{"gene":"DIAPH3","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2003,"finding":"DIAPH3 (Drf3/mDia2) contains a CRIB-like motif within its GTPase binding domain (GBD) that is required for Cdc42 binding; FRET analysis demonstrated this motif is necessary for Cdc42-mediated recruitment of DIAPH3 to the leading edge and to the microtubule organizing center (MTOC) of migrating fibroblasts. Inactive Drf3 variants and microinjected Drf3 antibodies interfered with Cdc42-induced filopodia, establishing Drf3 as a Cdc42 effector for actin cytoskeleton remodeling.","method":"FRET analysis, microinjection of dominant-negative variants and antibodies, gene targeting (Drf1 knockout), fluorescence microscopy","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — FRET-based binding validation combined with dominant-negative interference and genetic loss-of-function, multiple orthogonal methods in one study","pmids":["12676083"],"is_preprint":false},{"year":2008,"finding":"A constitutively active form of DIAPH3 (Drf3ΔDaD, lacking the C-terminal DAD regulatory region) induces filopodia formation and accumulates at filopodial tips, while full-length DIAPH3 remains cytosolic and does not affect cell behavior. The data support de novo actin filament nucleation as the mechanism of DIAPH3-driven filopodia, rather than convergent elongation from lamellipodia.","method":"Ectopic expression of GFP-tagged full-length vs. constitutively active DIAPH3 truncation, live-cell fluorescence microscopy, electron microscopy of actin filament architecture","journal":"Journal of microscopy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean gain-of-function with deletion construct and structural characterization of actin bundles, single lab","pmids":["18755006"],"is_preprint":false},{"year":2010,"finding":"Overexpression of DIAPH3 (via a 5' UTR mutation c.-172G>A that increases transcription) causes auditory neuropathy in humans; expression of a constitutively active diaphanous in the Drosophila auditory organ recapitulates impaired response to sound, establishing that excess DIAPH3 formin activity is causally sufficient for auditory dysfunction.","method":"Luciferase reporter assay for mutation-driven overexpression, quantitative immunoblotting of patient lymphoblastoid cell lines, transgenic Drosophila expression of constitutively active diaphanous, genome-wide expression arrays and qRT-PCR","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (reporter assay, quantitative protein measurement, Drosophila genetic model), replicated across human samples and model organism","pmids":["20624953"],"is_preprint":false},{"year":2010,"finding":"A missense variant Pro614Thr in a highly conserved domain of DIAPH3 significantly reduces the number of filopodia induced upon transfection into murine fibroblasts compared to wild-type DIAPH3, demonstrating that this residue is functionally required for DIAPH3-mediated filopodia formation.","method":"Transfection of wild-type vs. Pro614Thr mutant DIAPH3 in murine fibroblasts, quantification of filopodia","journal":"Molecular psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — site-directed mutagenesis with functional readout, single lab, single method","pmids":["20308993"],"is_preprint":false},{"year":2011,"finding":"DIAPH3 is required for plasma membrane blebbing during cell adhesion; isoform-specific roles were identified whereby the low-abundance isoform 1 specifically drives blebbing, while activation of isoform 7 (by DAD deletion) induces filopodia instead. The N-terminal GTPase-binding domain region determines subcellular localization and the type of protrusion formed. Dimerization and actin assembly activity are both essential for protrusion induction.","method":"siRNA screen against 21 actin nucleation factors, isoform-specific expression constructs, deletion mutagenesis of DAD domain, immunofluorescence, cell spreading/spreading assays","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — targeted siRNA screen followed by isoform-specific rescue experiments with deletion constructs, multiple orthogonal approaches in single lab","pmids":["22184005"],"is_preprint":false},{"year":2012,"finding":"DIAPH3 silencing destabilizes microtubules, induces defective endocytic trafficking, causes endosomal accumulation of EGFR, and hyperactivates EGFR/MEK/ERK signaling, leading to amoeboid cell behavior, increased invasion, and metastasis in mice. DIAPH3-silenced cells showed reduced sensitivity to EGFR inhibition but increased sensitivity to MEK inhibition.","method":"siRNA-mediated knockdown in human carcinoma cells, immunofluorescence of microtubules and EGFR trafficking, in vivo mouse metastasis assay, pharmacological inhibitor experiments","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple cellular and in vivo phenotypic readouts, pathway placement via pharmacological inhibitors, single lab with multiple orthogonal methods","pmids":["22593025"],"is_preprint":false},{"year":2015,"finding":"DIAPH3 directly interacts with microtubules; its loss alters microtubule dynamics parameters, decreases polarized force generation, contractility, and mechanosensing response to substrate stiffness. These changes render cells hypersensitive to taxane chemotherapy.","method":"Co-localization and interaction assays with microtubules, DIAPH3 silencing in prostate and breast cancer cell lines, live-cell imaging of microtubule dynamics, atomic force microscopy for mechanics, cytotoxicity assays with taxanes, NCI-60 drug sensitivity analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct MT interaction shown with silencing phenotype and MT dynamics measurements, single lab with multiple readouts","pmids":["26179371"],"is_preprint":false},{"year":2016,"finding":"DIAPH3 co-immunoprecipitates with BubR1 (a key spindle assembly checkpoint regulator) in embryonic brain extracts; Diaph3-deficient cortical progenitors have decreased BubR1 levels, fail to activate the spindle assembly checkpoint, and undergo anaphase despite incorrect chromosome segregation, generating aneuploidy and causing microcephaly.","method":"Co-immunoprecipitation from embryonic brain extracts, conditional Diaph3 knockout in mice, immunofluorescence of BubR1 and mitotic markers, live imaging, brain histology","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP from brain tissue combined with conditional KO mouse with defined cellular and developmental phenotype, rigorous mechanistic follow-up","pmids":["27848932"],"is_preprint":false},{"year":2016,"finding":"Overexpression of DIAPH3 (diap3) in inner hair cells causes selective aberrant targeting of microtubule meshwork into the cuticular plate, followed by stereociliary bundle collapse and eventual loss of inner hair cell capacity to transmit auditory stimuli; DIAPH3 was identified as a component of the hair cell apical pole in wild-type mice.","method":"Transgenic diap3-overexpressing mice, immunofluorescence and electron microscopy of inner hair cell apical structures, electrophysiology (neurotransmitter release and potassium conductance measurements)","journal":"eNeuro","confidence":"High","confidence_rationale":"Tier 2 / Moderate — transgenic mouse model with gain-of-function, multiple structural and functional readouts, mechanistic link to microtubule targeting","pmids":["28058271"],"is_preprint":false},{"year":2017,"finding":"DIAPH3 activates β-catenin/TCF signaling in hepatocellular carcinoma cells by binding HSP90 and disrupting the interaction between GSK3β and HSP90.","method":"Co-immunoprecipitation, siRNA knockdown and overexpression in HCC cells, western blotting, in vivo mouse metastasis model","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — Co-IP showing DIAPH3-HSP90 interaction with functional consequence on GSK3β-HSP90 complex, single lab, limited mechanistic depth","pmids":["28795316"],"is_preprint":false},{"year":2019,"finding":"DIAPH3 was identified as a binding protein of STK38 (NDR kinase); DIAPH3 impairs the interaction between STK38 and MEKK, thereby activating ERK signaling and promoting lung adenocarcinoma growth.","method":"Co-immunoprecipitation, siRNA knockdown and overexpression, in vivo nude mouse and de novo mouse tumor models, western blotting for ERK pathway markers","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — Co-IP identifying binding partner with functional downstream signaling consequence, single lab","pmids":["31586548"],"is_preprint":false},{"year":2020,"finding":"DIAPH3 interacts with the selenoprotein RPL6, a ribosome protein subunit involved in selenoamino acid metabolism, and promotes selenium content and TrxR1 (thioredoxin reductase 1) expression, thereby reducing cellular ROS levels in pancreatic cancer cells.","method":"Co-immunoprecipitation, overexpression and siRNA interference in pancreatic cancer cells, ROS measurement, nude mice xenograft model, TrxR1 western blotting","journal":"Journal of cellular and molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP identifying RPL6 binding partner, single lab, indirect mechanism for TrxR1 upregulation not fully established","pmids":["33345387"],"is_preprint":false},{"year":2020,"finding":"Loss of DIAPH3 (Diaph3 knockout) causes cytokinesis failure specifically at high temperature (39°C) but not at 32°C in mouse FM3A cells. This phenotype was rescued by re-expression of Diaph3 WT but not by Diaph1 or Diaph2. Diaph3 knockout cells at 39°C also showed significantly increased levels of acetylated α-tubulin (indicating stabilized microtubules), which was reduced by Diaph3 re-expression.","method":"Exome sequencing of temperature-sensitive mutants, Diaph3 knockout cells, rescue with Diaph1/Diaph2/Diaph3 re-expression, acetylated α-tubulin immunofluorescence and western blotting","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO plus isoform-specific rescue experiments with defined cytokinesis phenotype and molecular readout, single lab","pmids":["33187357"],"is_preprint":false},{"year":2021,"finding":"DIAPH3 localizes to the centrosome during mitosis and regulates assembly and bipolarity of the mitotic spindle; DIAPH3-deficient cells display multipolar spindles and disorganized cytoskeleton. DIAPH3 deficiency disrupts expression/stability of the kinetochore-associated protein SPAG5; knockdown of SPAG5 phenocopies DIAPH3 deficiency, and SPAG5 overexpression rescues DIAPH3 knockdown, placing SPAG5 downstream of DIAPH3 in spindle assembly.","method":"Immunofluorescence localization to centrosome, DIAPH3 conditional knockout in mouse cerebral cortex, siRNA knockdown, SPAG5 overexpression rescue experiments, live imaging, behavioral testing of mice","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — centrosomal localization combined with genetic epistasis (knockdown phenocopy + overexpression rescue), conditional mouse KO with brain phenotype, multiple orthogonal methods","pmids":["33899739"],"is_preprint":false},{"year":2023,"finding":"Under cellular stress conditions, DIAPH3 undergoes liquid-liquid phase separation (LLPS) to form cytosolic condensates (D-granules) that spatially sequester DIAPH3, thereby inhibiting actin filament assembly in filopodia. D-granules are distinct from stress granules and P-bodies (lacking G3BP1, G3BP2, TIA1, and DCP1A markers). This was demonstrated using overexpression, knockout, pharmacological interventions, and optogenetics.","method":"LLPS characterization (fluorescence recovery after photobleaching, droplet fusion assay), DIAPH3 overexpression and knockout, optogenetic induction of DIAPH3 condensation, pharmacological stress induction, marker colocalization studies","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — LLPS reconstitution with biophysical validation, optogenetics, and KO/OE experiments with defined actin phenotype, multiple orthogonal methods in single lab","pmids":["36640348"],"is_preprint":false},{"year":2023,"finding":"The lncRNA LINC01089 interacts with hnRNPM to cause hnRNPM-mediated skipping of DIAPH3 exon 3; inclusion of exon 3 contains an m6A modification site recognized by IGF2BP3 that stabilizes DIAPH3 mRNA. Knockdown of LINC01089 increased DIAPH3 protein levels, which suppressed ERK/Elk1/Snail signaling and inhibited EMT.","method":"RNA immunoprecipitation, alternative splicing analysis, m6A-seq, IGF2BP3 binding assays, siRNA knockdown, mRNA stability assays, in vivo HCC metastasis model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal RNA biology methods establishing the splicing-m6A-stability axis controlling DIAPH3 levels, single lab","pmids":["37756562"],"is_preprint":false},{"year":2024,"finding":"DIAPH3 β-actin networks (but not γ-actin networks generated by DIAPH1) are required for maintaining non-muscle myosin II and RhoA at the cytokinetic furrow. Expression of hybrid DIAPH1/DIAPH3 proteins with altered actin isoform specificity relocalized actin isoform networks and caused cytokinetic failure, demonstrating non-redundant, specialized roles for β- and γ-actin in cytokinesis.","method":"Expression of hybrid DIAPH1/DIAPH3 chimeric proteins with altered actin isoform specificity, immunofluorescence of myosin II and RhoA localization at cytokinetic furrow, live-cell imaging of cytokinesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — chimeric protein approach with defined functional rescue, mechanistic epistasis placing β-actin network upstream of RhoA/myosin II maintenance, rigorous domain-swap strategy","pmids":["38897998"],"is_preprint":false},{"year":2024,"finding":"The E3 ubiquitin ligase Stub1 ubiquitinates activated DIAPH3 at K1142/1143/1144 lysine residues and promotes its proteasomal degradation. DID-DAD intramolecular interaction stabilizes DIAPH3 against degradation; disruption of DID-DAD by RhoA binding or M1041A mutation activates DIAPH3 but simultaneously triggers accelerated Stub1-mediated ubiquitination and degradation. FH2-FH2 interaction promotes activity while DD-DD interaction inhibits it. Knockdown of Stub1 in mouse cochlea causes hair cell stereocilia defects and hearing loss resembling DIAPH3 overexpression phenotype.","method":"In vitro ubiquitination assay, site-directed mutagenesis (M1041A, K1142/1143/1144R), Co-IP of domain interactions, RhoA binding experiments, proteasome inhibitor (MG132) treatment, Stub1 knockdown in mouse cochlea, auditory phenotyping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination reconstitution combined with mutagenesis identifying specific ubiquitination sites and autoinhibitory domain interactions, validated in vivo in cochlea","pmids":["39322015"],"is_preprint":false},{"year":2024,"finding":"DIAPH3 interacts with FOXM1 (forkhead box M1) transcription factor, as determined by Co-IP; loss of DIAPH3 downregulates FOXM1 and blocks Wnt/β-catenin signaling in anaplastic thyroid carcinoma cells. FOXM1 overexpression rescues the anti-proliferative, pro-apoptotic effects of DIAPH3 depletion.","method":"Co-immunoprecipitation, siRNA knockdown, FOXM1 overexpression rescue, western blotting for Wnt/β-catenin pathway markers, cell proliferation/migration/invasion assays","journal":"Endokrynologia Polska","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with functional rescue experiment, single lab, limited mechanistic depth for the DIAPH3-FOXM1 interaction","pmids":["39376176"],"is_preprint":false},{"year":2025,"finding":"DIAPH3 reduces RPL6 protein levels by disrupting the interaction between RPL6 and the deubiquitinase OTUD4, thereby reducing RPL6-mediated activation of cGAS-STING signaling in pancreatic cancer cells.","method":"Co-immunoprecipitation of RPL6-OTUD4 interaction, forced expression of RPL6, DIAPH3 overexpression experiments, interferon-β production measurement","journal":"European journal of medical research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP-based interaction mapping with functional readout, single lab, limited mechanistic validation of the DIAPH3-OTUD4-RPL6 axis","pmids":["41318619"],"is_preprint":false},{"year":2025,"finding":"DIAPH3 silencing suppresses migration and invasion of colorectal cancer cells; mass spectrometry identified KRT19 as a downstream target of DIAPH3. Knockdown of DIAPH3 reduces KRT19 protein levels through proteasome-dependent degradation (blocked by MG132), and KRT19 overexpression rescues the invasion defect caused by DIAPH3 silencing, establishing a DIAPH3-KRT19 signaling axis.","method":"Mass spectrometry identification of downstream targets, siRNA knockdown, KRT19 overexpression rescue, proteasome inhibitor (MG132) treatment, wound healing and Transwell invasion assays, xenograft metastatic model","journal":"Clinical & experimental metastasis","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — MS-based target identification with rescue experiment and proteasomal mechanism, single lab","pmids":["39843730"],"is_preprint":false}],"current_model":"DIAPH3 is a Diaphanous-related formin that functions as an actin nucleator/elongator downstream of Rho GTPases (RhoA-C and Cdc42), regulated by autoinhibitory DID-DAD intramolecular interaction and subject to Stub1-mediated ubiquitination and proteasomal degradation when activated; it localizes to the centrosome during mitosis, maintains spindle bipolarity and spindle assembly checkpoint activity (via BubR1 and SPAG5), specifically nucleates β-actin networks required for RhoA and myosin II retention at the cytokinetic furrow, stabilizes microtubule dynamics to regulate amoeboid-to-mesenchymal transition, and forms stress-induced phase-separated condensates (D-granules) that sequester DIAPH3 to inhibit filopodial actin assembly, with overexpression causing inner hair cell microtubule meshwork disruption and auditory neuropathy."},"narrative":{"mechanistic_narrative":"DIAPH3 (Drf3/mDia2) is a Diaphanous-related formin that nucleates and elongates actin filaments downstream of Rho-family GTPases to drive membrane protrusion, mitotic spindle integrity, and cytokinesis [PMID:12676083, PMID:22184005, PMID:38897998]. It acts as a Cdc42 effector: a CRIB-like motif in its GTPase-binding domain mediates Cdc42-dependent recruitment to the leading edge and the MTOC, where it promotes filopodia formation [PMID:12676083]. Its activity is governed by autoinhibition — full-length DIAPH3 is cytosolic and inert, whereas deletion of the C-terminal DAD region or RhoA-mediated disruption of the DID–DAD interaction yields a constitutively active protein that nucleates de novo actin filaments at filopodial tips [PMID:18755006, PMID:39322015]; the same activating events that relieve autoinhibition simultaneously expose DIAPH3 to Stub1-mediated ubiquitination at K1142/1143/1144 and proteasomal degradation, coupling activation to turnover [PMID:39322015]. Isoform identity dictates protrusion outcome, with distinct isoforms driving membrane blebbing versus filopodia [PMID:22184005]. Beyond actin, DIAPH3 binds and stabilizes microtubule dynamics, and its loss destabilizes microtubules, perturbs endocytic EGFR trafficking, and hyperactivates EGFR/MEK/ERK signaling to promote amoeboid invasion and metastasis [PMID:22593025, PMID:26179371]. During mitosis DIAPH3 localizes to the centrosome and maintains spindle bipolarity and the spindle assembly checkpoint through BubR1 and SPAG5, with deficiency causing aneuploidy and microcephaly [PMID:27848932, PMID:33899739]. In cytokinesis it specifically generates β-actin networks required to retain RhoA and non-muscle myosin II at the furrow, a role non-redundant with the γ-actin networks made by DIAPH1 [PMID:38897998, PMID:33187357]. Under stress, DIAPH3 undergoes liquid–liquid phase separation into D-granules that sequester it and inhibit filopodial actin assembly [PMID:36640348]. Overexpression of DIAPH3, including via a 5'UTR mutation that elevates transcription, causes auditory neuropathy in humans and mislocalizes the microtubule meshwork in inner hair cells, establishing excess formin activity as causally sufficient for hearing loss [PMID:20624953, PMID:28058271].","teleology":[{"year":2003,"claim":"Established DIAPH3 as a Cdc42 effector, answering how it is recruited and activated to remodel the actin cytoskeleton.","evidence":"FRET binding analysis, dominant-negative microinjection, and gene targeting in migrating fibroblasts","pmids":["12676083"],"confidence":"High","gaps":["Did not resolve how RhoA versus Cdc42 differentially activate the formin","Structural basis of CRIB-like motif engagement not defined"]},{"year":2008,"claim":"Defined the autoinhibitory logic, showing the DAD region restrains actin nucleation and that its removal converts DIAPH3 into a de novo filopodial actin nucleator.","evidence":"Gain-of-function expression of full-length vs DAD-truncated constructs with live-cell and electron microscopy","pmids":["18755006"],"confidence":"Medium","gaps":["Physiological signal relieving autoinhibition not identified in this study","In vitro nucleation kinetics not measured"]},{"year":2010,"claim":"Linked DIAPH3 dosage to human disease, showing overexpression is causally sufficient for auditory neuropathy.","evidence":"Luciferase reporter of a 5'UTR mutation, quantitative immunoblotting of patient cells, and constitutively active diaphanous in Drosophila auditory organ; plus mutagenesis showing Pro614Thr reduces filopodia","pmids":["20624953","20308993"],"confidence":"High","gaps":["Cellular target tissue of overexpression not yet defined in 2010","Mechanism connecting formin activity to hair cell dysfunction unresolved"]},{"year":2012,"claim":"Revealed a microtubule- and trafficking-based function, showing DIAPH3 loss destabilizes microtubules and hyperactivates EGFR/MEK/ERK to drive amoeboid invasion.","evidence":"siRNA knockdown in carcinoma cells with EGFR trafficking imaging, in vivo metastasis, and pharmacological pathway placement","pmids":["22593025"],"confidence":"High","gaps":["Direct molecular link between DIAPH3 and microtubule stabilization not yet shown","How a formin restrains EGFR endosomal trafficking unclear"]},{"year":2015,"claim":"Demonstrated direct microtubule interaction, connecting DIAPH3 to mechanosensing, force generation, and taxane sensitivity.","evidence":"MT co-localization/interaction assays, silencing with live MT-dynamics imaging and atomic force microscopy in cancer lines","pmids":["26179371"],"confidence":"Medium","gaps":["Binding interface on microtubules not mapped","Whether MT effects are independent of actin nucleation unresolved"]},{"year":2016,"claim":"Placed DIAPH3 in mitotic fidelity control, showing it sustains the spindle assembly checkpoint via BubR1 to prevent aneuploidy and microcephaly.","evidence":"Co-IP from embryonic brain, conditional Diaph3 knockout mice, and mitotic imaging; plus gain-of-function transgenic mice mislocalizing the hair cell MT meshwork","pmids":["27848932","28058271"],"confidence":"High","gaps":["Whether DIAPH3-BubR1 interaction is direct not established","How a centrosomal formin regulates checkpoint protein stability unclear"]},{"year":2021,"claim":"Mapped the centrosomal spindle role, placing SPAG5 downstream of DIAPH3 in maintaining spindle bipolarity.","evidence":"Centrosomal localization, conditional cortical knockout, and SPAG5 knockdown-phenocopy plus overexpression-rescue epistasis","pmids":["33899739"],"confidence":"High","gaps":["Mechanism by which DIAPH3 controls SPAG5 stability unresolved","Relationship between centrosomal actin and spindle bipolarity not defined"]},{"year":2024,"claim":"Resolved actin-isoform specificity in cytokinesis, showing DIAPH3-generated β-actin networks uniquely retain RhoA and myosin II at the furrow.","evidence":"DIAPH1/DIAPH3 chimeric domain-swap proteins with furrow imaging and live cytokinesis assays; temperature-sensitive KO with isoform-specific rescue","pmids":["38897998","33187357"],"confidence":"High","gaps":["Molecular basis of β- vs γ-actin discrimination by the FH2 domain not fully defined","How β-actin networks physically retain RhoA unresolved"]},{"year":2024,"claim":"Defined the activation–degradation coupling, showing Stub1 ubiquitinates activated DIAPH3 and DID-DAD interaction protects it from turnover.","evidence":"In vitro ubiquitination, mutagenesis of ubiquitination and autoinhibition residues, domain-interaction Co-IP, and Stub1 knockdown in cochlea","pmids":["39322015"],"confidence":"High","gaps":["Upstream signals controlling Stub1 recruitment not identified","Quantitative kinetics of activation versus degradation not measured"]},{"year":2023,"claim":"Identified a stress-responsive regulatory mode, showing DIAPH3 phase-separates into D-granules that sequester it to suppress filopodial actin.","evidence":"FRAP, droplet fusion, optogenetic condensation, KO/OE, and marker colocalization distinguishing D-granules from stress granules and P-bodies","pmids":["36640348"],"confidence":"High","gaps":["Sequence determinants driving DIAPH3 LLPS not mapped","Physiological stresses triggering D-granules in vivo not defined"]},{"year":2025,"claim":"Extended DIAPH3 into multiple cancer signaling axes through reported protein interactions controlling proliferation, EMT, redox, and innate immune signaling.","evidence":"Co-IP and rescue studies implicating HSP90/GSK3β, STK38/ERK, FOXM1/Wnt, RPL6/OTUD4/cGAS-STING, and KRT19; plus LINC01089-hnRNPM-m6A control of DIAPH3 mRNA","pmids":["28795316","31586548","39376176","33345387","41318619","39843730","37756562"],"confidence":"Low","gaps":["Most interactions rest on single Co-IPs without reciprocal or direct-binding validation","Whether these signaling roles depend on actin/microtubule activity unclear","Mechanistic depth of the RPL6/OTUD4/cGAS-STING axis limited"]},{"year":null,"claim":"How DIAPH3's distinct activities — actin nucleation, microtubule regulation, spindle/checkpoint control, and stress-induced phase separation — are coordinated by a single regulatory state remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model linking RhoA/Cdc42 input, Stub1 turnover, and LLPS to specific cellular outputs","Structural basis of actin-isoform and microtubule selectivity undetermined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,4,16,6]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[17]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,14]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,13]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[6,16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,13,16,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,0]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[7,13]}],"complexes":[],"partners":["BUBR1","SPAG5","STUB1","RHOA","CDC42","HSP90","STK38","FOXM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NSV4","full_name":"Protein diaphanous homolog 3","aliases":["Diaphanous-related formin-3","DRF3","MDia2"],"length_aa":1193,"mass_kda":136.9,"function":"Actin nucleation and elongation factor required for the assembly of F-actin structures, such as actin cables and stress fibers. Required for cytokinesis, stress fiber formation and transcriptional activation of the serum response factor. Binds to GTP-bound form of Rho and to profilin: acts in a Rho-dependent manner to recruit profilin to the membrane, where it promotes actin polymerization. DFR proteins couple Rho and Src tyrosine kinase during signaling and the regulation of actin dynamics. Also acts as an actin nucleation and elongation factor in the nucleus by promoting nuclear actin polymerization inside the nucleus to drive serum-dependent SRF-MRTFA activity","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NSV4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DIAPH3","classification":"Not Classified","n_dependent_lines":110,"n_total_lines":1208,"dependency_fraction":0.09105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DIAPH3","total_profiled":1310},"omim":[{"mim_id":"614567","title":"DIAPHANOUS-RELATED FORMIN 3; DIAPH3","url":"https://www.omim.org/entry/614567"},{"mim_id":"609129","title":"AUDITORY NEUROPATHY, AUTOSOMAL DOMINANT 1; AUNA1","url":"https://www.omim.org/entry/609129"},{"mim_id":"605551","title":"NITRIC OXIDE SYNTHASE 1 (NEURONAL) ADAPTOR PROTEIN; NOS1AP","url":"https://www.omim.org/entry/605551"},{"mim_id":"601428","title":"RNA, U4ATAC SMALL NUCLEAR; RNU4ATAC","url":"https://www.omim.org/entry/601428"},{"mim_id":"263450","title":"POLYDACTYLY, POSTAXIAL, TYPE A5; PAPA5","url":"https://www.omim.org/entry/263450"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Microtubules","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"bone marrow","ntpm":6.7},{"tissue":"testis","ntpm":17.7}],"url":"https://www.proteinatlas.org/search/DIAPH3"},"hgnc":{"alias_symbol":["DRF3","FLJ34705","AN","NSDAN"],"prev_symbol":["AUNA1"]},"alphafold":{"accession":"Q9NSV4","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NSV4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NSV4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NSV4-F1-predicted_aligned_error_v6.png","plddt_mean":71.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DIAPH3","jax_strain_url":"https://www.jax.org/strain/search?query=DIAPH3"},"sequence":{"accession":"Q9NSV4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NSV4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NSV4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NSV4"}},"corpus_meta":[{"pmid":"12676083","id":"PMC_12676083","title":"Disruption of the Diaphanous-related formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42.","date":"2003","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/12676083","citation_count":205,"is_preprint":false},{"pmid":"20624953","id":"PMC_20624953","title":"Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila.","date":"2010","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/20624953","citation_count":93,"is_preprint":false},{"pmid":"22593025","id":"PMC_22593025","title":"DIAPH3 governs the cellular transition to the amoeboid tumour phenotype.","date":"2012","source":"EMBO molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/22593025","citation_count":87,"is_preprint":false},{"pmid":"18755006","id":"PMC_18755006","title":"Filopodia formation induced by active mDia2/Drf3.","date":"2008","source":"Journal of microscopy","url":"https://pubmed.ncbi.nlm.nih.gov/18755006","citation_count":79,"is_preprint":false},{"pmid":"37756562","id":"PMC_37756562","title":"Super Enhancer-Regulated LncRNA LINC01089 Induces Alternative Splicing of DIAPH3 to Drive Hepatocellular Carcinoma Metastasis.","date":"2023","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/37756562","citation_count":71,"is_preprint":false},{"pmid":"20308993","id":"PMC_20308993","title":"A double hit implicates DIAPH3 as an autism risk gene.","date":"2010","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/20308993","citation_count":55,"is_preprint":false},{"pmid":"33345387","id":"PMC_33345387","title":"DIAPH3 promotes pancreatic cancer progression by activating selenoprotein TrxR1-mediated antioxidant effects.","date":"2020","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33345387","citation_count":44,"is_preprint":false},{"pmid":"14767582","id":"PMC_14767582","title":"Identification and characterization of human DIAPH3 gene in silico.","date":"2004","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/14767582","citation_count":43,"is_preprint":false},{"pmid":"26179371","id":"PMC_26179371","title":"Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26179371","citation_count":38,"is_preprint":false},{"pmid":"28795316","id":"PMC_28795316","title":"DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/TCF signaling.","date":"2017","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28795316","citation_count":37,"is_preprint":false},{"pmid":"27848932","id":"PMC_27848932","title":"Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27848932","citation_count":35,"is_preprint":false},{"pmid":"33899739","id":"PMC_33899739","title":"DIAPH3 deficiency links microtubules to mitotic errors, defective neurogenesis, and brain dysfunction.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/33899739","citation_count":33,"is_preprint":false},{"pmid":"22184005","id":"PMC_22184005","title":"Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation.","date":"2011","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/22184005","citation_count":21,"is_preprint":false},{"pmid":"36640348","id":"PMC_36640348","title":"DIAPH3 condensates formed by liquid-liquid phase separation act as a regulatory hub for stress-induced actin cytoskeleton remodeling.","date":"2023","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/36640348","citation_count":20,"is_preprint":false},{"pmid":"28058271","id":"PMC_28058271","title":"Remodeling of the Inner Hair Cell Microtubule Meshwork in a Mouse Model of Auditory Neuropathy AUNA1.","date":"2016","source":"eNeuro","url":"https://pubmed.ncbi.nlm.nih.gov/28058271","citation_count":19,"is_preprint":false},{"pmid":"31586548","id":"PMC_31586548","title":"DIAPH3 promotes the tumorigenesis of lung adenocarcinoma.","date":"2019","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/31586548","citation_count":14,"is_preprint":false},{"pmid":"19353688","id":"PMC_19353688","title":"Pure monosomy and pure trisomy of 13q21.2-31.1 consequent to a familial insertional translocation: exclusion of PCDH9 as the responsible gene for autosomal dominant auditory neuropathy (AUNA1).","date":"2009","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/19353688","citation_count":11,"is_preprint":false},{"pmid":"34659408","id":"PMC_34659408","title":"Knockdown of DIAPH3 Inhibits the Proliferation of Cervical Cancer Cells through Inactivating mTOR Signaling Pathway.","date":"2021","source":"Journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/34659408","citation_count":10,"is_preprint":false},{"pmid":"27455002","id":"PMC_27455002","title":"[Analysis of DIAPH3 gene mutation in a boy with autism spectrum disorder].","date":"2016","source":"Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27455002","citation_count":7,"is_preprint":false},{"pmid":"38897998","id":"PMC_38897998","title":"The DIAPH3 linker specifies a β-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38897998","citation_count":6,"is_preprint":false},{"pmid":"36750200","id":"PMC_36750200","title":"The pan-cancer analysis identified DIAPH3 as a diagnostic biomarker of clinical cancer.","date":"2023","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/36750200","citation_count":5,"is_preprint":false},{"pmid":"38454929","id":"PMC_38454929","title":"DIAPH3 predicts survival of patients with MGMT-methylated glioblastoma.","date":"2024","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38454929","citation_count":4,"is_preprint":false},{"pmid":"35380294","id":"PMC_35380294","title":"Diaph3 underlines tumor cell heterogeneity in glioblastoma with implications for treatment modalities resistance.","date":"2022","source":"Journal of neuro-oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35380294","citation_count":4,"is_preprint":false},{"pmid":"38728194","id":"PMC_38728194","title":"Role of MEK1 and DIAPH3 expression in colorectal adenoma-carcinoma sequence.","date":"2024","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38728194","citation_count":2,"is_preprint":false},{"pmid":"33187357","id":"PMC_33187357","title":"Loss of DIAPH3, a Formin Family Protein, Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33187357","citation_count":2,"is_preprint":false},{"pmid":"39843730","id":"PMC_39843730","title":"m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19.","date":"2025","source":"Clinical & experimental metastasis","url":"https://pubmed.ncbi.nlm.nih.gov/39843730","citation_count":1,"is_preprint":false},{"pmid":"39322015","id":"PMC_39322015","title":"Stub1 promotes degradation of the activated Diaph3: A negative feedback regulatory mechanism of the actin nucleator.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39322015","citation_count":1,"is_preprint":false},{"pmid":"41318619","id":"PMC_41318619","title":"The DIAPH3/RPL6 axis regulates the cGAS-STING pathway in pancreatic cancer.","date":"2025","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/41318619","citation_count":0,"is_preprint":false},{"pmid":"41473744","id":"PMC_41473744","title":"DIAPH3 is upregulated in high-grade gliomas and linked to chromosomal instability.","date":"2025","source":"Neuro-oncology advances","url":"https://pubmed.ncbi.nlm.nih.gov/41473744","citation_count":0,"is_preprint":false},{"pmid":"40040362","id":"PMC_40040362","title":"Advances in Understanding the Molecular Dynamics of Autosomal Dominant Auditory Neuropathy: Unveiling a Novel DIAPH3 Gene Variant Associated With Sensorineural Hearing Loss and Bilateral Vestibular Aqueduct Enlargement.","date":"2025","source":"Journal of audiology & otology","url":"https://pubmed.ncbi.nlm.nih.gov/40040362","citation_count":0,"is_preprint":false},{"pmid":"39376176","id":"PMC_39376176","title":"The functional DIAPH3-FOXM1 interaction modulates the aggressive transformation of anaplastic thyroid carcinoma cells and Wnt/β-catenin signalling.","date":"2024","source":"Endokrynologia Polska","url":"https://pubmed.ncbi.nlm.nih.gov/39376176","citation_count":0,"is_preprint":false},{"pmid":"41872154","id":"PMC_41872154","title":"Loss of DIAPH3 accelerates glioma genesis in mice.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41872154","citation_count":0,"is_preprint":false},{"pmid":"41511813","id":"PMC_41511813","title":"Identification of a Novel Likely Pathogenic Variant of DIAPH3 Associated With New Phenotype of Sensorineural Hearing Loss.","date":"2026","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41511813","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18522,"output_tokens":5886,"usd":0.071928,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14590,"output_tokens":4152,"usd":0.088375,"stage2_stop_reason":"end_turn"},"total_usd":0.160303,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"DIAPH3 (Drf3/mDia2) contains a CRIB-like motif within its GTPase binding domain (GBD) that is required for Cdc42 binding; FRET analysis demonstrated this motif is necessary for Cdc42-mediated recruitment of DIAPH3 to the leading edge and to the microtubule organizing center (MTOC) of migrating fibroblasts. Inactive Drf3 variants and microinjected Drf3 antibodies interfered with Cdc42-induced filopodia, establishing Drf3 as a Cdc42 effector for actin cytoskeleton remodeling.\",\n      \"method\": \"FRET analysis, microinjection of dominant-negative variants and antibodies, gene targeting (Drf1 knockout), fluorescence microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — FRET-based binding validation combined with dominant-negative interference and genetic loss-of-function, multiple orthogonal methods in one study\",\n      \"pmids\": [\"12676083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A constitutively active form of DIAPH3 (Drf3ΔDaD, lacking the C-terminal DAD regulatory region) induces filopodia formation and accumulates at filopodial tips, while full-length DIAPH3 remains cytosolic and does not affect cell behavior. The data support de novo actin filament nucleation as the mechanism of DIAPH3-driven filopodia, rather than convergent elongation from lamellipodia.\",\n      \"method\": \"Ectopic expression of GFP-tagged full-length vs. constitutively active DIAPH3 truncation, live-cell fluorescence microscopy, electron microscopy of actin filament architecture\",\n      \"journal\": \"Journal of microscopy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean gain-of-function with deletion construct and structural characterization of actin bundles, single lab\",\n      \"pmids\": [\"18755006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Overexpression of DIAPH3 (via a 5' UTR mutation c.-172G>A that increases transcription) causes auditory neuropathy in humans; expression of a constitutively active diaphanous in the Drosophila auditory organ recapitulates impaired response to sound, establishing that excess DIAPH3 formin activity is causally sufficient for auditory dysfunction.\",\n      \"method\": \"Luciferase reporter assay for mutation-driven overexpression, quantitative immunoblotting of patient lymphoblastoid cell lines, transgenic Drosophila expression of constitutively active diaphanous, genome-wide expression arrays and qRT-PCR\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (reporter assay, quantitative protein measurement, Drosophila genetic model), replicated across human samples and model organism\",\n      \"pmids\": [\"20624953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A missense variant Pro614Thr in a highly conserved domain of DIAPH3 significantly reduces the number of filopodia induced upon transfection into murine fibroblasts compared to wild-type DIAPH3, demonstrating that this residue is functionally required for DIAPH3-mediated filopodia formation.\",\n      \"method\": \"Transfection of wild-type vs. Pro614Thr mutant DIAPH3 in murine fibroblasts, quantification of filopodia\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — site-directed mutagenesis with functional readout, single lab, single method\",\n      \"pmids\": [\"20308993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"DIAPH3 is required for plasma membrane blebbing during cell adhesion; isoform-specific roles were identified whereby the low-abundance isoform 1 specifically drives blebbing, while activation of isoform 7 (by DAD deletion) induces filopodia instead. The N-terminal GTPase-binding domain region determines subcellular localization and the type of protrusion formed. Dimerization and actin assembly activity are both essential for protrusion induction.\",\n      \"method\": \"siRNA screen against 21 actin nucleation factors, isoform-specific expression constructs, deletion mutagenesis of DAD domain, immunofluorescence, cell spreading/spreading assays\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — targeted siRNA screen followed by isoform-specific rescue experiments with deletion constructs, multiple orthogonal approaches in single lab\",\n      \"pmids\": [\"22184005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DIAPH3 silencing destabilizes microtubules, induces defective endocytic trafficking, causes endosomal accumulation of EGFR, and hyperactivates EGFR/MEK/ERK signaling, leading to amoeboid cell behavior, increased invasion, and metastasis in mice. DIAPH3-silenced cells showed reduced sensitivity to EGFR inhibition but increased sensitivity to MEK inhibition.\",\n      \"method\": \"siRNA-mediated knockdown in human carcinoma cells, immunofluorescence of microtubules and EGFR trafficking, in vivo mouse metastasis assay, pharmacological inhibitor experiments\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple cellular and in vivo phenotypic readouts, pathway placement via pharmacological inhibitors, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22593025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DIAPH3 directly interacts with microtubules; its loss alters microtubule dynamics parameters, decreases polarized force generation, contractility, and mechanosensing response to substrate stiffness. These changes render cells hypersensitive to taxane chemotherapy.\",\n      \"method\": \"Co-localization and interaction assays with microtubules, DIAPH3 silencing in prostate and breast cancer cell lines, live-cell imaging of microtubule dynamics, atomic force microscopy for mechanics, cytotoxicity assays with taxanes, NCI-60 drug sensitivity analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct MT interaction shown with silencing phenotype and MT dynamics measurements, single lab with multiple readouts\",\n      \"pmids\": [\"26179371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DIAPH3 co-immunoprecipitates with BubR1 (a key spindle assembly checkpoint regulator) in embryonic brain extracts; Diaph3-deficient cortical progenitors have decreased BubR1 levels, fail to activate the spindle assembly checkpoint, and undergo anaphase despite incorrect chromosome segregation, generating aneuploidy and causing microcephaly.\",\n      \"method\": \"Co-immunoprecipitation from embryonic brain extracts, conditional Diaph3 knockout in mice, immunofluorescence of BubR1 and mitotic markers, live imaging, brain histology\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP from brain tissue combined with conditional KO mouse with defined cellular and developmental phenotype, rigorous mechanistic follow-up\",\n      \"pmids\": [\"27848932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Overexpression of DIAPH3 (diap3) in inner hair cells causes selective aberrant targeting of microtubule meshwork into the cuticular plate, followed by stereociliary bundle collapse and eventual loss of inner hair cell capacity to transmit auditory stimuli; DIAPH3 was identified as a component of the hair cell apical pole in wild-type mice.\",\n      \"method\": \"Transgenic diap3-overexpressing mice, immunofluorescence and electron microscopy of inner hair cell apical structures, electrophysiology (neurotransmitter release and potassium conductance measurements)\",\n      \"journal\": \"eNeuro\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic mouse model with gain-of-function, multiple structural and functional readouts, mechanistic link to microtubule targeting\",\n      \"pmids\": [\"28058271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DIAPH3 activates β-catenin/TCF signaling in hepatocellular carcinoma cells by binding HSP90 and disrupting the interaction between GSK3β and HSP90.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown and overexpression in HCC cells, western blotting, in vivo mouse metastasis model\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP showing DIAPH3-HSP90 interaction with functional consequence on GSK3β-HSP90 complex, single lab, limited mechanistic depth\",\n      \"pmids\": [\"28795316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DIAPH3 was identified as a binding protein of STK38 (NDR kinase); DIAPH3 impairs the interaction between STK38 and MEKK, thereby activating ERK signaling and promoting lung adenocarcinoma growth.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown and overexpression, in vivo nude mouse and de novo mouse tumor models, western blotting for ERK pathway markers\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP identifying binding partner with functional downstream signaling consequence, single lab\",\n      \"pmids\": [\"31586548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DIAPH3 interacts with the selenoprotein RPL6, a ribosome protein subunit involved in selenoamino acid metabolism, and promotes selenium content and TrxR1 (thioredoxin reductase 1) expression, thereby reducing cellular ROS levels in pancreatic cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and siRNA interference in pancreatic cancer cells, ROS measurement, nude mice xenograft model, TrxR1 western blotting\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP identifying RPL6 binding partner, single lab, indirect mechanism for TrxR1 upregulation not fully established\",\n      \"pmids\": [\"33345387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of DIAPH3 (Diaph3 knockout) causes cytokinesis failure specifically at high temperature (39°C) but not at 32°C in mouse FM3A cells. This phenotype was rescued by re-expression of Diaph3 WT but not by Diaph1 or Diaph2. Diaph3 knockout cells at 39°C also showed significantly increased levels of acetylated α-tubulin (indicating stabilized microtubules), which was reduced by Diaph3 re-expression.\",\n      \"method\": \"Exome sequencing of temperature-sensitive mutants, Diaph3 knockout cells, rescue with Diaph1/Diaph2/Diaph3 re-expression, acetylated α-tubulin immunofluorescence and western blotting\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO plus isoform-specific rescue experiments with defined cytokinesis phenotype and molecular readout, single lab\",\n      \"pmids\": [\"33187357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DIAPH3 localizes to the centrosome during mitosis and regulates assembly and bipolarity of the mitotic spindle; DIAPH3-deficient cells display multipolar spindles and disorganized cytoskeleton. DIAPH3 deficiency disrupts expression/stability of the kinetochore-associated protein SPAG5; knockdown of SPAG5 phenocopies DIAPH3 deficiency, and SPAG5 overexpression rescues DIAPH3 knockdown, placing SPAG5 downstream of DIAPH3 in spindle assembly.\",\n      \"method\": \"Immunofluorescence localization to centrosome, DIAPH3 conditional knockout in mouse cerebral cortex, siRNA knockdown, SPAG5 overexpression rescue experiments, live imaging, behavioral testing of mice\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — centrosomal localization combined with genetic epistasis (knockdown phenocopy + overexpression rescue), conditional mouse KO with brain phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"33899739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Under cellular stress conditions, DIAPH3 undergoes liquid-liquid phase separation (LLPS) to form cytosolic condensates (D-granules) that spatially sequester DIAPH3, thereby inhibiting actin filament assembly in filopodia. D-granules are distinct from stress granules and P-bodies (lacking G3BP1, G3BP2, TIA1, and DCP1A markers). This was demonstrated using overexpression, knockout, pharmacological interventions, and optogenetics.\",\n      \"method\": \"LLPS characterization (fluorescence recovery after photobleaching, droplet fusion assay), DIAPH3 overexpression and knockout, optogenetic induction of DIAPH3 condensation, pharmacological stress induction, marker colocalization studies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — LLPS reconstitution with biophysical validation, optogenetics, and KO/OE experiments with defined actin phenotype, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"36640348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The lncRNA LINC01089 interacts with hnRNPM to cause hnRNPM-mediated skipping of DIAPH3 exon 3; inclusion of exon 3 contains an m6A modification site recognized by IGF2BP3 that stabilizes DIAPH3 mRNA. Knockdown of LINC01089 increased DIAPH3 protein levels, which suppressed ERK/Elk1/Snail signaling and inhibited EMT.\",\n      \"method\": \"RNA immunoprecipitation, alternative splicing analysis, m6A-seq, IGF2BP3 binding assays, siRNA knockdown, mRNA stability assays, in vivo HCC metastasis model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal RNA biology methods establishing the splicing-m6A-stability axis controlling DIAPH3 levels, single lab\",\n      \"pmids\": [\"37756562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DIAPH3 β-actin networks (but not γ-actin networks generated by DIAPH1) are required for maintaining non-muscle myosin II and RhoA at the cytokinetic furrow. Expression of hybrid DIAPH1/DIAPH3 proteins with altered actin isoform specificity relocalized actin isoform networks and caused cytokinetic failure, demonstrating non-redundant, specialized roles for β- and γ-actin in cytokinesis.\",\n      \"method\": \"Expression of hybrid DIAPH1/DIAPH3 chimeric proteins with altered actin isoform specificity, immunofluorescence of myosin II and RhoA localization at cytokinetic furrow, live-cell imaging of cytokinesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — chimeric protein approach with defined functional rescue, mechanistic epistasis placing β-actin network upstream of RhoA/myosin II maintenance, rigorous domain-swap strategy\",\n      \"pmids\": [\"38897998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The E3 ubiquitin ligase Stub1 ubiquitinates activated DIAPH3 at K1142/1143/1144 lysine residues and promotes its proteasomal degradation. DID-DAD intramolecular interaction stabilizes DIAPH3 against degradation; disruption of DID-DAD by RhoA binding or M1041A mutation activates DIAPH3 but simultaneously triggers accelerated Stub1-mediated ubiquitination and degradation. FH2-FH2 interaction promotes activity while DD-DD interaction inhibits it. Knockdown of Stub1 in mouse cochlea causes hair cell stereocilia defects and hearing loss resembling DIAPH3 overexpression phenotype.\",\n      \"method\": \"In vitro ubiquitination assay, site-directed mutagenesis (M1041A, K1142/1143/1144R), Co-IP of domain interactions, RhoA binding experiments, proteasome inhibitor (MG132) treatment, Stub1 knockdown in mouse cochlea, auditory phenotyping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination reconstitution combined with mutagenesis identifying specific ubiquitination sites and autoinhibitory domain interactions, validated in vivo in cochlea\",\n      \"pmids\": [\"39322015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DIAPH3 interacts with FOXM1 (forkhead box M1) transcription factor, as determined by Co-IP; loss of DIAPH3 downregulates FOXM1 and blocks Wnt/β-catenin signaling in anaplastic thyroid carcinoma cells. FOXM1 overexpression rescues the anti-proliferative, pro-apoptotic effects of DIAPH3 depletion.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, FOXM1 overexpression rescue, western blotting for Wnt/β-catenin pathway markers, cell proliferation/migration/invasion assays\",\n      \"journal\": \"Endokrynologia Polska\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with functional rescue experiment, single lab, limited mechanistic depth for the DIAPH3-FOXM1 interaction\",\n      \"pmids\": [\"39376176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DIAPH3 reduces RPL6 protein levels by disrupting the interaction between RPL6 and the deubiquitinase OTUD4, thereby reducing RPL6-mediated activation of cGAS-STING signaling in pancreatic cancer cells.\",\n      \"method\": \"Co-immunoprecipitation of RPL6-OTUD4 interaction, forced expression of RPL6, DIAPH3 overexpression experiments, interferon-β production measurement\",\n      \"journal\": \"European journal of medical research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP-based interaction mapping with functional readout, single lab, limited mechanistic validation of the DIAPH3-OTUD4-RPL6 axis\",\n      \"pmids\": [\"41318619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DIAPH3 silencing suppresses migration and invasion of colorectal cancer cells; mass spectrometry identified KRT19 as a downstream target of DIAPH3. Knockdown of DIAPH3 reduces KRT19 protein levels through proteasome-dependent degradation (blocked by MG132), and KRT19 overexpression rescues the invasion defect caused by DIAPH3 silencing, establishing a DIAPH3-KRT19 signaling axis.\",\n      \"method\": \"Mass spectrometry identification of downstream targets, siRNA knockdown, KRT19 overexpression rescue, proteasome inhibitor (MG132) treatment, wound healing and Transwell invasion assays, xenograft metastatic model\",\n      \"journal\": \"Clinical & experimental metastasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — MS-based target identification with rescue experiment and proteasomal mechanism, single lab\",\n      \"pmids\": [\"39843730\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DIAPH3 is a Diaphanous-related formin that functions as an actin nucleator/elongator downstream of Rho GTPases (RhoA-C and Cdc42), regulated by autoinhibitory DID-DAD intramolecular interaction and subject to Stub1-mediated ubiquitination and proteasomal degradation when activated; it localizes to the centrosome during mitosis, maintains spindle bipolarity and spindle assembly checkpoint activity (via BubR1 and SPAG5), specifically nucleates β-actin networks required for RhoA and myosin II retention at the cytokinetic furrow, stabilizes microtubule dynamics to regulate amoeboid-to-mesenchymal transition, and forms stress-induced phase-separated condensates (D-granules) that sequester DIAPH3 to inhibit filopodial actin assembly, with overexpression causing inner hair cell microtubule meshwork disruption and auditory neuropathy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DIAPH3 (Drf3/mDia2) is a Diaphanous-related formin that nucleates and elongates actin filaments downstream of Rho-family GTPases to drive membrane protrusion, mitotic spindle integrity, and cytokinesis [#0, #4, #16]. It acts as a Cdc42 effector: a CRIB-like motif in its GTPase-binding domain mediates Cdc42-dependent recruitment to the leading edge and the MTOC, where it promotes filopodia formation [#0]. Its activity is governed by autoinhibition — full-length DIAPH3 is cytosolic and inert, whereas deletion of the C-terminal DAD region or RhoA-mediated disruption of the DID–DAD interaction yields a constitutively active protein that nucleates de novo actin filaments at filopodial tips [#1, #17]; the same activating events that relieve autoinhibition simultaneously expose DIAPH3 to Stub1-mediated ubiquitination at K1142/1143/1144 and proteasomal degradation, coupling activation to turnover [#17]. Isoform identity dictates protrusion outcome, with distinct isoforms driving membrane blebbing versus filopodia [#4]. Beyond actin, DIAPH3 binds and stabilizes microtubule dynamics, and its loss destabilizes microtubules, perturbs endocytic EGFR trafficking, and hyperactivates EGFR/MEK/ERK signaling to promote amoeboid invasion and metastasis [#5, #6]. During mitosis DIAPH3 localizes to the centrosome and maintains spindle bipolarity and the spindle assembly checkpoint through BubR1 and SPAG5, with deficiency causing aneuploidy and microcephaly [#7, #13]. In cytokinesis it specifically generates β-actin networks required to retain RhoA and non-muscle myosin II at the furrow, a role non-redundant with the γ-actin networks made by DIAPH1 [#16, #12]. Under stress, DIAPH3 undergoes liquid–liquid phase separation into D-granules that sequester it and inhibit filopodial actin assembly [#14]. Overexpression of DIAPH3, including via a 5'UTR mutation that elevates transcription, causes auditory neuropathy in humans and mislocalizes the microtubule meshwork in inner hair cells, establishing excess formin activity as causally sufficient for hearing loss [#2, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established DIAPH3 as a Cdc42 effector, answering how it is recruited and activated to remodel the actin cytoskeleton.\",\n      \"evidence\": \"FRET binding analysis, dominant-negative microinjection, and gene targeting in migrating fibroblasts\",\n      \"pmids\": [\"12676083\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how RhoA versus Cdc42 differentially activate the formin\", \"Structural basis of CRIB-like motif engagement not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the autoinhibitory logic, showing the DAD region restrains actin nucleation and that its removal converts DIAPH3 into a de novo filopodial actin nucleator.\",\n      \"evidence\": \"Gain-of-function expression of full-length vs DAD-truncated constructs with live-cell and electron microscopy\",\n      \"pmids\": [\"18755006\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological signal relieving autoinhibition not identified in this study\", \"In vitro nucleation kinetics not measured\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linked DIAPH3 dosage to human disease, showing overexpression is causally sufficient for auditory neuropathy.\",\n      \"evidence\": \"Luciferase reporter of a 5'UTR mutation, quantitative immunoblotting of patient cells, and constitutively active diaphanous in Drosophila auditory organ; plus mutagenesis showing Pro614Thr reduces filopodia\",\n      \"pmids\": [\"20624953\", \"20308993\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular target tissue of overexpression not yet defined in 2010\", \"Mechanism connecting formin activity to hair cell dysfunction unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed a microtubule- and trafficking-based function, showing DIAPH3 loss destabilizes microtubules and hyperactivates EGFR/MEK/ERK to drive amoeboid invasion.\",\n      \"evidence\": \"siRNA knockdown in carcinoma cells with EGFR trafficking imaging, in vivo metastasis, and pharmacological pathway placement\",\n      \"pmids\": [\"22593025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular link between DIAPH3 and microtubule stabilization not yet shown\", \"How a formin restrains EGFR endosomal trafficking unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated direct microtubule interaction, connecting DIAPH3 to mechanosensing, force generation, and taxane sensitivity.\",\n      \"evidence\": \"MT co-localization/interaction assays, silencing with live MT-dynamics imaging and atomic force microscopy in cancer lines\",\n      \"pmids\": [\"26179371\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface on microtubules not mapped\", \"Whether MT effects are independent of actin nucleation unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed DIAPH3 in mitotic fidelity control, showing it sustains the spindle assembly checkpoint via BubR1 to prevent aneuploidy and microcephaly.\",\n      \"evidence\": \"Co-IP from embryonic brain, conditional Diaph3 knockout mice, and mitotic imaging; plus gain-of-function transgenic mice mislocalizing the hair cell MT meshwork\",\n      \"pmids\": [\"27848932\", \"28058271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DIAPH3-BubR1 interaction is direct not established\", \"How a centrosomal formin regulates checkpoint protein stability unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped the centrosomal spindle role, placing SPAG5 downstream of DIAPH3 in maintaining spindle bipolarity.\",\n      \"evidence\": \"Centrosomal localization, conditional cortical knockout, and SPAG5 knockdown-phenocopy plus overexpression-rescue epistasis\",\n      \"pmids\": [\"33899739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which DIAPH3 controls SPAG5 stability unresolved\", \"Relationship between centrosomal actin and spindle bipolarity not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved actin-isoform specificity in cytokinesis, showing DIAPH3-generated β-actin networks uniquely retain RhoA and myosin II at the furrow.\",\n      \"evidence\": \"DIAPH1/DIAPH3 chimeric domain-swap proteins with furrow imaging and live cytokinesis assays; temperature-sensitive KO with isoform-specific rescue\",\n      \"pmids\": [\"38897998\", \"33187357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of β- vs γ-actin discrimination by the FH2 domain not fully defined\", \"How β-actin networks physically retain RhoA unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the activation–degradation coupling, showing Stub1 ubiquitinates activated DIAPH3 and DID-DAD interaction protects it from turnover.\",\n      \"evidence\": \"In vitro ubiquitination, mutagenesis of ubiquitination and autoinhibition residues, domain-interaction Co-IP, and Stub1 knockdown in cochlea\",\n      \"pmids\": [\"39322015\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling Stub1 recruitment not identified\", \"Quantitative kinetics of activation versus degradation not measured\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified a stress-responsive regulatory mode, showing DIAPH3 phase-separates into D-granules that sequester it to suppress filopodial actin.\",\n      \"evidence\": \"FRAP, droplet fusion, optogenetic condensation, KO/OE, and marker colocalization distinguishing D-granules from stress granules and P-bodies\",\n      \"pmids\": [\"36640348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sequence determinants driving DIAPH3 LLPS not mapped\", \"Physiological stresses triggering D-granules in vivo not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended DIAPH3 into multiple cancer signaling axes through reported protein interactions controlling proliferation, EMT, redox, and innate immune signaling.\",\n      \"evidence\": \"Co-IP and rescue studies implicating HSP90/GSK3β, STK38/ERK, FOXM1/Wnt, RPL6/OTUD4/cGAS-STING, and KRT19; plus LINC01089-hnRNPM-m6A control of DIAPH3 mRNA\",\n      \"pmids\": [\"28795316\", \"31586548\", \"39376176\", \"33345387\", \"41318619\", \"39843730\", \"37756562\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Most interactions rest on single Co-IPs without reciprocal or direct-binding validation\", \"Whether these signaling roles depend on actin/microtubule activity unclear\", \"Mechanistic depth of the RPL6/OTUD4/cGAS-STING axis limited\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DIAPH3's distinct activities — actin nucleation, microtubule regulation, spindle/checkpoint control, and stress-induced phase separation — are coordinated by a single regulatory state remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model linking RhoA/Cdc42 input, Stub1 turnover, and LLPS to specific cellular outputs\", \"Structural basis of actin-isoform and microtubule selectivity undetermined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 4, 16, 6]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 13]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [6, 16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 13, 16, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 0]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [7, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"BubR1\", \"SPAG5\", \"STUB1\", \"RhoA\", \"Cdc42\", \"HSP90\", \"STK38\", \"FOXM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}