plus Co-IP plus epistasis with multiple formin mutants plus MT dynamics measurements; multiple orthogonal methods\",\n      \"pmids\": [\"27030671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPOP (acting as adaptor of the CUL3-RBX1 E3 ubiquitin ligase) recognizes a Ser/Thr-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. This ubiquitination does not cause INF2 degradation but reduces INF2 ER localization and mitochondrially associated DRP1 puncta formation, thereby inhibiting INF2-mediated mitochondrial fission. INF2 mutants that escape SPOP-mediated ubiquitination promote more 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 assays, confocal microscopy (ER localization, DRP1 puncta), site-directed mutagenesis of SPOP-binding motif, SPOP knockout/knockdown\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, in-cell ubiquitination assays, functional readout (fission/DRP1 puncta), mutagenesis of binding motif, multiple orthogonal methods\",\n      \"pmids\": [\"28448495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"INF2 is inhibited by a complex of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin). This inhibition is dependent on the INF2 DID domain. Treatment with HDAC6 releases INF2 inhibition by deacetylating the CAP-KAc-actin complex; HDAC6 inhibitors block cellular INF2 activation. FSGS/CMT disease-associated INF2 DID mutants are poorly inhibited by CAP-KAc-actin, suggesting these diseases result from reduced CAP-KAc-actin binding.\",\n      \"method\": \"Biochemical purification from mouse brain tissue, in vitro actin polymerization assays, HDAC6 enzymatic treatment, HDAC6 inhibitor treatment, disease mutant analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purification of endogenous inhibitor, reconstitution of inhibition in vitro, pharmacological manipulation, disease mutant validation; rigorous single study with multiple orthogonal methods\",\n      \"pmids\": [\"30962575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Lysine residues K50 and K61 on actin are the relevant acetylation sites for CAP/KAc-actin-mediated INF2 inhibition. K50Q- and K61Q-actin (acetylmimetics), when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. The CAP WH2 domain binds INF2-DID with submicromolar affinity. INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. A model is proposed whereby CAP/KAc-actin bridges INF2 DID and DAD to enforce 'facilitated autoinhibition'.\",\n      \"method\": \"K→Q acetylmimetic mutations in actin, in vitro polymerization assays, direct-binding assays (fluorescence polarization), cell expression of mutant actins, TIRF microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific mutagenesis, in vitro reconstitution, quantitative binding assays, cellular validation; rigorous mechanistic dissection\",\n      \"pmids\": [\"31871199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2-CAAX is the predominant podocyte isoform and is proteolytically cleaved by cathepsin proteases, liberating the N-terminal DID to function independently. The N-terminal fragment localizes to podocyte foot processes (dependent on cleavage), binds mDIA1, and promotes cell spreading; these functions are impaired by the FSGS-associated R218Q mutation without affecting cleavage itself.\",\n      \"method\": \"Immunoblot (cleavage detection), cathepsin inhibitor treatment, subcellular fractionation, co-immunoprecipitation (mDIA1), cell spreading assay, INF2 disease mutant analysis, human kidney disease tissue immunostaining\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, protease inhibition, localization, functional assay), disease mutant mechanistic validation\",\n      \"pmids\": [\"31924668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2 mutations cause a deregulated calcium-mediated actin reset (CaAR) stress response in cells, resulting in constitutive formin activation. Mutations linked exclusively to FSGS can be distinguished from those causing FSGS+CMT based on degree of INF2 activation. This was validated in primary patient cells and Drosophila nephrocytes.\",\n      \"method\": \"Quantitative live-cell imaging of CaAR, expression of >50 INF2 mutant variants, validation in primary patient cells and Drosophila nephrocyte model\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative functional assay across large panel of mutants, multiple systems; but single study with cell imaging as primary readout\",\n      \"pmids\": [\"32444357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"INF2 interacts with CFTR and regulates CFTR plasma membrane levels. Reduction of INF2 levels promotes CFTR trafficking to the plasma membrane under EPAC1 activation, indicating INF2 acts as a negative regulator of CFTR PM stability after cAMP/EPAC1 signaling.\",\n      \"method\": \"Protein interaction profiling (Co-IP/pulldown), siRNA knockdown of INF2, surface biotinylation assay for CFTR PM levels, EPAC1 activation with cAMP analog\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP interaction plus functional KD with defined readout; single study, limited mechanistic depth\",\n      \"pmids\": [\"32573649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FSGS-causing INF2 mutation R218Q disrupts an interaction of INF2 with dynein light chain 1 (DYNLL1). This mutation diverts dynein-mediated post-endocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport rescues this effect.\",\n      \"method\": \"Yeast two-hybrid (INF2-DYNLL1 interaction), live cell imaging, fluorescent and surface biotinylation trafficking assays in cultured podocytes, dynein inhibitor treatment, R218Q knockin mouse model with puromycin nephropathy\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Y2H interaction, trafficking assays, in vivo mouse model, pharmacological rescue; multiple orthogonal methods\",\n      \"pmids\": [\"33443052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The N-terminal extension of INF2 contains a calmodulin-binding site (CaMBS) organized into two α-helices; the first helix binds Ca2+/calmodulin (CaM) directly through its C-terminal lobe, with residues W11, L14, and L18 (1-4-8 motif) being critical. This CaM interaction is required for INF2 activation: INF2 KO cells lack perinuclear F-actin ring, have low cytosolic F-actin, and do not respond to Ca2+ by making F-actin. Expression of INF2 with inactivated CaMBS fails to restore these defects and fails to translocate MRTF to the nucleus.\",\n      \"method\": \"NMR structure determination, biochemical binding assays, site-directed mutagenesis (W11, L14, L18), INF2 KO cell complementation, F-actin quantification, MRTF localization assay, Ca2+ stimulation experiments\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure combined with mutagenesis, biochemical binding, KO complementation, and multiple functional readouts in single study\",\n      \"pmids\": [\"36306014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"INF2 activation mediates actin reorganization (actinification) in neurons in response to NMDA receptor hyperactivation and ischemia: F-actin depolymerizes from dendritic spines and polymerizes into stable filaments in dendrite shafts and soma within <5 min. INF2 silencing renders neurons vulnerable to cell death; INF2 overexpression is protective against ischemia-induced death. Na+, Cl-, water, and Ca2+ influx combined with spine F-actin depolymerization are required together to activate INF2.\",\n      \"method\": \"siRNA knockdown of INF2 in cultured neurons, INF2 overexpression, bath-applied NMDA, photothrombotic stroke in mice, live F-actin imaging, cell viability assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KD and OE with defined phenotype, in vitro and in vivo models, ionic requirements dissected systematically\",\n      \"pmids\": [\"36229429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"INF2 R218Q is a gain-of-function mutation: R218Q knockin mice develop FSGS upon PAN injury while INF2 knockout mice show minimal phenotype. Co-immunoprecipitation and cellular actin measurements show R218Q INF2 confers increased actin cytoskeleton activity. RNA expression analysis shows adhesion and mitochondria-related pathways are enriched in R218Q PAN-treated mice. Both R218Q mouse podocytes and human INF2-S186P organoid podocytes recapitulate adhesion and mitochondrial phenotypes.\",\n      \"method\": \"Knockin vs knockout mouse comparison with PAN-induced injury model, co-immunoprecipitation, actin content measurement, RNA-seq, human kidney organoids with INF2 patient mutation\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct knockin vs KO comparison in vivo, molecular mechanism (Co-IP, actin measurement), transcriptomics, human organoid validation; multiple orthogonal approaches\",\n      \"pmids\": [\"39536114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AMPK phosphorylates INF2 at Ser1077 under energy stress conditions, leading to increased INF2 localization to the ER and enhanced DRP1 recruitment to mitochondria, thereby promoting mitochondrial fission and cancer cell growth.\",\n      \"method\": \"In vitro kinase assay, phosphorylation site mutagenesis (S1077A), confocal microscopy (ER localization, DRP1 puncta), immunohistochemistry of patient specimens correlating AMPK activity with phospho-INF2\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus mutagenesis plus cell imaging; single study with multiple methods but limited independent validation\",\n      \"pmids\": [\"38233384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO7 E3 ubiquitin ligase mediates ubiquitination and degradation of INF2, thereby restraining INF2-mediated DRP1 recruitment and mitochondrial division. Cancer-associated FBXO7 mutants are defective in INF2 ubiquitination and degradation, promoting mitochondrial hyper-division. The FBXO7-INF2-DRP1 axis controls endometrial cancer cell proliferation and apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, in-cell ubiquitination assays, INF2 and DRP1 knockdown, DRP1 inhibitor Mdivi-1, immunohistochemistry of patient specimens\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, functional KD with phenotypic readouts; single lab study\",\n      \"pmids\": [\"37344480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton (both F-actin and microtubules). FSGS variants induce sheet-like ER, while CMT-FSGS variants cause severe ER dysmorphism with fragmented ER and INF2 aggregates. Pathogenic variants also induce mitochondrial fragmentation and dysregulate mitochondrial distribution, more severely in CMT-FSGS than FSGS-only variants.\",\n      \"method\": \"High-resolution live imaging in HeLa cells expressing WT and pathogenic INF2 variants, actin and microtubule inhibitor treatment, quantification of ER tubule/sheet ratio, mitochondrial morphology analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live cell imaging with pharmacological probes, comparative analysis of multiple variants; single study\",\n      \"pmids\": [\"39337270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Pathogenic INF2 DID variants trigger translocation of the transcriptional cofactor MRTF into the nucleus, causing profound transcriptome reprogramming primarily through sustained MRTF-SRF complex activation. This leads to surplus microtubule-organizing centers, multipolar spindle assembly, multi-micronucleation, mitotic catastrophe, and cell death. Inactivation of INF2's catalytic domain prevents aberrant nuclei formation; reducing MRTF-SRF activation mitigates multi-micronucleation.\",\n      \"method\": \"Retrovirus and plasmid expression of WT and pathogenic INF2 variants, immunofluorescence microscopy, RNA sequencing, MRTF localization assay, INF2 catalytic domain inactivation, MRTF-SRF pathway inhibition\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-seq plus imaging with multiple controls including catalytic mutant and pathway inhibitor; single lab study\",\n      \"pmids\": [\"38916773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The R218Q INF2 mutation disrupts sequestration of DYNLL1 by INF2, allowing DYNLL1 to interact with PI31, which promotes dynein-mediated transport of nephrin to the proteasome for degradation. Knockdown of DYNLL1 or PI31, dynein inactivation, or proteasome inhibition (bortezomib) each restore nephrin proteostasis in R218Q knockin podocytes. Bortezomib ameliorates R218Q podocytopathy and FSGS in vivo.\",\n      \"method\": \"Co-immunoprecipitation (DYNLL1-INF2, DYNLL1-PI31), siRNA knockdown (DYNLL1, PI31), dynein inhibitor, bortezomib treatment, R218Q knockin mouse PAN model, nephrin trafficking assays\",\n      \"journal\": \"Kidney360\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, multiple genetic and pharmacological interventions with defined molecular readout, in vivo validation; multiple orthogonal methods\",\n      \"pmids\": [\"39621430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"INF2-mediated actin polymerization at ER-organelle contacts regulates morphology and mobility of mitochondria, endosomes, and lysosomes. ER-associated actin consistently marks mitochondrial, endosomal, and lysosomal fission sites. INF2 (ER-anchored isoform) is a key regulator of morphology and mobility of these organelles.\",\n      \"method\": \"Live-cell imaging, INF2 knockdown/knockout, ER and organelle co-localization analysis, organelle morphology and motility quantification\",\n      \"journal\": \"Research square (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — live imaging with KD/KO and defined organelle phenotypes; preprint, single study\",\n      \"pmids\": [\"39184068\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"INF2 is activated by calcium-bound calmodulin (CALM) through binding to the N-terminus. INF2 inhibition does not require CAP proteins (contradicting prior reports) but does require actin 'buffering' by monomer-binding proteins such as profilin or thymosin. The N-terminus plays roles in INF2 regulation beyond CALM binding.\",\n      \"method\": \"Novel cell-free reconstitution assay, calmodulin binding assays, actin polymerization assays with profilin/thymosin, CAP protein titration, N-terminal deletion mutants\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution assay with multiple conditions; single study that contradicts prior CAP requirement finding, awaiting independent replication\",\n      \"pmids\": [\"41498749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Piezo1-mediated Ca2+ influx activates INF2, causing widespread actin cytoskeletal remodeling that promotes de-adhesion and drives mesenchymal-to-amoeboid transition (MAT) in confined environments in melanoma cells.\",\n      \"method\": \"Piezo1 knockdown/inhibition, INF2 knockdown, intracellular Ca2+ measurement, confinement assays (microchannels), cell migration quantification, actin staining\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD of both Piezo1 and INF2 with defined migration phenotype, multiple confinement conditions; single study\",\n      \"pmids\": [\"40120583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"INF2 co-localizes with HDAC6 and interacts with it, affecting tubulin acetylation and microtubule stability for mitochondrial transport during oocyte meiosis. INF2 co-localizes with cytoplasmic actin and its depletion reduces actin polymerization, causing spindle migration failure in mouse and porcine oocytes. INF2 associates with DRP1 and depletion disturbs mitochondrial distribution and fission. Exogenous INF2 mRNA rescues meiotic maturation defects in obese mouse oocytes.\",\n      \"method\": \"siRNA/morpholino knockdown in mouse and porcine oocytes, immunofluorescence microscopy, co-immunoprecipitation (INF2-HDAC6, INF2-DRP1), mRNA rescue experiment, JC-1 mitochondrial potential assay\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for two interactions, functional KD in two species with defined phenotypes, mRNA rescue; single study\",\n      \"pmids\": [\"40180021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"INF2-mediated actin filaments facilitate agonist-induced IP3R activity and regulate the formation and/or stability of IP3R clusters on the ER. INF2 physically interacts with IP3R isoforms, often at IP3R clusters. INF2-IP3R interaction is independent of INF2 actin polymerization activity, but the ability of INF2 to mediate IP3R clusters requires its actin polymerization activity. INF2 on the ER regulates IP3R cluster positioning to mediate ER-mitochondrial contacts and facilitate ER-to-mitochondrial calcium transfer.\",\n      \"method\": \"Co-immunoprecipitation (INF2-IP3R), INF2 knockdown, live-cell calcium imaging, IP3R cluster imaging, ER-mitochondria contact site analysis, INF2 activity mutants\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KD with functional readout, activity mutant dissection; single study with multiple orthogonal methods\",\n      \"pmids\": [\"42259291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"INF2 and actin form an 8S complex (4 actin monomers : 2 INF2 dimer molecules) in which actin monomers are in a parallel orientation, as shown by chemical crosslinking and negative-staining electron microscopy 2D class averages. 8S particles can seed rapid actin assembly. INF2 also accelerates disassembly of oxidized Mox-F-actin.\",\n      \"method\": \"Negative-staining electron microscopy (2D class averages), chemical crosslinking, pyrene-actin assembly/disassembly assays, stoichiometry determination\",\n      \"journal\": \"Journal of biomolecular structure & dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural EM plus biochemical assays; single study with limited resolution (no atomic structure)\",\n      \"pmids\": [\"35343388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CAP1 and CAP2 inhibit INF2 to induce dendritic spine maturation in hippocampal neurons. INF2 overactivation causes an impaired spine maturation phenotype similar to CAP1/CAP2 double KO. INF2 inactivation largely rescues spine maturation defects in CAP-deficient neurons. CAPs act as a molecular switch controlling the transition from filopodia-like to mature spines partly by inhibiting INF2.\",\n      \"method\": \"Genetic inactivation of CAP1 and/or CAP2 in hippocampal neurons, INF2 overexpression and inactivation constructs, dendritic spine morphology analysis, epistasis experiments\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with CAP and INF2 manipulations plus defined synaptic phenotype; single study\",\n      \"pmids\": [\"39154297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The WH2/DAD motif of INF2 forms a single α-helix (determined by NMR in H2O and TFE). The hydrophobic N-terminal region of the WH2/DAD is essential for INF2-mediated actin polymerization; cells expressing INF2 with only the hydrophobic region of WH2/DAD show higher F-actin and nuclear abnormalities phenocopying pathogenic DID variants. Deletion of the entire WH2/DAD or hydrophobic region alone abolishes INF2 activity.\",\n      \"method\": \"NMR structure determination, site-directed deletion/truncation of WH2/DAD subregions, cell F-actin quantification, nuclear abnormality scoring\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus cell functional mutagenesis; single study\",\n      \"pmids\": [\"40993919\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"INF2 is an ER-anchored formin (CAAX isoform, farnesylated) that both polymerizes and severs/depolymerizes actin filaments: it nucleates actin through its FH2 domain, processively elongates barbed ends, and severs filaments by encircling them with FH2 while the C-terminal WH2/DAD motif disrupts filament structure after phosphate release; its regulation involves facilitated autoinhibition whereby a complex of CAP protein bound to lysine-acetylated actin (KAc-actin at K50/K61) bridges the DID and DAD domains, and is relieved by HDAC6-mediated actin deacetylation or by Ca2+/calmodulin binding to an N-terminal CaM-binding site; INF2-driven actin polymerization at ER–mitochondria contacts drives initial mitochondrial constriction upstream of Drp1-mediated fission, and INF2 also stabilizes microtubules, organizes IP3R clusters to facilitate ER-to-mitochondria calcium transfer, and regulates vesicular trafficking (transcytosis, Lck targeting via MAL, nephrin recycling via dynein light chain DYNLL1); all FSGS- and CMT-causing mutations cluster in the DID, act as gain-of-function alleles that constitutively activate actin polymerization, disrupt interactions with mDia DADs, CAP/KAc-actin, DYNLL1, and mDIA1, and ultimately drive MRTF-SRF transcriptional reprogramming, mitotic catastrophe, and progressive podocyte/Schwann cell loss.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"INF2 is an ER-anchored formin that uniquely combines actin filament nucleation, processive barbed-end elongation, and filament severing/depolymerization to drive rapid actin turnover at organelle membranes [#0, #11, #13]. Its catalytic core nucleates and elongates actin, while the FH2 domain encircles filament sides and, together with a C-terminal WH2/DAD motif, severs filaments after phosphate release [#11, #38]; this generates short, fast-turning-over filaments whose disassembly is rate-limited by nucleotide exchange and accelerated by profilin [#13]. INF2 is held inactive by intramolecular DID-DAD autoinhibition that selectively suppresses depolymerization [#0], and is activated by Ca2+/calmodulin binding to an N-terminal CaM-binding site required for stress-induced actin assembly and MRTF nuclear translocation [#23, #32]. The CAAX/farnesylated isoform anchors to the ER cytoplasmic face, where INF2-driven actin polymerization at ER-mitochondria contacts mediates initial mitochondrial constriction upstream of Drp1 fission [#0, #10], a function tuned by SPOP/CUL3 and FBXO7 ubiquitination and by AMPK phosphorylation [#16, #26, #27], and extended to endosomal and lysosomal fission and to IP3R cluster organization that supports ER-to-mitochondria calcium transfer [#35]. INF2 also bundles and stabilizes microtubules through its C-terminus, acting downstream of an mDia1\\u2192IQGAP1\\u2192INF2 cascade to build detyrosinated microtubules and reorient the centrosome [#7, #9, #15], and regulates polarized membrane trafficking through MAL/MAL2 and Cdc42 [#2, #3]. INF2 functions as a node in Rho/mDia and MRTF-SRF transcriptional signaling, where it inhibits mDia-driven actin polymerization via DID-DAD contacts [#5, #12]. Mutations in the DID cause autosomal-dominant focal segmental glomerulosclerosis and Charcot-Marie-Tooth neuropathy [#1, #4]; these are gain-of-function alleles that escape CAP/acetylated-actin- and mDia-mediated inhibition, constitutively activate actin polymerization, and disrupt DYNLL1-dependent nephrin trafficking, ultimately driving MRTF-SRF reprogramming and mitotic catastrophe [#17, #22, #25, #29].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established INF2 as an ER-localized formin whose distinctive depolymerization activity is held in check by an intramolecular DID-DAD interaction, defining its core autoregulatory architecture.\",\n      \"evidence\": \"Subcellular fractionation, farnesylation inhibition, in vitro binding/Kd measurement, and pyrene-actin assays with DAD/WH2 mutants\",\n      \"pmids\": [\"19366733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the physiological trigger that relieves autoinhibition\", \"Structural basis of FH2-mediated severing not yet defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linked INF2 to human disease by showing DID missense mutations cause autosomal-dominant FSGS, focusing attention on the autoregulatory domain in podocyte actin control.\",\n      \"evidence\": \"Linkage analysis and candidate gene sequencing across 11 FSGS families\",\n      \"pmids\": [\"20023659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro functional consequence of the mutations demonstrated in this study\", \"Cell type-specific mechanism not addressed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed INF2 in polarized membrane trafficking by identifying MAL/MAL2 and GTP-Cdc42 as partners required for transcytosis, lumen formation, and Lck-dependent immunological synapse assembly.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA knockdown, GTP-loading assays, and activity-dead mutant rescue in epithelial and T cells\",\n      \"pmids\": [\"20493814\", \"20881207\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether trafficking roles depend on ER versus non-CAAX isoform unresolved\", \"Direct substrate filaments for transcytosis not visualized\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined INF2's signaling and cytoskeletal cross-talk: its DID binds mDia DADs to restrain Rho/SRF-driven actin polymerization, and its C-terminus bundles and stabilizes microtubules.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, Co-IP, SRF reporter assays, and microtubule co-sedimentation/bundling assays\",\n      \"pmids\": [\"21278336\", \"21998204\", \"21998196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DID-mDia inhibition is integrated with INF2's own actin activity unclear\", \"Non-CAAX isoform role at Golgi mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Refined the catalytic mechanism and a microtubule-stabilization function: specific FH2 residues separate capping/severing from elongation, and INF2 is required for detyrosinated microtubule formation and centrosome reorientation.\",\n      \"evidence\": \"Pyrene-actin and TIRF assays with FH2 point mutants plus knockdown and FH2 rescue in T cells\",\n      \"pmids\": [\"22879592\", \"22986496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coupling between actin and microtubule activities at the molecular level unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified INF2's signature cellular role: ER-anchored actin polymerization at ER-mitochondria contacts drives initial mitochondrial constriction upstream of Drp1, establishing actin's role in fission.\",\n      \"evidence\": \"siRNA knockdown, super-resolution and live imaging, and Drp1 epistasis\",\n      \"pmids\": [\"23349293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal initiating contact-site actin polymerization not defined\", \"Quantitative contribution relative to Drp1 not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the severing mechanism structurally, showing FH2 encircles filament sides and requires phosphate release, with the WH2/DAD boosting severing potency.\",\n      \"evidence\": \"20 \\u00c5 helical 3D EM reconstruction, two-color TIRF, and phosphate-release/truncation assays\",\n      \"pmids\": [\"24412206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of the severing complex not obtained\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided in vivo validation that INF2 acts upstream of RhoA/Dia in the glomerulus and that FSGS DID mutants are loss-of-rescue alleles in this context.\",\n      \"evidence\": \"Zebrafish morpholino knockdown with human INF2 WT/mutant rescue and RhoA/Dia2 genetic epistasis\",\n      \"pmids\": [\"26086034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single model system\", \"Mechanistic reconciliation with gain-of-function findings unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Characterized INF2's rapid filament turnover kinetics, identifying nucleotide exchange as rate-limiting and profilin as an accelerator, and confirmed conserved in vivo function through the C. elegans ortholog.\",\n      \"evidence\": \"Pyrene/TIRF kinetics with profilin titration and C. elegans exc-6 rescue with human disease variants\",\n      \"pmids\": [\"26124273\", \"25771894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of short-filament steady state not directly tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined a formin activation cascade in which IQGAP1 scaffolds mDia1 to INF2 to build stable detyrosinated microtubules, linking actin and microtubule regulation hierarchically.\",\n      \"evidence\": \"Direct binding assays, Co-IP, knockdown, and MT dynamics measurements with interaction-disrupting mutants\",\n      \"pmids\": [\"27030671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether INF2 microtubule and actin pools are spatially distinct unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed INF2 mitochondrial fission activity is restrained post-translationally by SPOP/CUL3 atypical ubiquitination that controls its ER localization rather than its stability.\",\n      \"evidence\": \"Co-IP, in-cell ubiquitination assays, ER/DRP1 imaging, and SPOP-binding motif mutagenesis\",\n      \"pmids\": [\"28448495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling SPOP-INF2 engagement not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Discovered facilitated autoinhibition: a CAP\\u00b7lysine-acetylated-actin complex bridges DID and DAD to inhibit INF2, relieved by HDAC6 deacetylation, and impaired by disease DID mutants.\",\n      \"evidence\": \"Endogenous inhibitor purification, in vitro reconstitution with K50/K61 acetylmimetics, HDAC6 treatment, and disease-mutant binding assays\",\n      \"pmids\": [\"30962575\", \"31871199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular dynamics of acetylation-controlled switching not fully mapped\", \"Later contradicted regarding CAP requirement for inhibition\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated that diverse FSGS/CMT mutations are gain-of-function alleles producing graded constitutive formin activation, and uncovered additional disease-relevant interactions with DYNLL1, CFTR, mDIA1, and a cathepsin-cleaved DID fragment.\",\n      \"evidence\": \"Quantitative CaAR live imaging across >50 mutants, Y2H, surface trafficking assays, protease inhibition, and a R218Q knockin mouse\",\n      \"pmids\": [\"32444357\", \"31924668\", \"32573649\", \"33443052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single DID determines both mitochondrial and trafficking phenotypes unresolved\", \"Quantitative activation thresholds distinguishing FSGS from CMT not mechanistically explained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified Ca2+/calmodulin binding at an N-terminal site as a direct activating input required for stress-induced actin assembly and MRTF nuclear translocation, and extended INF2 activation to neuronal injury (actinification).\",\n      \"evidence\": \"NMR structure with 1-4-8 motif mutagenesis, KO complementation, MRTF assays, and neuronal NMDA/ischemia models\",\n      \"pmids\": [\"36306014\", \"36229429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of CaM activation with DID-DAD/CAP autoinhibition not resolved\", \"8S INF2:actin complex stoichiometry resolved only at low EM resolution (#36)\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established R218Q as a gain-of-function allele driving FSGS in vivo and dissected its downstream effects on actin, adhesion, mitochondria, nephrin proteostasis, MRTF-SRF reprogramming, and mitotic catastrophe.\",\n      \"evidence\": \"Knockin-versus-knockout mouse comparisons, organoids, RNA-seq, Co-IP, DYNLL1-PI31 trafficking dissection with bortezomib rescue, and pathogenic-variant ER/mitochondria imaging\",\n      \"pmids\": [\"39536114\", \"39621430\", \"38916773\", \"39337270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue specificity of podocyte/Schwann vulnerability not fully explained\", \"Causal ordering of mitochondrial versus adhesion phenotypes unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded regulatory and physiological scope: AMPK phosphorylation and FBXO7 ubiquitination tune INF2-DRP1 fission in cancer, CAP1/CAP2 inhibit INF2 during dendritic spine maturation, and ER-actin marks endosome/lysosome fission.\",\n      \"evidence\": \"Kinase and ubiquitination assays, neuronal genetic epistasis, and live organelle imaging (one preprint)\",\n      \"pmids\": [\"38233384\", \"37344480\", \"39154297\", \"39184068\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether multiple regulatory inputs converge on the same INF2 pool unknown\", \"Organelle-fission generality not yet peer-reviewed (#31)\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected mechanosensory and meiotic contexts and revised the autoinhibition model, proposing actin buffering by profilin/thymosin rather than CAP is required, alongside refined structural definition of the WH2/DAD helix.\",\n      \"evidence\": \"Cell-free reconstitution, Piezo1/INF2 confinement migration assays, oocyte knockdown with Co-IP, and NMR of the WH2/DAD\",\n      \"pmids\": [\"41498749\", \"40120583\", \"40180021\", \"40993919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CAP-requirement contradiction (#32 vs #17/#18) awaits independent reconciliation\", \"Single-study mechanistic claims for migration and meiosis\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined an INF2-IP3R axis in which INF2 actin activity organizes IP3R clusters to position ER-mitochondria contacts and facilitate calcium transfer, integrating its fission and calcium-handling roles.\",\n      \"evidence\": \"Co-IP, knockdown, calcium and IP3R cluster imaging, and activity-mutant dissection\",\n      \"pmids\": [\"42259291\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study\", \"Whether IP3R clustering precedes or follows fission machinery recruitment unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple activating (Ca2+/CaM, HDAC6, mechanical/ionic) and inhibitory (CAP/acetyl-actin, profilin buffering, ubiquitination, phosphorylation) inputs are integrated in space and time to select between mitochondrial, microtubule, trafficking, and transcriptional outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling CAP-dependent versus actin-buffering-dependent autoinhibition\", \"No atomic structure of activated full-length INF2 on a filament\", \"Mechanism dictating cell-type-specific disease vulnerability undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 11, 13]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [7, 11, 36]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 12]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [22, 30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 10, 16, 28]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 23]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7, 15]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [9, 29]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [10, 16, 27, 31]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [2, 3, 22, 30]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 12, 23, 29]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 4, 25, 29]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 23, 29]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MAL\", \"MAL2\", \"CDC42\", \"DIAPH1\", \"IQGAP1\", \"DYNLL1\", \"DNM1L\", \"ITPR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}