{"gene":"CARMIL1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2001,"finding":"Dictyostelium CARMIL (p116) physically links type I myosins (myoB, myoC), capping protein (CP alpha/beta), and the Arp2/3 complex into a single in vivo complex: myosins bind CARMIL through their SH3 domains, and CP and Arp2/3 complex bind to CARMIL's N-terminal region; a region containing an acidic stretch activates Arp2/3-dependent actin nucleation. Cells lacking p116 show defective macropinocytosis, reduced pinocytosis, impaired chemotaxis, and decreased F-actin content.","method":"SH3-domain pulldown, co-immunoprecipitation, fusion-protein binding assays, Arp2/3 nucleation assay, p116-null cell phenotypic analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal biochemical methods plus genetic null phenotype, foundational paper with 146 citations","pmids":["11425877"],"is_preprint":false},{"year":2003,"finding":"Acanthamoeba CARMIL binds capping protein (CP) with a Kd of ~0.4 µM via its C-terminal ~200-residue proline-rich domain; CARMIL self-associates in a monomer-dimer equilibrium (Ka ~1×10^6 M^-1) and is asymmetric, as shown by analytical ultracentrifugation and rotary-shadow EM.","method":"Co-purification, chemical cross-linking, analytical ultracentrifugation, surface plasmon resonance, SH3-domain pulldown, rotary-shadow electron microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple quantitative biophysical methods in a single study","pmids":["14594951"],"is_preprint":false},{"year":2005,"finding":"Mammalian CARMIL (mCARMIL/CARMIL1) binds CP with high affinity and reduces CP's affinity for actin filament barbed ends (anti-capping activity); addition to cell extracts enhances Arp2/3- and spectrin-actin-seed-induced polymerization. In cells, mCARMIL concentrates in lamellipodia; siRNA knockdown decreases F-actin and slows cell migration via reduced lamellipodial protrusion, a phenotype rescued by full-length but not CP-binding-deficient mCARMIL.","method":"In vitro barbed-end capping assay, actin polymerization assay with cell extracts, GFP localization, siRNA knockdown with rescue","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro assay plus loss-of-function with domain-specific rescue, 109 citations","pmids":["16054028"],"is_preprint":false},{"year":2006,"finding":"The conserved C-terminal CAH3 domain of CARMIL is necessary and sufficient for potent anti-capping protein activity: it inhibits CP's barbed-end capping activity and drives uncapping of CP-capped filaments. Point mutations in conserved CAH3 residues abolish these activities. Full-length CARMIL is partially autoinhibited relative to C-terminal fragments.","method":"In vitro actin polymerization/uncapping assays, GST-fusion protein binding, site-directed mutagenesis, proteolytic cleavage","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus mutagenesis with multiple domain deletions","pmids":["16434392"],"is_preprint":false},{"year":2009,"finding":"CARMIL1 localizes to lamellipodia and macropinosomes; CARMIL1 knockdown causes loss of lamellipodial actin, defects in protrusion/ruffling/macropinocytosis, and loss of Rac1 activation. CARMIL1 co-immunoprecipitates with the dual-GEF Trio. CARMIL2 colocalizes with vimentin intermediate filaments and loss decreases myosin-IIB levels, causing a multipolar phenotype. The two isoforms have distinct, non-redundant functions.","method":"siRNA knockdown, GFP localization, co-immunoprecipitation with Trio, Rac1 activation assay, rescue experiments","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function with defined cellular phenotypes and biochemical interaction, 61 citations","pmids":["19846667"],"is_preprint":false},{"year":2009,"finding":"C. elegans CARMIL ortholog (CRML-1) negatively regulates neuronal cell and axon growth cone migration by inhibiting the Rac GEF activity of UNC-73/Trio; CRML-1 and UNC-73 form a complex in vivo, and the antagonism between them controls SAX-3/Robo guidance receptor levels.","method":"Genetic epistasis (suppressor screens, double mutants), co-immunoprecipitation in vivo","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis plus in vivo co-IP in C. elegans ortholog","pmids":["19244282"],"is_preprint":false},{"year":2009,"finding":"CARMIL contains unstructured membrane-binding sites with basic and hydrophobic amino acid character; synthetic peptides and protein domains containing these sites bind acidic phospholipids in vitro.","method":"Peptide synthesis, lipid vesicle binding assay, computational hydrophobicity analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct lipid-binding assay but with synthetic peptides and computational prediction","pmids":["20018884"],"is_preprint":false},{"year":2009,"finding":"Direct single-molecule TIRF microscopy demonstrates that the isolated CAH3 domain of mouse CARMIL-1 (mCAH3) physically removes mGFP-tagged CP from individual capped actin filament barbed ends (uncapping), reducing CP half-life at the barbed end from ~30 min to ~10 s at saturating mCAH3; the CP:CAH3 complex retains weak barbed-end affinity.","method":"Total internal reflection fluorescence microscopy (TIRF/TIRFM), single-molecule imaging, mGFP-tagged CP","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct single-molecule visualization of uncapping mechanism","pmids":["19926785"],"is_preprint":false},{"year":2010,"finding":"NMR structural analysis shows the highly basic CAH3a subdomain of mouse CARMIL-1 binds an 'acidic groove' on CP opposite its actin-binding surface, orienting CAH3b adjacent to the basic patch of CP required for barbed-end interaction; mutagenesis of both proteins confirmed specific residue contacts and provided a mechanistic explanation for uncapping.","method":"NMR (solution structure), site-directed mutagenesis, actin polymerization/uncapping assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structural data plus mutagenesis validation","pmids":["20630878"],"is_preprint":false},{"year":2012,"finding":"CARMIL inhibits CP via an allosteric mechanism: molecular dynamics shows CBR binding induces conformational changes in CP's actin-binding surface; a steric-blocking model was ruled out because CP actin-binding mutants still bind CBR normally. The CARMIL-specific interaction (CSI) motif in the CP-binding region is required for high-affinity binding and uncapping.","method":"Mutagenesis of CP actin-binding surface, CP-CBR binding assays, molecular dynamics simulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis plus molecular dynamics, ruling out alternative model","pmids":["22411988"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of CARMIL1-668 reveals an N-terminal non-canonical pleckstrin homology (PH) domain connected to a 16-leucine-rich repeat (LRR) domain; small-angle X-ray scattering shows the central helical domain mediates antiparallel dimerization, positioning PH domains for simultaneous membrane interaction. Deletion of the PH domain in cells impairs leading-edge localization.","method":"X-ray crystallography, SAXS, lipid-binding assays, GFP cell imaging with deletion mutants","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus SAXS plus functional cell imaging validation","pmids":["24071777"],"is_preprint":false},{"year":2013,"finding":"The CARMIL1-CP interaction is required for lamellipodia assembly, ruffle formation, and macropinocytosis, but is dispensable for CARMIL1 membrane localization, Rac1 activation, and wound-healing cell migration, as demonstrated by a point mutant (CARMIL1-AA) that specifically disrupts CP binding.","method":"Point mutagenesis, siRNA knockdown with rescue, Rac1 activation assay, phalloidin staining, live-cell imaging","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — domain-specific point mutant rescue with multiple orthogonal phenotypic readouts","pmids":["23904264"],"is_preprint":false},{"year":2014,"finding":"CARMIL retrieves CP from the inactive CP:V-1 complex via complex exchange, generating a CP:CARMIL complex with moderate (weak) barbed-end capping activity; CARMIL is recruited only to the plasma membrane at actively protruding cell edges, suggesting a spatially restricted regulatory cycle where V-1-sequestered CP is converted to weakly capping CP:CARMIL complexes specifically at protrusion sites to promote Arp2/3-dependent actin network assembly.","method":"In vitro actin polymerization assays, stopped-flow kinetics, quantitative analytical ultracentrifugation, live-cell TIRF imaging of CARMIL recruitment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — quantitative in vitro assays plus live-cell imaging, 61 citations","pmids":["24778263"],"is_preprint":false},{"year":2018,"finding":"Hydrogen-deuterium exchange mass spectrometry (HDX-MS) shows that both V-1 and CARMIL binding to CP induce changes in structural dynamics at their respective binding sites on CP and at the CP ββ subunit 'tentacle', a second distal actin-binding site, demonstrating allosteric coupling between CP modulator and actin binding sites.","method":"Hydrogen-deuterium exchange mass spectrometry (HDX-MS)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 — HDX-MS structural dynamics providing mechanistic allosteric model","pmids":["29847807"],"is_preprint":false},{"year":2019,"finding":"The leucine-rich repeat (LRR) region of CARMIL1 associates with IL-1 receptor type 1 (IL-1R1) and IL-1 receptor-associated kinase (IRAK), as shown by co-immunoprecipitation with CARMIL1 LRR-deletion mutants. CARMIL1 knockout (CRISPR-Cas9) reduces IL-1-induced ERK activation by 72% and MMP3 expression by 40%; a cell-permeable TAT-CARMIL1 LRR peptide reduces collagen degradation by 43%.","method":"Tandem mass tag mass spectrometry, co-immunoprecipitation with domain mutants, CRISPR-Cas9 knockout, TAT-peptide competition, ERK phosphorylation assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (MS, Co-IP, KO, peptide competition) with defined signaling phenotypes","pmids":["32610117"],"is_preprint":false},{"year":2022,"finding":"Dictyostelium CARMIL-GAP, a CARMIL isoform with a GAP domain insert, binds Dictyostelium Rac1a and accelerates its GTP hydrolysis rate; CARMIL-GAP-null cells show defects in phagocytosis and chemotactic streaming rescued by full-length but not GAP-activity-deficient or CP-regulation-deficient versions, demonstrating that both GAP and CP-regulatory activities are required.","method":"GTPase assay (in vitro), genetic null cells, rescue with domain mutants, fluorescence localization","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro GTPase assay plus genetic null rescue with domain mutants","pmids":["35583107"],"is_preprint":false},{"year":2024,"finding":"CARMIL at a membrane surface (reconstituted with lipid-coated beads) promotes and enhances Arp2/3-nucleated actin assembly by counteracting V-1 inhibition of CP, effectively activating CP at the membrane surface for Arp2/3-mediated assembly; this reconstitution used purified Arp2/3 activator, Arp2/3 complex, V-1, CP, profilin, and actin.","method":"In vitro reconstitution with purified proteins on lipid-coated beads, pyrene-actin polymerization assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 — full reconstitution with purified components","pmids":["39437783"],"is_preprint":false},{"year":2024,"finding":"CARMIL1-AA (CP-binding mutant) selectively inhibits macropinocytosis without affecting proliferation, Rac1 activation, or autophagy, confirming that the CARMIL1-CP interaction is specifically required for macropinocytosis but not these other cellular processes.","method":"CRISPR-Cas9 knockout, expression of CARMIL1-AA point mutant, macropinocytosis assay, autophagy assay, Rac1 activation assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — domain-specific mutant with multiple orthogonal phenotypic readouts","pmids":["39602282"],"is_preprint":false},{"year":2024,"finding":"CARMIL1 binds TRIM27 (co-immunoprecipitation), which in turn binds p53, and CARMIL1 promotes p53 degradation through TRIM27; CARMIL1 knockdown reduces ERK and mTOR phosphorylation in liver cancer cells.","method":"Co-immunoprecipitation, western blotting, siRNA knockdown, subcutaneous tumor model","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 3 — single co-IP plus knockdown phenotype, single lab","pmids":["38739978"],"is_preprint":false},{"year":2026,"finding":"Cryo-EM/crystal structure of CP-bound CARMIL1(1-1046) reveals a dimeric assembly with PH-LRR on a plane flanked by the helical dimerization domain and CBR-bound CP; an 'antenna' motif connecting the helical domain to CBR-CP contacts the LRR-LRR dimer interface and mediates partial autoinhibition. Disruption of the antenna partially relieves autoinhibition in vitro and increases cell area in knockout cells; deletion of the proline-rich (myosin-I-binding) domain induces membrane spikes.","method":"Crystal structure, in vitro uncapping assay, CARMIL1 knockout rescue with mutants, cell area imaging","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 — structural determination plus mutagenesis with in vitro and cell-based validation","pmids":["41861019"],"is_preprint":false},{"year":2026,"finding":"The membrane-binding (MB) domain of CARMIL targets the CPI and CSI motifs to lipid membranes (lipid-coated beads), enabling activation of CP for Arp2/3-mediated actin assembly; upon CP binding, the MB domain can dissociate from the membrane, enhancing uncapping of capped barbed ends and further activating soluble CP beyond what is seen in the membrane-attached state.","method":"In vitro reconstitution with lipid-coated beads, pyrene-actin polymerization assay, domain deletion mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with defined domain mutants revealing dual membrane release mechanism","pmids":["42031174"],"is_preprint":false}],"current_model":"CARMIL1 is a large multidomain scaffold that localizes to the plasma membrane at the leading edge via a non-canonical PH domain and dimerizes via a helical domain; there it recruits and allosterically inhibits capping protein (CP) through its CAH3/CBR domain (containing CPI and CSI motifs), directly uncapping CP-capped actin filament barbed ends and converting V-1-sequestered inactive CP into weakly capping CP:CARMIL complexes that promote Arp2/3-dependent actin network assembly, while its LRR domain additionally engages IL-1R1/IRAK for inflammatory ERK signaling and Rac GEF Trio for Rac1 activation, collectively driving lamellipodia formation, macropinocytosis, and cell migration."},"narrative":{"teleology":[{"year":2001,"claim":"Identification of CARMIL as a physical hub linking capping protein, Arp2/3 complex, and type I myosins established that a single scaffold coordinates barbed-end capping, actin nucleation, and motor-driven membrane dynamics, and that its loss impairs macropinocytosis and chemotaxis.","evidence":"SH3-domain pulldown, co-IP, Arp2/3 nucleation assay, and p116-null Dictyostelium phenotypic analysis","pmids":["11425877"],"confidence":"High","gaps":["Mammalian ortholog function unknown","Direct binding affinities not measured","Mechanism of CP regulation (inhibition vs. uncapping) not resolved"]},{"year":2003,"claim":"Quantitative biophysical characterization revealed that CARMIL self-associates as a monomer-dimer equilibrium and binds CP with submicromolar affinity through a C-terminal proline-rich region, establishing the basic architecture and stoichiometry of the CARMIL-CP complex.","evidence":"Analytical ultracentrifugation, surface plasmon resonance, rotary-shadow EM of Acanthamoeba CARMIL","pmids":["14594951"],"confidence":"High","gaps":["Binding mechanism (steric vs. allosteric) unresolved","Mammalian CARMIL not yet characterized"]},{"year":2005,"claim":"Transfer of CARMIL function to mammalian cells showed that CARMIL1 concentrates in lamellipodia, anti-caps CP to promote barbed-end polymerization, and is required for lamellipodial protrusion and cell migration—establishing its conserved role in mammalian cell motility.","evidence":"In vitro barbed-end capping assay, siRNA knockdown with full-length vs. CP-binding-deficient rescue in cultured cells","pmids":["16054028"],"confidence":"High","gaps":["Uncapping activity not directly demonstrated","Membrane-targeting mechanism unknown","Rac1 connection not yet established"]},{"year":2006,"claim":"Mapping the minimal anti-capping and uncapping activity to the conserved CAH3 domain, and showing that full-length CARMIL is partially autoinhibited, defined the functional module within the multidomain protein and introduced the concept of intramolecular regulation.","evidence":"In vitro reconstituted capping/uncapping assays with GST-fusion domain deletions and point mutants","pmids":["16434392"],"confidence":"High","gaps":["Structural basis of autoinhibition unknown","Mechanism of uncapping (steric vs. allosteric) unresolved"]},{"year":2009,"claim":"Multiple advances resolved CARMIL1's uncapping mechanism, membrane association, and signaling connections: single-molecule TIRF showed CAH3 physically removes CP from barbed ends reducing its dwell time from ~30 min to ~10 s; lipid-binding sites were mapped; and CARMIL1 was linked to Rac1 activation via Trio, while C. elegans genetics placed CARMIL upstream of Trio in guidance signaling.","evidence":"Single-molecule TIRF of mGFP-CP uncapping; peptide-lipid vesicle binding; siRNA knockdown with Rac1 pull-down and co-IP of Trio; C. elegans genetic epistasis and in vivo co-IP","pmids":["19926785","20018884","19846667","19244282"],"confidence":"High","gaps":["Atomic-resolution structure of full-length CARMIL unavailable","Whether CARMIL activates or inhibits Trio GEF activity unclear across species","Lipid specificity and PH domain structure not resolved"]},{"year":2010,"claim":"NMR structural analysis of the CAH3-CP interface revealed that CAH3a binds an acidic groove on CP opposite its actin-binding surface and positions CAH3b near CP's basic patch, providing the first atomic model explaining how CARMIL disrupts CP-barbed-end interaction without occupying the actin-binding site.","evidence":"NMR solution structure of CAH3-CP complex with confirmatory mutagenesis","pmids":["20630878"],"confidence":"High","gaps":["Whether uncapping proceeds by allosteric or steric mechanism still debated","Full-length structural context missing"]},{"year":2012,"claim":"Ruling out steric blockade and demonstrating that CBR binding induces conformational changes in CP's actin-binding surface established that CARMIL inhibits and uncaps CP via an allosteric mechanism; the CSI motif was identified as essential for high-affinity binding and uncapping.","evidence":"CP actin-binding surface mutants retaining CBR binding; molecular dynamics simulations","pmids":["22411988"],"confidence":"High","gaps":["Experimental demonstration of allosteric conformational change (e.g., HDX-MS) not yet performed","Role of CSI motif in vivo not tested"]},{"year":2013,"claim":"Crystal structure of CARMIL1 N-terminal half revealed a non-canonical PH domain and 16-repeat LRR domain; SAXS showed the helical domain mediates antiparallel dimerization positioning PH domains for membrane binding. Separately, a CP-binding point mutant (CARMIL1-AA) dissected CP-dependent (lamellipodia, macropinocytosis) from CP-independent (membrane localization, Rac1 activation, wound migration) functions.","evidence":"X-ray crystallography, SAXS, GFP imaging with PH-deletion mutants; siRNA rescue with CARMIL1-AA point mutant","pmids":["24071777","23904264"],"confidence":"High","gaps":["Structure of C-terminal CBR-containing region unresolved","Identity of LRR-binding partners unknown","Autoinhibition mechanism structurally undefined"]},{"year":2014,"claim":"Demonstrating that CARMIL retrieves CP from the inhibitory CP:V-1 complex to generate weakly capping CP:CARMIL complexes at protrusion sites established a spatially restricted regulatory cycle explaining how inactive cytoplasmic CP is converted to a functional form specifically at the leading edge to support Arp2/3-dependent actin assembly.","evidence":"Stopped-flow kinetics, analytical ultracentrifugation, in vitro actin polymerization, live-cell TIRF of CARMIL recruitment","pmids":["24778263"],"confidence":"High","gaps":["Reconstitution with all components on a membrane not yet achieved","In vivo concentrations and fluxes of CP, V-1, and CARMIL not measured"]},{"year":2018,"claim":"HDX-MS experimentally confirmed allosteric coupling between the CARMIL-binding site and CP's distal actin-binding β-tentacle, providing direct biophysical evidence for the long-range conformational changes underlying CARMIL-mediated uncapping.","evidence":"Hydrogen-deuterium exchange mass spectrometry of CP alone and in complex with CARMIL peptide and V-1","pmids":["29847807"],"confidence":"High","gaps":["Full-length CARMIL-CP structural model still lacking","Dynamics of allosteric change at single-molecule resolution not captured"]},{"year":2019,"claim":"Discovery that the LRR domain of CARMIL1 associates with IL-1R1 and IRAK and is required for IL-1-induced ERK activation and MMP3 expression revealed a signaling function for CARMIL1 beyond actin regulation, linking it to inflammatory pathways.","evidence":"Tandem mass tag MS, co-IP with LRR-deletion mutants, CRISPR-Cas9 knockout, TAT-peptide competition in chondrocytes","pmids":["32610117"],"confidence":"High","gaps":["Whether CARMIL1 is a direct scaffold vs. indirect adapter for IRAK unclear","Relevance to in vivo inflammatory disease not tested"]},{"year":2022,"claim":"Identification of a Dictyostelium CARMIL isoform with intrinsic Rac GAP activity demonstrated that CARMIL proteins can directly regulate Rho-family GTPases, and that both GAP and CP-regulatory activities are independently required for phagocytosis and chemotaxis.","evidence":"In vitro GTPase assay, genetic null rescue with GAP-dead and CP-regulation-deficient mutants in Dictyostelium","pmids":["35583107"],"confidence":"High","gaps":["Mammalian CARMIL1 lacks a GAP domain; whether it regulates Rac through an analogous direct mechanism is unknown","Structural basis of GAP domain insertion not resolved"]},{"year":2024,"claim":"Full reconstitution on lipid-coated beads with purified Arp2/3 activator, Arp2/3, V-1, CP, profilin, and CARMIL demonstrated that membrane-anchored CARMIL counteracts V-1 inhibition and activates CP for Arp2/3-nucleated actin assembly, and that CP-binding by CARMIL1 is specifically required for macropinocytosis but dispensable for proliferation and autophagy.","evidence":"Reconstitution on lipid-coated beads with pyrene-actin assay; CRISPR-Cas9 KO cells with CARMIL1-AA rescue and macropinocytosis/autophagy assays","pmids":["39437783","39602282"],"confidence":"High","gaps":["Reconstitution lacks Trio/Rac module","Contribution of CARMIL membrane release to in vivo dynamics not tested"]},{"year":2026,"claim":"Cryo-EM/crystal structures of dimeric CP-bound CARMIL1(1–1046) revealed the full-length architecture including an 'antenna' motif mediating autoinhibition at the LRR dimer interface, and reconstitution showed that the membrane-binding domain targets CPI/CSI motifs to membranes and that upon CP binding, CARMIL can release from the membrane to enhance cytoplasmic uncapping—establishing a dual-mode mechanism.","evidence":"Crystal structure of dimeric CARMIL1-CP complex; mutagenesis of antenna with in vitro uncapping and cell area assays; lipid-coated bead reconstitution with MB domain deletions","pmids":["41861019","42031174"],"confidence":"High","gaps":["Dynamic structural transitions during membrane release not captured in real time","How autoinhibition is relieved by upstream signals in vivo remains unclear","Structure of the proline-rich/myosin-I-binding C-terminal tail still missing"]},{"year":null,"claim":"Key open questions include: how CARMIL1 autoinhibition is relieved by physiological signals; how LRR-mediated IL-1R1/IRAK and Trio interactions are coordinated with CP regulation at the leading edge; and whether the membrane-release uncapping mode operates in vivo during protrusion cycles.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No upstream activating signal for autoinhibition relief identified","Spatial and temporal coordination of CP-regulatory and signaling functions not resolved in vivo","Complete reconstitution including Trio/Rac module and myosin-I not achieved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,7,9,12,13,16,20]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,2,3,7,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6,10,20]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,4,10,11,12,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,14]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[2,4,11]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[14]}],"complexes":["CARMIL1 homodimer","CP:CARMIL1 complex"],"partners":["CAPZA1","CAPZB","TRIO","ACTR2","IL1R1","IRAK1","TRIM27","MYO1B"],"other_free_text":[]},"mechanistic_narrative":"CARMIL1 is a large multidomain scaffold protein that coordinates actin barbed-end dynamics, membrane protrusion, and signaling at the leading edge of migrating cells. It dimerizes via a central helical domain and localizes to the plasma membrane through a non-canonical PH domain; its C-terminal CAH3/CBR domain allosterically inhibits capping protein (CP) and directly uncaps CP-capped actin filament barbed ends—reducing CP barbed-end half-life from ~30 min to ~10 s—thereby converting V-1-sequestered inactive CP into weakly capping CP:CARMIL complexes that promote Arp2/3-dependent actin network assembly at membrane protrusion sites [PMID:19926785, PMID:24778263, PMID:39437783]. An intramolecular 'antenna' motif connecting the helical domain to the CBR contacts the LRR dimer interface and mediates partial autoinhibition, while the LRR domain independently engages IL-1R1/IRAK to support IL-1-induced ERK signaling and associates with the Rac GEF Trio to activate Rac1, collectively driving lamellipodia formation, macropinocytosis, and cell migration [PMID:41861019, PMID:32610117, PMID:19846667, PMID:39602282]."},"prefetch_data":{"uniprot":{"accession":"Q5VZK9","full_name":"F-actin-uncapping protein LRRC16A","aliases":["CARMIL homolog","Capping protein regulator and myosin 1 linker protein 1","Capping protein, Arp2/3 and myosin-I linker homolog 1","Capping protein, Arp2/3 and myosin-I linker protein 1","Leucine-rich repeat-containing protein 16A"],"length_aa":1371,"mass_kda":151.6,"function":"Cell membrane-cytoskeleton-associated protein that plays a role in the regulation of actin polymerization at the barbed end of actin filaments. Prevents F-actin heterodimeric capping protein (CP) activity at the leading edges of migrating cells, and hence generates uncapped barbed ends and enhances actin polymerization, however, seems unable to nucleate filaments (PubMed:16054028). Plays a role in lamellipodial protrusion formations and cell migration (PubMed:19846667)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton; Cell membrane; Cell projection, lamellipodium","url":"https://www.uniprot.org/uniprotkb/Q5VZK9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CARMIL1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CARMIL1","total_profiled":1310},"omim":[{"mim_id":"609593","title":"CAPPING PROTEIN REGULATOR AND MYOSIN 1 LINKER 1; CARMIL1","url":"https://www.omim.org/entry/609593"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CARMIL1"},"hgnc":{"alias_symbol":["dJ501N12.1","FLJ20048","CARMIL"],"prev_symbol":["LRRC16","LRRC16A"]},"alphafold":{"accession":"Q5VZK9","domains":[{"cath_id":"2.30.29.30","chopping":"11-152","consensus_level":"high","plddt":88.4496,"start":11,"end":152},{"cath_id":"-","chopping":"673-741_756-868","consensus_level":"high","plddt":75.8039,"start":673,"end":868}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VZK9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VZK9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VZK9-F1-predicted_aligned_error_v6.png","plddt_mean":67.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CARMIL1","jax_strain_url":"https://www.jax.org/strain/search?query=CARMIL1"},"sequence":{"accession":"Q5VZK9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5VZK9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5VZK9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VZK9"}},"corpus_meta":[{"pmid":"11425877","id":"PMC_11425877","title":"The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11425877","citation_count":146,"is_preprint":false},{"pmid":"16054028","id":"PMC_16054028","title":"Mammalian CARMIL inhibits actin filament capping by capping protein.","date":"2005","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/16054028","citation_count":109,"is_preprint":false},{"pmid":"19846667","id":"PMC_19846667","title":"Distinct roles for CARMIL isoforms in cell migration.","date":"2009","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/19846667","citation_count":61,"is_preprint":false},{"pmid":"24778263","id":"PMC_24778263","title":"Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges.","date":"2014","source":"Proceedings of the National Academy of Sciences 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role of the interaction between CARMIL1 and capping protein.","date":"2013","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/23904264","citation_count":31,"is_preprint":false},{"pmid":"19244282","id":"PMC_19244282","title":"C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development.","date":"2009","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/19244282","citation_count":31,"is_preprint":false},{"pmid":"20630878","id":"PMC_20630878","title":"Molecular basis for barbed end uncapping by CARMIL homology domain 3 of mouse CARMIL-1.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20630878","citation_count":30,"is_preprint":false},{"pmid":"14594951","id":"PMC_14594951","title":"CARMIL is a bona fide capping protein interactant.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14594951","citation_count":28,"is_preprint":false},{"pmid":"29847807","id":"PMC_29847807","title":"Allosteric Coupling of CARMIL and V-1 Binding to Capping Protein Revealed by Hydrogen-Deuterium Exchange.","date":"2018","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/29847807","citation_count":21,"is_preprint":false},{"pmid":"32610117","id":"PMC_32610117","title":"The Leucine-Rich Repeat Region of CARMIL1 Regulates IL-1-Mediated ERK Activation, MMP Expression, and Collagen Degradation.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32610117","citation_count":17,"is_preprint":false},{"pmid":"21572169","id":"PMC_21572169","title":"Actin capping protein and its inhibitor CARMIL: how intrinsically disordered regions function.","date":"2011","source":"Physical 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Cells lacking p116 show defective macropinocytosis, reduced pinocytosis, impaired chemotaxis, and decreased F-actin content.\",\n      \"method\": \"SH3-domain pulldown, co-immunoprecipitation, fusion-protein binding assays, Arp2/3 nucleation assay, p116-null cell phenotypic analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal biochemical methods plus genetic null phenotype, foundational paper with 146 citations\",\n      \"pmids\": [\"11425877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Acanthamoeba CARMIL binds capping protein (CP) with a Kd of ~0.4 µM via its C-terminal ~200-residue proline-rich domain; CARMIL self-associates in a monomer-dimer equilibrium (Ka ~1×10^6 M^-1) and is asymmetric, as shown by analytical ultracentrifugation and rotary-shadow EM.\",\n      \"method\": \"Co-purification, chemical cross-linking, analytical ultracentrifugation, surface plasmon resonance, SH3-domain pulldown, rotary-shadow electron microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple quantitative biophysical methods in a single study\",\n      \"pmids\": [\"14594951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mammalian CARMIL (mCARMIL/CARMIL1) binds CP with high affinity and reduces CP's affinity for actin filament barbed ends (anti-capping activity); addition to cell extracts enhances Arp2/3- and spectrin-actin-seed-induced polymerization. In cells, mCARMIL concentrates in lamellipodia; siRNA knockdown decreases F-actin and slows cell migration via reduced lamellipodial protrusion, a phenotype rescued by full-length but not CP-binding-deficient mCARMIL.\",\n      \"method\": \"In vitro barbed-end capping assay, actin polymerization assay with cell extracts, GFP localization, siRNA knockdown with rescue\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro assay plus loss-of-function with domain-specific rescue, 109 citations\",\n      \"pmids\": [\"16054028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The conserved C-terminal CAH3 domain of CARMIL is necessary and sufficient for potent anti-capping protein activity: it inhibits CP's barbed-end capping activity and drives uncapping of CP-capped filaments. Point mutations in conserved CAH3 residues abolish these activities. Full-length CARMIL is partially autoinhibited relative to C-terminal fragments.\",\n      \"method\": \"In vitro actin polymerization/uncapping assays, GST-fusion protein binding, site-directed mutagenesis, proteolytic cleavage\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus mutagenesis with multiple domain deletions\",\n      \"pmids\": [\"16434392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CARMIL1 localizes to lamellipodia and macropinosomes; CARMIL1 knockdown causes loss of lamellipodial actin, defects in protrusion/ruffling/macropinocytosis, and loss of Rac1 activation. CARMIL1 co-immunoprecipitates with the dual-GEF Trio. CARMIL2 colocalizes with vimentin intermediate filaments and loss decreases myosin-IIB levels, causing a multipolar phenotype. The two isoforms have distinct, non-redundant functions.\",\n      \"method\": \"siRNA knockdown, GFP localization, co-immunoprecipitation with Trio, Rac1 activation assay, rescue experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined cellular phenotypes and biochemical interaction, 61 citations\",\n      \"pmids\": [\"19846667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"C. elegans CARMIL ortholog (CRML-1) negatively regulates neuronal cell and axon growth cone migration by inhibiting the Rac GEF activity of UNC-73/Trio; CRML-1 and UNC-73 form a complex in vivo, and the antagonism between them controls SAX-3/Robo guidance receptor levels.\",\n      \"method\": \"Genetic epistasis (suppressor screens, double mutants), co-immunoprecipitation in vivo\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis plus in vivo co-IP in C. elegans ortholog\",\n      \"pmids\": [\"19244282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CARMIL contains unstructured membrane-binding sites with basic and hydrophobic amino acid character; synthetic peptides and protein domains containing these sites bind acidic phospholipids in vitro.\",\n      \"method\": \"Peptide synthesis, lipid vesicle binding assay, computational hydrophobicity analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct lipid-binding assay but with synthetic peptides and computational prediction\",\n      \"pmids\": [\"20018884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Direct single-molecule TIRF microscopy demonstrates that the isolated CAH3 domain of mouse CARMIL-1 (mCAH3) physically removes mGFP-tagged CP from individual capped actin filament barbed ends (uncapping), reducing CP half-life at the barbed end from ~30 min to ~10 s at saturating mCAH3; the CP:CAH3 complex retains weak barbed-end affinity.\",\n      \"method\": \"Total internal reflection fluorescence microscopy (TIRF/TIRFM), single-molecule imaging, mGFP-tagged CP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct single-molecule visualization of uncapping mechanism\",\n      \"pmids\": [\"19926785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NMR structural analysis shows the highly basic CAH3a subdomain of mouse CARMIL-1 binds an 'acidic groove' on CP opposite its actin-binding surface, orienting CAH3b adjacent to the basic patch of CP required for barbed-end interaction; mutagenesis of both proteins confirmed specific residue contacts and provided a mechanistic explanation for uncapping.\",\n      \"method\": \"NMR (solution structure), site-directed mutagenesis, actin polymerization/uncapping assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural data plus mutagenesis validation\",\n      \"pmids\": [\"20630878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CARMIL inhibits CP via an allosteric mechanism: molecular dynamics shows CBR binding induces conformational changes in CP's actin-binding surface; a steric-blocking model was ruled out because CP actin-binding mutants still bind CBR normally. The CARMIL-specific interaction (CSI) motif in the CP-binding region is required for high-affinity binding and uncapping.\",\n      \"method\": \"Mutagenesis of CP actin-binding surface, CP-CBR binding assays, molecular dynamics simulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis plus molecular dynamics, ruling out alternative model\",\n      \"pmids\": [\"22411988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of CARMIL1-668 reveals an N-terminal non-canonical pleckstrin homology (PH) domain connected to a 16-leucine-rich repeat (LRR) domain; small-angle X-ray scattering shows the central helical domain mediates antiparallel dimerization, positioning PH domains for simultaneous membrane interaction. Deletion of the PH domain in cells impairs leading-edge localization.\",\n      \"method\": \"X-ray crystallography, SAXS, lipid-binding assays, GFP cell imaging with deletion mutants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus SAXS plus functional cell imaging validation\",\n      \"pmids\": [\"24071777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CARMIL1-CP interaction is required for lamellipodia assembly, ruffle formation, and macropinocytosis, but is dispensable for CARMIL1 membrane localization, Rac1 activation, and wound-healing cell migration, as demonstrated by a point mutant (CARMIL1-AA) that specifically disrupts CP binding.\",\n      \"method\": \"Point mutagenesis, siRNA knockdown with rescue, Rac1 activation assay, phalloidin staining, live-cell imaging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific point mutant rescue with multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"23904264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CARMIL retrieves CP from the inactive CP:V-1 complex via complex exchange, generating a CP:CARMIL complex with moderate (weak) barbed-end capping activity; CARMIL is recruited only to the plasma membrane at actively protruding cell edges, suggesting a spatially restricted regulatory cycle where V-1-sequestered CP is converted to weakly capping CP:CARMIL complexes specifically at protrusion sites to promote Arp2/3-dependent actin network assembly.\",\n      \"method\": \"In vitro actin polymerization assays, stopped-flow kinetics, quantitative analytical ultracentrifugation, live-cell TIRF imaging of CARMIL recruitment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — quantitative in vitro assays plus live-cell imaging, 61 citations\",\n      \"pmids\": [\"24778263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Hydrogen-deuterium exchange mass spectrometry (HDX-MS) shows that both V-1 and CARMIL binding to CP induce changes in structural dynamics at their respective binding sites on CP and at the CP ββ subunit 'tentacle', a second distal actin-binding site, demonstrating allosteric coupling between CP modulator and actin binding sites.\",\n      \"method\": \"Hydrogen-deuterium exchange mass spectrometry (HDX-MS)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — HDX-MS structural dynamics providing mechanistic allosteric model\",\n      \"pmids\": [\"29847807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The leucine-rich repeat (LRR) region of CARMIL1 associates with IL-1 receptor type 1 (IL-1R1) and IL-1 receptor-associated kinase (IRAK), as shown by co-immunoprecipitation with CARMIL1 LRR-deletion mutants. CARMIL1 knockout (CRISPR-Cas9) reduces IL-1-induced ERK activation by 72% and MMP3 expression by 40%; a cell-permeable TAT-CARMIL1 LRR peptide reduces collagen degradation by 43%.\",\n      \"method\": \"Tandem mass tag mass spectrometry, co-immunoprecipitation with domain mutants, CRISPR-Cas9 knockout, TAT-peptide competition, ERK phosphorylation assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (MS, Co-IP, KO, peptide competition) with defined signaling phenotypes\",\n      \"pmids\": [\"32610117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Dictyostelium CARMIL-GAP, a CARMIL isoform with a GAP domain insert, binds Dictyostelium Rac1a and accelerates its GTP hydrolysis rate; CARMIL-GAP-null cells show defects in phagocytosis and chemotactic streaming rescued by full-length but not GAP-activity-deficient or CP-regulation-deficient versions, demonstrating that both GAP and CP-regulatory activities are required.\",\n      \"method\": \"GTPase assay (in vitro), genetic null cells, rescue with domain mutants, fluorescence localization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro GTPase assay plus genetic null rescue with domain mutants\",\n      \"pmids\": [\"35583107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CARMIL at a membrane surface (reconstituted with lipid-coated beads) promotes and enhances Arp2/3-nucleated actin assembly by counteracting V-1 inhibition of CP, effectively activating CP at the membrane surface for Arp2/3-mediated assembly; this reconstitution used purified Arp2/3 activator, Arp2/3 complex, V-1, CP, profilin, and actin.\",\n      \"method\": \"In vitro reconstitution with purified proteins on lipid-coated beads, pyrene-actin polymerization assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — full reconstitution with purified components\",\n      \"pmids\": [\"39437783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CARMIL1-AA (CP-binding mutant) selectively inhibits macropinocytosis without affecting proliferation, Rac1 activation, or autophagy, confirming that the CARMIL1-CP interaction is specifically required for macropinocytosis but not these other cellular processes.\",\n      \"method\": \"CRISPR-Cas9 knockout, expression of CARMIL1-AA point mutant, macropinocytosis assay, autophagy assay, Rac1 activation assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific mutant with multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"39602282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CARMIL1 binds TRIM27 (co-immunoprecipitation), which in turn binds p53, and CARMIL1 promotes p53 degradation through TRIM27; CARMIL1 knockdown reduces ERK and mTOR phosphorylation in liver cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, western blotting, siRNA knockdown, subcutaneous tumor model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP plus knockdown phenotype, single lab\",\n      \"pmids\": [\"38739978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM/crystal structure of CP-bound CARMIL1(1-1046) reveals a dimeric assembly with PH-LRR on a plane flanked by the helical dimerization domain and CBR-bound CP; an 'antenna' motif connecting the helical domain to CBR-CP contacts the LRR-LRR dimer interface and mediates partial autoinhibition. Disruption of the antenna partially relieves autoinhibition in vitro and increases cell area in knockout cells; deletion of the proline-rich (myosin-I-binding) domain induces membrane spikes.\",\n      \"method\": \"Crystal structure, in vitro uncapping assay, CARMIL1 knockout rescue with mutants, cell area imaging\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural determination plus mutagenesis with in vitro and cell-based validation\",\n      \"pmids\": [\"41861019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The membrane-binding (MB) domain of CARMIL targets the CPI and CSI motifs to lipid membranes (lipid-coated beads), enabling activation of CP for Arp2/3-mediated actin assembly; upon CP binding, the MB domain can dissociate from the membrane, enhancing uncapping of capped barbed ends and further activating soluble CP beyond what is seen in the membrane-attached state.\",\n      \"method\": \"In vitro reconstitution with lipid-coated beads, pyrene-actin polymerization assay, domain deletion mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with defined domain mutants revealing dual membrane release mechanism\",\n      \"pmids\": [\"42031174\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CARMIL1 is a large multidomain scaffold that localizes to the plasma membrane at the leading edge via a non-canonical PH domain and dimerizes via a helical domain; there it recruits and allosterically inhibits capping protein (CP) through its CAH3/CBR domain (containing CPI and CSI motifs), directly uncapping CP-capped actin filament barbed ends and converting V-1-sequestered inactive CP into weakly capping CP:CARMIL complexes that promote Arp2/3-dependent actin network assembly, while its LRR domain additionally engages IL-1R1/IRAK for inflammatory ERK signaling and Rac GEF Trio for Rac1 activation, collectively driving lamellipodia formation, macropinocytosis, and cell migration.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CARMIL1 is a large multidomain scaffold protein that coordinates actin barbed-end dynamics, membrane protrusion, and signaling at the leading edge of migrating cells. It dimerizes via a central helical domain and localizes to the plasma membrane through a non-canonical PH domain; its C-terminal CAH3/CBR domain allosterically inhibits capping protein (CP) and directly uncaps CP-capped actin filament barbed ends—reducing CP barbed-end half-life from ~30 min to ~10 s—thereby converting V-1-sequestered inactive CP into weakly capping CP:CARMIL complexes that promote Arp2/3-dependent actin network assembly at membrane protrusion sites [PMID:19926785, PMID:24778263, PMID:39437783]. An intramolecular 'antenna' motif connecting the helical domain to the CBR contacts the LRR dimer interface and mediates partial autoinhibition, while the LRR domain independently engages IL-1R1/IRAK to support IL-1-induced ERK signaling and associates with the Rac GEF Trio to activate Rac1, collectively driving lamellipodia formation, macropinocytosis, and cell migration [PMID:41861019, PMID:32610117, PMID:19846667, PMID:39602282].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of CARMIL as a physical hub linking capping protein, Arp2/3 complex, and type I myosins established that a single scaffold coordinates barbed-end capping, actin nucleation, and motor-driven membrane dynamics, and that its loss impairs macropinocytosis and chemotaxis.\",\n      \"evidence\": \"SH3-domain pulldown, co-IP, Arp2/3 nucleation assay, and p116-null Dictyostelium phenotypic analysis\",\n      \"pmids\": [\"11425877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian ortholog function unknown\", \"Direct binding affinities not measured\", \"Mechanism of CP regulation (inhibition vs. uncapping) not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Quantitative biophysical characterization revealed that CARMIL self-associates as a monomer-dimer equilibrium and binds CP with submicromolar affinity through a C-terminal proline-rich region, establishing the basic architecture and stoichiometry of the CARMIL-CP complex.\",\n      \"evidence\": \"Analytical ultracentrifugation, surface plasmon resonance, rotary-shadow EM of Acanthamoeba CARMIL\",\n      \"pmids\": [\"14594951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding mechanism (steric vs. allosteric) unresolved\", \"Mammalian CARMIL not yet characterized\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Transfer of CARMIL function to mammalian cells showed that CARMIL1 concentrates in lamellipodia, anti-caps CP to promote barbed-end polymerization, and is required for lamellipodial protrusion and cell migration—establishing its conserved role in mammalian cell motility.\",\n      \"evidence\": \"In vitro barbed-end capping assay, siRNA knockdown with full-length vs. CP-binding-deficient rescue in cultured cells\",\n      \"pmids\": [\"16054028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Uncapping activity not directly demonstrated\", \"Membrane-targeting mechanism unknown\", \"Rac1 connection not yet established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapping the minimal anti-capping and uncapping activity to the conserved CAH3 domain, and showing that full-length CARMIL is partially autoinhibited, defined the functional module within the multidomain protein and introduced the concept of intramolecular regulation.\",\n      \"evidence\": \"In vitro reconstituted capping/uncapping assays with GST-fusion domain deletions and point mutants\",\n      \"pmids\": [\"16434392\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of autoinhibition unknown\", \"Mechanism of uncapping (steric vs. allosteric) unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Multiple advances resolved CARMIL1's uncapping mechanism, membrane association, and signaling connections: single-molecule TIRF showed CAH3 physically removes CP from barbed ends reducing its dwell time from ~30 min to ~10 s; lipid-binding sites were mapped; and CARMIL1 was linked to Rac1 activation via Trio, while C. elegans genetics placed CARMIL upstream of Trio in guidance signaling.\",\n      \"evidence\": \"Single-molecule TIRF of mGFP-CP uncapping; peptide-lipid vesicle binding; siRNA knockdown with Rac1 pull-down and co-IP of Trio; C. elegans genetic epistasis and in vivo co-IP\",\n      \"pmids\": [\"19926785\", \"20018884\", \"19846667\", \"19244282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of full-length CARMIL unavailable\", \"Whether CARMIL activates or inhibits Trio GEF activity unclear across species\", \"Lipid specificity and PH domain structure not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"NMR structural analysis of the CAH3-CP interface revealed that CAH3a binds an acidic groove on CP opposite its actin-binding surface and positions CAH3b near CP's basic patch, providing the first atomic model explaining how CARMIL disrupts CP-barbed-end interaction without occupying the actin-binding site.\",\n      \"evidence\": \"NMR solution structure of CAH3-CP complex with confirmatory mutagenesis\",\n      \"pmids\": [\"20630878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether uncapping proceeds by allosteric or steric mechanism still debated\", \"Full-length structural context missing\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Ruling out steric blockade and demonstrating that CBR binding induces conformational changes in CP's actin-binding surface established that CARMIL inhibits and uncaps CP via an allosteric mechanism; the CSI motif was identified as essential for high-affinity binding and uncapping.\",\n      \"evidence\": \"CP actin-binding surface mutants retaining CBR binding; molecular dynamics simulations\",\n      \"pmids\": [\"22411988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Experimental demonstration of allosteric conformational change (e.g., HDX-MS) not yet performed\", \"Role of CSI motif in vivo not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Crystal structure of CARMIL1 N-terminal half revealed a non-canonical PH domain and 16-repeat LRR domain; SAXS showed the helical domain mediates antiparallel dimerization positioning PH domains for membrane binding. Separately, a CP-binding point mutant (CARMIL1-AA) dissected CP-dependent (lamellipodia, macropinocytosis) from CP-independent (membrane localization, Rac1 activation, wound migration) functions.\",\n      \"evidence\": \"X-ray crystallography, SAXS, GFP imaging with PH-deletion mutants; siRNA rescue with CARMIL1-AA point mutant\",\n      \"pmids\": [\"24071777\", \"23904264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of C-terminal CBR-containing region unresolved\", \"Identity of LRR-binding partners unknown\", \"Autoinhibition mechanism structurally undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that CARMIL retrieves CP from the inhibitory CP:V-1 complex to generate weakly capping CP:CARMIL complexes at protrusion sites established a spatially restricted regulatory cycle explaining how inactive cytoplasmic CP is converted to a functional form specifically at the leading edge to support Arp2/3-dependent actin assembly.\",\n      \"evidence\": \"Stopped-flow kinetics, analytical ultracentrifugation, in vitro actin polymerization, live-cell TIRF of CARMIL recruitment\",\n      \"pmids\": [\"24778263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconstitution with all components on a membrane not yet achieved\", \"In vivo concentrations and fluxes of CP, V-1, and CARMIL not measured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"HDX-MS experimentally confirmed allosteric coupling between the CARMIL-binding site and CP's distal actin-binding β-tentacle, providing direct biophysical evidence for the long-range conformational changes underlying CARMIL-mediated uncapping.\",\n      \"evidence\": \"Hydrogen-deuterium exchange mass spectrometry of CP alone and in complex with CARMIL peptide and V-1\",\n      \"pmids\": [\"29847807\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length CARMIL-CP structural model still lacking\", \"Dynamics of allosteric change at single-molecule resolution not captured\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Discovery that the LRR domain of CARMIL1 associates with IL-1R1 and IRAK and is required for IL-1-induced ERK activation and MMP3 expression revealed a signaling function for CARMIL1 beyond actin regulation, linking it to inflammatory pathways.\",\n      \"evidence\": \"Tandem mass tag MS, co-IP with LRR-deletion mutants, CRISPR-Cas9 knockout, TAT-peptide competition in chondrocytes\",\n      \"pmids\": [\"32610117\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CARMIL1 is a direct scaffold vs. indirect adapter for IRAK unclear\", \"Relevance to in vivo inflammatory disease not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of a Dictyostelium CARMIL isoform with intrinsic Rac GAP activity demonstrated that CARMIL proteins can directly regulate Rho-family GTPases, and that both GAP and CP-regulatory activities are independently required for phagocytosis and chemotaxis.\",\n      \"evidence\": \"In vitro GTPase assay, genetic null rescue with GAP-dead and CP-regulation-deficient mutants in Dictyostelium\",\n      \"pmids\": [\"35583107\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian CARMIL1 lacks a GAP domain; whether it regulates Rac through an analogous direct mechanism is unknown\", \"Structural basis of GAP domain insertion not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Full reconstitution on lipid-coated beads with purified Arp2/3 activator, Arp2/3, V-1, CP, profilin, and CARMIL demonstrated that membrane-anchored CARMIL counteracts V-1 inhibition and activates CP for Arp2/3-nucleated actin assembly, and that CP-binding by CARMIL1 is specifically required for macropinocytosis but dispensable for proliferation and autophagy.\",\n      \"evidence\": \"Reconstitution on lipid-coated beads with pyrene-actin assay; CRISPR-Cas9 KO cells with CARMIL1-AA rescue and macropinocytosis/autophagy assays\",\n      \"pmids\": [\"39437783\", \"39602282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconstitution lacks Trio/Rac module\", \"Contribution of CARMIL membrane release to in vivo dynamics not tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Cryo-EM/crystal structures of dimeric CP-bound CARMIL1(1–1046) revealed the full-length architecture including an 'antenna' motif mediating autoinhibition at the LRR dimer interface, and reconstitution showed that the membrane-binding domain targets CPI/CSI motifs to membranes and that upon CP binding, CARMIL can release from the membrane to enhance cytoplasmic uncapping—establishing a dual-mode mechanism.\",\n      \"evidence\": \"Crystal structure of dimeric CARMIL1-CP complex; mutagenesis of antenna with in vitro uncapping and cell area assays; lipid-coated bead reconstitution with MB domain deletions\",\n      \"pmids\": [\"41861019\", \"42031174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamic structural transitions during membrane release not captured in real time\", \"How autoinhibition is relieved by upstream signals in vivo remains unclear\", \"Structure of the proline-rich/myosin-I-binding C-terminal tail still missing\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: how CARMIL1 autoinhibition is relieved by physiological signals; how LRR-mediated IL-1R1/IRAK and Trio interactions are coordinated with CP regulation at the leading edge; and whether the membrane-release uncapping mode operates in vivo during protrusion cycles.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No upstream activating signal for autoinhibition relief identified\", \"Spatial and temporal coordination of CP-regulatory and signaling functions not resolved in vivo\", \"Complete reconstitution including Trio/Rac module and myosin-I not achieved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 7, 9, 12, 13, 16, 20]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 2, 3, 7, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6, 10, 20]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 4, 10, 11, 12, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 14]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [2, 4, 11]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"complexes\": [\n      \"CARMIL1 homodimer\",\n      \"CP:CARMIL1 complex\"\n    ],\n    \"partners\": [\n      \"CAPZA1\",\n      \"CAPZB\",\n      \"TRIO\",\n      \"ACTR2\",\n      \"IL1R1\",\n      \"IRAK1\",\n      \"TRIM27\",\n      \"MYO1B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}