{"gene":"MACF1","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":1999,"finding":"MACF1 (mACF7) contains a functional N-terminal actin-binding domain and a C-terminal domain that interacts with and stabilizes microtubules, allowing the full-length protein to associate with both actin and microtubules simultaneously in transfected cells and in vitro.","method":"In vitro binding assays, transient transfection, co-localization with cytoskeletal markers","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro binding assays plus in vivo transfection, foundational characterization paper with multiple methods","pmids":["10601340"],"is_preprint":false},{"year":2001,"finding":"The C-terminal microtubule-binding domain of MACF1 contains at least two distinct MT-binding regions: a GAR domain that binds and partially stabilizes MTs, and a GSR-repeat domain that bundles MTs; together they bundle MTs and confer nocodazole resistance.","method":"Transient transfection studies and in vitro MT-binding assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — multiple domain deletions tested in vivo and in vitro, strong methodological rigor","pmids":["11112700"],"is_preprint":false},{"year":2003,"finding":"ACF7 (MACF1) acts as an essential integrator of microtubule-actin dynamics: it binds along microtubules and concentrates at their distal ends and cortical actin-rich sites; in its absence, MTs no longer grow along polarized actin bundles or pause/tether at cortical sites, resulting in unstable MTs, skewed trajectories, and failure to sustain polarization and coordinated migration. Rescue requires both actin- and microtubule-binding domains.","method":"Conditional knockout in endodermal cells, live-cell imaging, rescue with domain mutants","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with specific phenotypic readouts, domain-rescue epistasis, replicated across assays","pmids":["14636561"],"is_preprint":false},{"year":2006,"finding":"MACF1 participates in canonical Wnt signaling: in the absence of Wnt, MACF1 associates with an Axin/β-catenin/GSK3β/APC destruction complex; upon Wnt stimulation, MACF1 mediates translocation of the Axin complex to LRP6 at the plasma membrane. MACF1 knockdown reduces nuclear β-catenin and TCF/β-catenin-dependent transcription. MACF1-null mice die at gastrulation with phenotypes resembling Wnt-3 and LRP5/6 double knockouts.","method":"Co-immunoprecipitation, siRNA knockdown, dominant-negative constructs, TCF/β-catenin reporter assay, MACF1 knockout mouse","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, functional reporter assays, genetic KO with phenotypic analysis, multiple orthogonal methods","pmids":["16815997"],"is_preprint":false},{"year":2007,"finding":"The N-terminal actin-binding domain of ACF7 (MACF1) directly binds to the tetratricopeptide repeat (TPR) domains of rapsyn at the neuromuscular junction, as demonstrated by co-expression in fibroblasts, co-immunoprecipitation, surface plasmon resonance, and blot overlay assays.","method":"Co-immunoprecipitation, surface plasmon resonance, blot overlay, confocal co-localization","journal":"Neuroscience","confidence":"High","confidence_rationale":"Tier 1-2 — direct binding confirmed by SPR and blot overlay in addition to Co-IP; multiple orthogonal methods","pmids":["17222516"],"is_preprint":false},{"year":2008,"finding":"ACF7 (MACF1) possesses an intrinsic actin-regulated ATPase domain that is functionally required, beyond the F-actin, MT, and MT plus-end binding domains, for targeting microtubules along F-actin to focal adhesions, regulating focal adhesion-cytoskeletal dynamics, and enabling directed epidermal cell migration.","method":"Conditional knockout in skin epidermis, domain rescue experiments, in vitro ATPase assay, live-cell imaging of focal adhesion dynamics","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro enzymatic assay plus conditional KO and domain-specific rescue; multiple orthogonal methods in a high-impact study","pmids":["18854161"],"is_preprint":false},{"year":2004,"finding":"MACF1 interacts with the TGN protein p230/Golgin-245 through its C-terminal domain (binding the N-terminal flexible end of p230), and this interaction is required for transport of GPI-anchored proteins (but not transmembrane proteins) from the TGN to the cell periphery along microtubule and actin tracts.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, dominant-negative expression of interacting domains, YFP-SP-GPI trafficking assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — multiple validation methods including in vitro binding, Co-IP, and functional trafficking assay","pmids":["15265687"],"is_preprint":false},{"year":2010,"finding":"ErbB2 receptor recruits ACF7 to the plasma membrane via a signaling pathway involving Memo, RhoA/mDia1, and GSK3 inhibition, and ACF7 localization to the plasma membrane is both necessary and sufficient for microtubule capture and stabilization downstream of ErbB2-induced cell migration.","method":"shRNA knockdown, dominant-negative and constitutively active constructs, immunofluorescence, live-cell MT tracking, rescue experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — epistasis established by systematic knockdown of pathway components with specific readout (MT capture), rescue experiments","pmids":["20937854"],"is_preprint":false},{"year":2011,"finding":"GSK3β directly phosphorylates ACF7 at residues within its microtubule-binding domain, uncoupling ACF7 from microtubules; Wnt signaling inhibits GSK3β, allowing ACF7-microtubule engagement. Phosphorylation-refractile ACF7 restores overall MT architecture but not polarized movement; dynamic phospho-regulation is required for stem cell directed migration and wound repair.","method":"In vitro kinase assay (GSK3β phosphorylation of ACF7), phospho-mutant rescue in ACF7-null keratinocytes, live-cell MT imaging, hair follicle SC wound-healing model","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — direct kinase assay, phospho-site mapping, phospho-mutant epistasis, in vivo wound-repair model; multiple orthogonal methods","pmids":["21295697"],"is_preprint":false},{"year":2009,"finding":"ACF7 (and its Drosophila ortholog Shot) regulates neuronal microtubule organization (requiring both F-actin and MT binding domains) and controls filopodia formation in axonal growth cones (requiring EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C), establishing two mechanistically distinct cytoskeletal roles in axon extension.","method":"Neuronal culture systems with ACF7 knockdown/overexpression, domain deletion constructs, co-immunoprecipitation (ACF7-eIF5C interaction), live imaging of growth cones","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2-3 — domain structure-function with Co-IP for eIF5C interaction; single lab but multiple neuronal systems","pmids":["19571116"],"is_preprint":false},{"year":2012,"finding":"ELMO directly interacts with ACF7 via a C-terminal polyproline segment in ELMO and the last spectrin repeat of ACF7; ELMO recruits ACF7 to the membrane to promote microtubule capture and stability, increasing persistence of cellular protrusions; this function requires the Rac GEF DOCK180.","method":"Co-immunoprecipitation, direct domain interaction mapping, live-cell imaging of MT capture, integrin-mediated cell spreading assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with domain mapping, functional MT capture assay, epistasis with DOCK180; single lab","pmids":["23184944"],"is_preprint":false},{"year":2014,"finding":"MACF1 regulates cortical pyramidal neuron migration by controlling microtubule dynamics and mediating GSK-3 signaling in developing neurons; MACF1 deletion causes aberrant neuronal positioning, abnormal leading process and centrosome dynamics, and reduced microtubule stability. MACF1 interacts with GSK-3 in developing neurons.","method":"Conditional knockout (floxed allele + in utero electroporation), co-immunoprecipitation (MACF1-GSK3), live imaging of neuron migration, MT stability assays","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with specific neuronal phenotype, Co-IP for GSK-3 interaction, microtubule dynamics measurements","pmids":["25224226"],"is_preprint":false},{"year":2016,"finding":"Src/FAK complex phosphorylates a key tyrosine residue in the calponin homology (CH) domain of ACF7's N-terminal domain, and this phosphorylation is essential for F-actin binding of ACF7 and for focal adhesion dynamics and epidermal migration in vitro and in vivo. Crystal structure of the ACF7 NT domain was resolved to inform this mechanism.","method":"X-ray crystallography of ACF7 NT domain (2.x Å), phospho-mutant rescue, Src/FAK kinase assay, in vitro and in vivo (skin epidermis) migration assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro phosphorylation assay plus phospho-mutant functional rescue in vivo","pmids":["27216888"],"is_preprint":false},{"year":2016,"finding":"ACF7 (MACF1) specifically binds CAMSAP3 and is required for apical localization of CAMSAP3-decorated microtubule minus ends in intestinal epithelial cells; loss of ACF7 impairs formation of polarized epithelial cysts and disrupts apico-basal polarity.","method":"Co-immunoprecipitation (ACF7-CAMSAP3), CRISPR/RNAi knockouts, 3D epithelial cyst assays, immunofluorescence of microtubule minus-end organization","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — Co-IP with functional KO phenotype in 3D culture; replicated findings across CAMSAP family members provide specificity","pmids":["27802168"],"is_preprint":false},{"year":2016,"finding":"In Caco2 epithelial cells, ACF7 interacts with CAMSAP3 (Nezha) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments; these minus-end MTs cooperate with actin retrograde flow to maintain MT orientation perpendicular to the cell edge and regulate focal adhesion size and cell migration.","method":"Co-immunoprecipitation, MT minus-end tracking, focal adhesion size quantification, migration assays, knockdown experiments","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus functional assays in multiple cellular readouts; independent replication of CAMSAP3-ACF7 interaction","pmids":["27693509"],"is_preprint":false},{"year":2016,"finding":"MACF1 is required for ciliogenesis: MACF1 ablation in the developing retina abolishes ciliogenesis, prevents basal body docking to ciliary vesicles, and blocks apical migration; MACF1 interacts with ciliary proteins MKKS and TALPID3, suggesting it coordinates microtubule-to-actin trafficking required for ciliogenesis.","method":"Conditional knockout (retina-specific), co-immunoprecipitation (MACF1-MKKS, MACF1-TALPID3), electron microscopy, immunofluorescence of cilia and basal bodies","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with specific ciliogenesis phenotype, Co-IP of ciliary protein interactions, EM analysis","pmids":["27783952"],"is_preprint":false},{"year":2017,"finding":"ACF7 loss leads to aberrant microtubule organization, tight junction stabilization, and impaired wound closure in intestinal epithelium; ablation of ACF7 in mice inhibits intestinal wound healing and increases susceptibility to experimental colitis.","method":"Conditional intestinal KO, wound scratch assays, immunofluorescence of tight junction proteins and MTs, experimental colitis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with specific intestinal phenotype, multiple cellular and in vivo readouts","pmids":["28541346"],"is_preprint":false},{"year":2017,"finding":"The 2.8 Å crystal structure of the hACF7 EF1-EF2-GAR MT-binding module revealed that the EF1-EF2 domain is an EFβ-scaffold with two Ca2+ ions and the GAR domain has a unique α/β sandwich fold coordinating Zn2+; the GAR domain alone is sufficient for MT binding via a conserved basic patch distal to the Zn2+-binding site, while EF1-EF2 alone is insufficient.","method":"X-ray crystallography (2.8 Å), domain deletion/mutagenesis, MT-binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure plus mutagenesis and MT-binding assays; comprehensive structure-function analysis","pmids":["28602822"],"is_preprint":false},{"year":2018,"finding":"The E3 ubiquitin ligase HectD1 targets ACF7 for proteasome-mediated degradation; HectD1 depletion stabilizes ACF7, promoting EMT and cell migration. ACF7 is essential for maintenance of the EMT program.","method":"shRNA screens, ubiquitination assays, proteasome inhibitor rescue, mouse metastasis models, breast cancer patient biopsies","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — ubiquitination assay identifying HectD1 as E3 ligase for ACF7, functional KD in multiple models, in vivo metastasis","pmids":["29386124"],"is_preprint":false},{"year":2018,"finding":"MACF1 variants encoding zinc-binding residues in the GAR (microtubule-binding) domain cause a distinctive lissencephaly and brainstem malformation with absent pontine crossing fibers, and affected cells show increased proportions of short cilia.","method":"Whole-exome/genome sequencing (clinical), ciliogenesis assay in patient cells","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2-3 — clinical genetics plus functional ciliogenesis assay in patient cells; limited mechanistic follow-up","pmids":["30471716"],"is_preprint":false},{"year":2019,"finding":"MACF1 is concentrated at neuromuscular synapses where it binds Rapsyn and serves as a synaptic organizer for MT-associated proteins EB1 and MAP1b and actin-associated protein Vinculin; MACF1 deficiency impairs synaptic differentiation and transmission in mice.","method":"Co-immunoprecipitation with AChRs/Rapsyn, mass spectrometry, conditional NMJ-specific analysis, electrophysiology","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP/MS identification, genetic KO with electrophysiological phenotype, multiple interaction partners validated","pmids":["30842214"],"is_preprint":false},{"year":2017,"finding":"MACF1 regulates GABAergic interneuron migration and positioning in the developing mouse brain; conditional MACF1 deletion reduces interneuron numbers and disrupts positioning, speed, and mode of migration, associated with decreased microtubule stability in interneurons.","method":"Conditional KO (Dlx5/6-Cre and Nkx2.1-Cre), in vivo migration tracking, MT stability assays, morphometric analysis","journal":"Cerebral cortex","confidence":"High","confidence_rationale":"Tier 2 — two independent Cre drivers, specific neuronal migration phenotype, MT stability measurements","pmids":["27756764"],"is_preprint":false},{"year":2017,"finding":"In zebrafish oocytes, Macf1a localizes to the Balbiani body and mediates its dissociation via its actin-binding domain (ABD); the ABD (not the plectin repeat domain) is required for linking cortical actin to the Balbiani body mRNP granule to drive its dissociation and nuclear positioning, establishing animal-vegetal polarity.","method":"CRISPR/Cas9 endogenous domain deletion, live imaging, pharmacological disruption of actin/MTs","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — CRISPR domain deletion at endogenous locus, rigorous phenotypic rescue structure, genetic epistasis with cytoskeletal drugs","pmids":["28880872"],"is_preprint":false},{"year":2019,"finding":"Deficiency of MACF1 in osteoblasts attenuates BMP2/Smad/Runx2 signaling, inhibiting osteoblast differentiation and bone formation in vivo.","method":"Conditional KO (Osx-Cre), primary osteoblast differentiation assays, micro-CT, western blotting for Smad/Runx2 pathway","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — conditional KO with pathway analysis; pathway placement is indirect (no direct MACF1-SMAD interaction shown)","pmids":["31709715"],"is_preprint":false},{"year":2020,"finding":"MACF1 directly interacts with SMAD7 and facilitates SMAD7 nuclear translocation to initiate downstream osteogenic pathways; mesenchymal-specific MACF1 deletion reduces nuclear SMAD7 and attenuates bone formation.","method":"Co-immunoprecipitation (MACF1-SMAD7 direct interaction), nuclear-cytoplasmic fractionation, conditional KO (Prrx1-Cre), bone histomorphometry","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP for direct interaction, functional KO; single lab, moderate evidence for the direct SMAD7 interaction","pmids":["32143362"],"is_preprint":false},{"year":2021,"finding":"MACF1 is an essential regulator of myonuclei positioning in skeletal muscle: it controls microtubule dynamics and density around myonuclei, regulates myonuclei motion, and its conditional muscle-specific KO causes extra-synaptic myonuclei mispositioning, NMJ fragmentation, and increased mitochondrial biogenesis.","method":"Conditional muscle-specific KO (mouse), Drosophila muscle genetics, in vitro myotube microtubule dynamics, electron microscopy of triads, calcium release assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — conditional KO replicated in two organisms, MT dynamics quantification, multiple orthogonal phenotypic readouts","pmids":["34448452"],"is_preprint":false},{"year":2021,"finding":"MACF1 interacts with transcription factors TCF12 and E2F6 (repressors of osteoblast differentiation) and with CDK12 and MEAF6; MACF1 sequesters TCF12 and E2F6 in the cytoplasm in a concentration-dependent manner, preventing their nuclear repressor activity and thereby promoting osteoblast differentiation.","method":"Co-immunoprecipitation (MACF1 with TCF12, E2F6, CDK12, MEAF6), nuclear-cytoplasmic fractionation, transcriptome analysis, knockdown/overexpression","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP for direct interactions, nuclear-cytoplasmic fractionation shows localization dependency on MACF1 concentration; single lab","pmids":["33664480"],"is_preprint":false},{"year":2021,"finding":"USP14 is a deubiquitinating enzyme that directly associates with ACF7 and inhibits its proteasomal degradation by removing ubiquitin; USP14-mediated stabilization of ACF7 promotes NSCLC cell migration.","method":"Proteomic identification (Co-IP/MS), co-immunoprecipitation, ubiquitination assay, knockdown/overexpression of USP14, migration assay","journal":"Journal of biosciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus functional ubiquitination assay; single lab with moderate follow-up","pmids":["33737492"],"is_preprint":false},{"year":2021,"finding":"MACF1 promotes osteoblastic cell migration by stabilizing microtubules and increasing total MAP1B mRNA expression through TCF7 transcriptional activity, while also decreasing GSK3β-mediated phosphorylation of MAP1B at T1265, thereby regulating MAP1B activity downstream of the GSK3β pathway.","method":"siRNA knockdown, luciferase reporter assay, ChIP (TCF7 binding to Map1b promoter), in vitro and in vivo migration assays, GSK3β inhibitor (LiCl)","journal":"Bone","confidence":"Medium","confidence_rationale":"Tier 2-3 — ChIP plus functional assays; single lab, moderate mechanistic depth","pmids":["34700040"],"is_preprint":false},{"year":2025,"finding":"De novo heterozygous missense variants in the EF-hand domains of MACF1 (in addition to the GAR domain) cause brain malformation; experimental evidence shows that EF-hand/GAR module variants increase microtubule binding, indicating a toxic gain-of-function mechanism. Bi-allelic non-GAR domain variants (mainly Plakin domain) cause a distinct complex neurodevelopmental syndrome.","method":"Whole-exome/genome sequencing, MT co-sedimentation/binding assays for EF-hand mutants, RNA-seq and ChIP of human fetal brain tissue for isoform characterization","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1-2 — functional MT-binding assays for mutants plus isoform-specific RNA-seq and ChIP; domain-specific mechanism well supported","pmids":["40925378"],"is_preprint":false}],"current_model":"MACF1 (ACF7) is a giant spectraplakin scaffold protein that crosslinks F-actin and microtubules through distinct N-terminal actin-binding (calponin homology) and C-terminal MT-binding (GAR/GSR) domains; it guides microtubule plus ends along actin filaments to focal adhesions using an intrinsic actin-regulated ATPase activity, is regulated by GSK3β phosphorylation (itself inhibited by Wnt) and Src/FAK phosphorylation at its CH domain, interacts with the Wnt destruction complex (Axin/β-catenin/GSK3β/APC) to facilitate LRP6 signaling, binds CAMSAP3 to anchor noncentrosomal microtubule minus ends, interacts with Rapsyn at the neuromuscular junction, is targeted for degradation by the E3 ligase HectD1 and stabilized by the deubiquitinase USP14, and is required for ciliogenesis, neuronal migration, epithelial polarity, and myonuclei positioning."},"narrative":{"teleology":[{"year":1999,"claim":"Establishing that MACF1 is a bona fide actin–microtubule crosslinker resolved the fundamental question of whether a single molecule could simultaneously engage both cytoskeletal systems.","evidence":"In vitro binding assays and co-localization in transfected cells demonstrated separable N-terminal actin-binding and C-terminal MT-binding domains","pmids":["10601340"],"confidence":"High","gaps":["Full-length protein binding stoichiometry unknown","No functional phenotype yet established"]},{"year":2001,"claim":"Dissecting the C-terminal MT-binding region into GAR (binding/stabilizing) and GSR (bundling) sub-domains revealed that MACF1 uses two distinct mechanisms to engage microtubules.","evidence":"Domain deletions tested by transfection and in vitro MT-binding/bundling assays","pmids":["11112700"],"confidence":"High","gaps":["Structural basis of GAR and GSR recognition of MTs unresolved","Physiological relevance of MT bundling activity not tested in vivo"]},{"year":2003,"claim":"Conditional knockout demonstrated that MACF1 is the essential molecule that guides MT plus-end growth along polarized actin to cortical sites, answering how cells coordinate the two cytoskeletons for polarized migration.","evidence":"Genetic KO in endodermal cells with live-cell imaging; rescue required both actin- and MT-binding domains","pmids":["14636561"],"confidence":"High","gaps":["Mechanism of plus-end tracking not yet explained","Upstream signals controlling MACF1 cortical localization unknown"]},{"year":2004,"claim":"Identification of p230/Golgin-245 as a MACF1 binding partner linked the crosslinker to Golgi-to-surface transport of GPI-anchored proteins, revealing a cargo-selective trafficking role.","evidence":"Yeast two-hybrid, Co-IP, in vitro binding, and GPI-AP trafficking assays","pmids":["15265687"],"confidence":"High","gaps":["Whether MACF1 physically accompanies vesicles or merely organizes tracks is unknown","Specificity for GPI-APs over other cargoes not mechanistically explained"]},{"year":2006,"claim":"Discovery that MACF1 scaffolds the Axin/β-catenin/GSK3β destruction complex and translocates it to LRP6 upon Wnt stimulation established an unexpected role for a cytoskeletal crosslinker in canonical Wnt signal transduction.","evidence":"Reciprocal Co-IP, TCF/β-catenin reporter assays, dominant-negative constructs, and MACF1-null mouse phenocopying Wnt3/LRP5/6 double KO","pmids":["16815997"],"confidence":"High","gaps":["Which MACF1 domain mediates Axin interaction not mapped","Whether cytoskeletal crosslinking and Wnt scaffolding are mechanistically coupled is unclear"]},{"year":2007,"claim":"Direct binding of the MACF1 actin-binding domain to Rapsyn at the neuromuscular junction extended MACF1's role to synaptic organization, linking cytoskeletal scaffolding to receptor clustering.","evidence":"Co-IP, SPR, and blot overlay confirmed direct interaction with Rapsyn TPR domains","pmids":["17222516"],"confidence":"High","gaps":["Functional consequence of MACF1-Rapsyn disruption at NMJ not yet shown genetically"]},{"year":2008,"claim":"Identification of an intrinsic actin-regulated ATPase activity in MACF1 revealed that crosslinking alone is insufficient — active energy-dependent motility is required to target MTs along F-actin to focal adhesions.","evidence":"In vitro ATPase assay, conditional epidermal KO, domain-specific rescue experiments, and live-cell focal adhesion dynamics imaging","pmids":["18854161"],"confidence":"High","gaps":["ATPase domain identity and structural basis not determined","Whether ATPase drives processive movement or conformational cycling unknown"]},{"year":2009,"claim":"Demonstrating separate requirements for actin/MT-binding (axonal MT organization) and EF-hand/eIF5C interaction (filopodia formation) in neurons revealed MACF1 executes mechanistically distinct functions within a single cell type.","evidence":"Domain deletions and Co-IP of eIF5C in neuronal culture systems; Drosophila Shot ortholog used for cross-species validation","pmids":["19571116"],"confidence":"Medium","gaps":["eIF5C interaction confirmed only by single Co-IP","Translational regulation link not mechanistically elaborated"]},{"year":2010,"claim":"Placing MACF1 downstream of ErbB2→Memo→RhoA/mDia1→GSK3 inhibition established how receptor tyrosine kinase signaling recruits MACF1 to the membrane for MT capture during directed migration.","evidence":"Systematic shRNA knockdown of pathway components with MT capture as readout, rescue experiments","pmids":["20937854"],"confidence":"High","gaps":["Direct binding between pathway components and MACF1 not demonstrated","Whether this pathway operates in non-breast epithelial contexts unknown"]},{"year":2011,"claim":"Direct phosphorylation of MACF1's MT-binding domain by GSK3β, inhibited by Wnt, explained how dynamic phospho-cycling controls polarized MT guidance — constitutive engagement restores MT architecture but not directionality.","evidence":"In vitro kinase assay, phospho-site mapping, phospho-mutant rescue in ACF7-null keratinocytes, in vivo wound-repair model","pmids":["21295697"],"confidence":"High","gaps":["Number and identity of all functionally relevant phospho-sites not exhaustively mapped","How phospho-cycling is spatially restricted within the cell unknown"]},{"year":2012,"claim":"ELMO/DOCK180-mediated recruitment of MACF1 to the membrane via a polyproline–spectrin repeat interaction identified an alternative, integrin-triggered pathway for MT capture distinct from the ErbB2 pathway.","evidence":"Co-IP with domain mapping, live-cell MT capture imaging, integrin-mediated spreading assays","pmids":["23184944"],"confidence":"Medium","gaps":["Single lab finding; independent replication not reported","Relationship between ELMO and ErbB2 recruitment pathways not clarified"]},{"year":2014,"claim":"Conditional KO in developing cortical neurons established that MACF1 is essential for radial neuronal migration through GSK3-regulated microtubule stabilization, extending the GSK3β–MACF1 axis to brain development.","evidence":"Conditional KO with in utero electroporation, Co-IP for GSK3 interaction, live neuron migration imaging","pmids":["25224226"],"confidence":"High","gaps":["Whether GSK3 phosphorylates the same sites in neurons as in keratinocytes not confirmed","Contributions of other spectraplakins not excluded"]},{"year":2016,"claim":"Multiple 2016 discoveries collectively resolved MACF1's structural basis and expanded its functional repertoire: crystal structures of the N-terminal and C-terminal domains revealed how Src/FAK phosphorylation activates actin binding and how the GAR domain coordinates Zn²⁺ for MT binding; CAMSAP3 interaction anchored noncentrosomal MT minus ends for epithelial polarity; and interaction with MKKS/TALPID3 established a requirement for ciliogenesis.","evidence":"X-ray crystallography of NT and EF1-EF2-GAR domains, phospho-mutant rescue in vivo, Co-IP of CAMSAP3 and ciliary proteins, CRISPR KO in epithelial and retinal systems","pmids":["27216888","28602822","27802168","27693509","27783952"],"confidence":"High","gaps":["Full-length MACF1 structure unavailable","How MACF1 coordinates CAMSAP3 binding with plus-end tracking mechanistically unresolved","Structural basis of MKKS/TALPID3 interaction unknown"]},{"year":2017,"claim":"Tissue-specific KO studies demonstrated MACF1's requirement for intestinal wound healing and GABAergic interneuron migration, broadening the phenotypic landscape and confirming MT stabilization as the common downstream mechanism.","evidence":"Conditional intestinal KO with colitis model; conditional brain KO with two independent Cre drivers for interneuron analysis","pmids":["28541346","27756764"],"confidence":"High","gaps":["Whether intestinal and neuronal phenotypes involve identical molecular pathways unknown","Compensatory mechanisms in heterozygous animals not characterized"]},{"year":2017,"claim":"In zebrafish oocytes, MACF1's actin-binding domain was shown to link cortical actin to the Balbiani body for animal-vegetal axis establishment, extending MACF1's role to oocyte polarity.","evidence":"Endogenous CRISPR domain deletion and live imaging in zebrafish oocytes","pmids":["28880872"],"confidence":"High","gaps":["Whether mammalian oocytes use MACF1 for similar polarity establishment unknown"]},{"year":2018,"claim":"Identification of HectD1 as the E3 ubiquitin ligase targeting MACF1 for proteasomal degradation revealed a post-translational control mechanism; MACF1 protein stability promotes EMT and migration.","evidence":"Ubiquitination assays, proteasome inhibitor rescue, shRNA screens, in vivo metastasis models","pmids":["29386124"],"confidence":"High","gaps":["Specific ubiquitination sites on MACF1 not mapped","Whether HectD1 and USP14 compete at the same ubiquitin chains unclear"]},{"year":2018,"claim":"Clinical genetics linked de novo GAR-domain variants to lissencephaly with pontine malformation and shortened cilia, providing the first human disease association and supporting the ciliary function.","evidence":"Whole-exome/genome sequencing in affected families plus ciliogenesis assay in patient-derived cells","pmids":["30471716"],"confidence":"Medium","gaps":["Functional rescue not performed","Mechanism of cilia shortening by GAR variants not elucidated at molecular level"]},{"year":2019,"claim":"NMJ-specific analysis confirmed that MACF1 organizes a cytoskeletal scaffold at the synapse (recruiting EB1, MAP1b, Vinculin via Rapsyn), and its loss impairs synaptic transmission, closing the loop opened by the 2007 Rapsyn binding discovery.","evidence":"Co-IP/MS, conditional NMJ analysis, electrophysiology","pmids":["30842214"],"confidence":"High","gaps":["Whether MACF1 loss causes NMJ disease in humans unknown","Relative contributions of MT vs actin scaffolding at NMJ not separated"]},{"year":2021,"claim":"Multiple discoveries in 2021 established MACF1 as a regulator of myonuclei positioning in muscle, identified USP14 as a stabilizing deubiquitinase opposing HectD1, and revealed cytoplasmic sequestration of transcriptional repressors TCF12/E2F6 as a mechanism for promoting osteoblast differentiation.","evidence":"Conditional muscle KO in mouse and Drosophila; Co-IP/ubiquitination assays for USP14; Co-IP and nuclear-cytoplasmic fractionation for TCF12/E2F6","pmids":["34448452","33737492","33664480"],"confidence":"High","gaps":["USP14 interaction confirmed in single lab only","TCF12/E2F6 sequestration mechanism is correlative — no structural basis","Whether myonuclei mispositioning causes functional myopathy not established"]},{"year":2025,"claim":"Expanding the genotype-phenotype map revealed that EF-hand domain variants cause brain malformation through gain-of-function increased MT binding, while bi-allelic Plakin domain variants cause a distinct neurodevelopmental syndrome, establishing domain-specific pathomechanisms.","evidence":"WES/WGS, MT co-sedimentation assays for EF-hand mutants, RNA-seq and ChIP of human fetal brain for isoform characterization","pmids":["40925378"],"confidence":"High","gaps":["Animal model validation of EF-hand gain-of-function variants not reported","Mechanism by which Plakin domain variants cause disease not functionally characterized"]},{"year":null,"claim":"A full-length structural model of MACF1 is lacking, and it remains unknown how its multiple functional domains are spatially coordinated, how its ATPase activity is structurally organized, and how tissue-specific isoforms dictate distinct cellular functions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length cryo-EM or AlphaFold-validated structure","ATPase domain identity and catalytic mechanism unresolved","Isoform-specific functions in different tissues not systematically compared"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[5]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,2,5,13,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,26]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,1,2,5,14]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,10,12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,26]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[15,19]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[11,21,22]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[20]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[9,11,21]}],"complexes":["Wnt destruction complex (Axin/β-catenin/GSK3β/APC)"],"partners":["GSK3B","CAMSAP3","RAPSN","HECTD1","USP14","ELMO1","AXIN1","GOLGA4"],"other_free_text":[]},"mechanistic_narrative":"MACF1 (also called ACF7) is a giant spectraplakin that crosslinks the actin and microtubule cytoskeletons to coordinate cell polarity, migration, and differentiation across diverse tissues. Its N-terminal calponin homology domains bind F-actin (regulated by Src/FAK phosphorylation) while its C-terminal GAR and GSR domains bind and bundle microtubules (regulated by GSK3β phosphorylation downstream of Wnt); an intrinsic ATPase activity guides microtubule plus ends along actin filaments toward focal adhesions, and interaction with CAMSAP3 anchors noncentrosomal microtubule minus ends to establish epithelial apico-basal polarity [PMID:10601340, PMID:18854161, PMID:21295697, PMID:27802168]. MACF1 also functions as a scaffold in canonical Wnt signaling by associating with the Axin/β-catenin/GSK3β/APC destruction complex and mediating its Wnt-dependent translocation to LRP6, and it is required for ciliogenesis through interaction with ciliary proteins MKKS and TALPID3 [PMID:16815997, PMID:27783952]. De novo missense variants in the GAR or EF-hand domains cause lissencephaly and brain malformations through gain-of-function increased microtubule binding, while bi-allelic variants in the Plakin domain cause a distinct complex neurodevelopmental syndrome [PMID:30471716, PMID:40925378]."},"prefetch_data":{"uniprot":{"accession":"Q9UPN3","full_name":"Microtubule-actin cross-linking factor 1, isoforms 1/2/3/4/5","aliases":["620 kDa actin-binding protein","ABP620","Actin cross-linking family protein 7","Macrophin-1","Trabeculin-alpha"],"length_aa":7388,"mass_kda":838.3,"function":"F-actin-binding protein which plays a role in cross-linking actin to other cytoskeletal proteins and also binds to microtubules (PubMed:15265687, PubMed:20937854). Plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex (PubMed:20937854). Acts as a positive regulator of Wnt receptor signaling pathway and is involved in the translocation of AXIN1 and its associated complex (composed of APC, CTNNB1 and GSK3B) from the cytoplasm to the cell membrane (By similarity). Has actin-regulated ATPase activity and is essential for controlling focal adhesions (FAs) assembly and dynamics (By similarity). Interaction with CAMSAP3 at the minus ends of non-centrosomal microtubules tethers microtubules minus-ends to actin filaments, regulating focal adhesion size and cell migration (PubMed:27693509). May play role in delivery of transport vesicles containing GPI-linked proteins from the trans-Golgi network through its interaction with GOLGA4 (PubMed:15265687). Plays a key role in wound healing and epidermal cell migration (By similarity). Required for efficient upward migration of bulge cells in response to wounding and this function is primarily rooted in its ability to coordinate microtubule dynamics and polarize hair follicle stem cells (By similarity). As a regulator of actin and microtubule arrangement and stabilization, it plays an essential role in neurite outgrowth, branching and spine formation during brain development (By similarity)","subcellular_location":"Cytoplasm; Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/Q9UPN3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MACF1","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLASP1","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MACF1","total_profiled":1310},"omim":[{"mim_id":"618325","title":"LISSENCEPHALY 9 WITH COMPLEX BRAINSTEM MALFORMATION; LIS9","url":"https://www.omim.org/entry/618325"},{"mim_id":"614453","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 7; LRRC7","url":"https://www.omim.org/entry/614453"},{"mim_id":"610861","title":"SPECTRIN REPEAT-CONTAINING NUCLEAR ENVELOPE PROTEIN 3; SYNE3","url":"https://www.omim.org/entry/610861"},{"mim_id":"608271","title":"MICROTUBULE-ACTIN CROSS-LINKING FACTOR 1; MACF1","url":"https://www.omim.org/entry/608271"},{"mim_id":"607432","title":"LISSENCEPHALY 1; LIS1","url":"https://www.omim.org/entry/607432"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Actin filaments","reliability":"Supported"},{"location":"Cell Junctions","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MACF1"},"hgnc":{"alias_symbol":["KIAA0465","ACF7","ABP620","KIAA1251","MACF","FLJ45612","FLJ46776","Lnc-PMIF"],"prev_symbol":["KIAA0754"]},"alphafold":{"accession":"Q9UPN3","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPN3","model_url":"","pae_url":"","plddt_mean":null},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MACF1","jax_strain_url":"https://www.jax.org/strain/search?query=MACF1"},"sequence":{"accession":"Q9UPN3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UPN3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UPN3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPN3"}},"corpus_meta":[{"pmid":"14636561","id":"PMC_14636561","title":"ACF7: an essential integrator of microtubule 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neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/33871731","citation_count":6,"is_preprint":false},{"pmid":"24225818","id":"PMC_24225818","title":"Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.).","date":"1989","source":"TAG. Theoretical and applied genetics. 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in<i>MACF1</i>","date":"2025-06-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.26.25330137","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.11.11.684658","title":"mRNA isoform switching plays a crucial role in mural cumulus differentiation","date":"2025-11-13","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.11.684658","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":40018,"output_tokens":7105,"usd":0.113314},"stage2":{"model":"claude-opus-4-6","input_tokens":10855,"output_tokens":4579,"usd":0.253125},"total_usd":0.366439,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"MACF1 (mACF7) contains a functional N-terminal actin-binding domain and a C-terminal domain that interacts with and stabilizes microtubules, allowing the full-length protein to associate with both actin and microtubules simultaneously in transfected cells and in vitro.\",\n      \"method\": \"In vitro binding assays, transient transfection, co-localization with cytoskeletal markers\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro binding assays plus in vivo transfection, foundational characterization paper with multiple methods\",\n      \"pmids\": [\"10601340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-terminal microtubule-binding domain of MACF1 contains at least two distinct MT-binding regions: a GAR domain that binds and partially stabilizes MTs, and a GSR-repeat domain that bundles MTs; together they bundle MTs and confer nocodazole resistance.\",\n      \"method\": \"Transient transfection studies and in vitro MT-binding assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple domain deletions tested in vivo and in vitro, strong methodological rigor\",\n      \"pmids\": [\"11112700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ACF7 (MACF1) acts as an essential integrator of microtubule-actin dynamics: it binds along microtubules and concentrates at their distal ends and cortical actin-rich sites; in its absence, MTs no longer grow along polarized actin bundles or pause/tether at cortical sites, resulting in unstable MTs, skewed trajectories, and failure to sustain polarization and coordinated migration. Rescue requires both actin- and microtubule-binding domains.\",\n      \"method\": \"Conditional knockout in endodermal cells, live-cell imaging, rescue with domain mutants\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with specific phenotypic readouts, domain-rescue epistasis, replicated across assays\",\n      \"pmids\": [\"14636561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MACF1 participates in canonical Wnt signaling: in the absence of Wnt, MACF1 associates with an Axin/β-catenin/GSK3β/APC destruction complex; upon Wnt stimulation, MACF1 mediates translocation of the Axin complex to LRP6 at the plasma membrane. MACF1 knockdown reduces nuclear β-catenin and TCF/β-catenin-dependent transcription. MACF1-null mice die at gastrulation with phenotypes resembling Wnt-3 and LRP5/6 double knockouts.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, dominant-negative constructs, TCF/β-catenin reporter assay, MACF1 knockout mouse\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, functional reporter assays, genetic KO with phenotypic analysis, multiple orthogonal methods\",\n      \"pmids\": [\"16815997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The N-terminal actin-binding domain of ACF7 (MACF1) directly binds to the tetratricopeptide repeat (TPR) domains of rapsyn at the neuromuscular junction, as demonstrated by co-expression in fibroblasts, co-immunoprecipitation, surface plasmon resonance, and blot overlay assays.\",\n      \"method\": \"Co-immunoprecipitation, surface plasmon resonance, blot overlay, confocal co-localization\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct binding confirmed by SPR and blot overlay in addition to Co-IP; multiple orthogonal methods\",\n      \"pmids\": [\"17222516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ACF7 (MACF1) possesses an intrinsic actin-regulated ATPase domain that is functionally required, beyond the F-actin, MT, and MT plus-end binding domains, for targeting microtubules along F-actin to focal adhesions, regulating focal adhesion-cytoskeletal dynamics, and enabling directed epidermal cell migration.\",\n      \"method\": \"Conditional knockout in skin epidermis, domain rescue experiments, in vitro ATPase assay, live-cell imaging of focal adhesion dynamics\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro enzymatic assay plus conditional KO and domain-specific rescue; multiple orthogonal methods in a high-impact study\",\n      \"pmids\": [\"18854161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MACF1 interacts with the TGN protein p230/Golgin-245 through its C-terminal domain (binding the N-terminal flexible end of p230), and this interaction is required for transport of GPI-anchored proteins (but not transmembrane proteins) from the TGN to the cell periphery along microtubule and actin tracts.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, dominant-negative expression of interacting domains, YFP-SP-GPI trafficking assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple validation methods including in vitro binding, Co-IP, and functional trafficking assay\",\n      \"pmids\": [\"15265687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ErbB2 receptor recruits ACF7 to the plasma membrane via a signaling pathway involving Memo, RhoA/mDia1, and GSK3 inhibition, and ACF7 localization to the plasma membrane is both necessary and sufficient for microtubule capture and stabilization downstream of ErbB2-induced cell migration.\",\n      \"method\": \"shRNA knockdown, dominant-negative and constitutively active constructs, immunofluorescence, live-cell MT tracking, rescue experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established by systematic knockdown of pathway components with specific readout (MT capture), rescue experiments\",\n      \"pmids\": [\"20937854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GSK3β directly phosphorylates ACF7 at residues within its microtubule-binding domain, uncoupling ACF7 from microtubules; Wnt signaling inhibits GSK3β, allowing ACF7-microtubule engagement. Phosphorylation-refractile ACF7 restores overall MT architecture but not polarized movement; dynamic phospho-regulation is required for stem cell directed migration and wound repair.\",\n      \"method\": \"In vitro kinase assay (GSK3β phosphorylation of ACF7), phospho-mutant rescue in ACF7-null keratinocytes, live-cell MT imaging, hair follicle SC wound-healing model\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct kinase assay, phospho-site mapping, phospho-mutant epistasis, in vivo wound-repair model; multiple orthogonal methods\",\n      \"pmids\": [\"21295697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ACF7 (and its Drosophila ortholog Shot) regulates neuronal microtubule organization (requiring both F-actin and MT binding domains) and controls filopodia formation in axonal growth cones (requiring EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C), establishing two mechanistically distinct cytoskeletal roles in axon extension.\",\n      \"method\": \"Neuronal culture systems with ACF7 knockdown/overexpression, domain deletion constructs, co-immunoprecipitation (ACF7-eIF5C interaction), live imaging of growth cones\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — domain structure-function with Co-IP for eIF5C interaction; single lab but multiple neuronal systems\",\n      \"pmids\": [\"19571116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ELMO directly interacts with ACF7 via a C-terminal polyproline segment in ELMO and the last spectrin repeat of ACF7; ELMO recruits ACF7 to the membrane to promote microtubule capture and stability, increasing persistence of cellular protrusions; this function requires the Rac GEF DOCK180.\",\n      \"method\": \"Co-immunoprecipitation, direct domain interaction mapping, live-cell imaging of MT capture, integrin-mediated cell spreading assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with domain mapping, functional MT capture assay, epistasis with DOCK180; single lab\",\n      \"pmids\": [\"23184944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MACF1 regulates cortical pyramidal neuron migration by controlling microtubule dynamics and mediating GSK-3 signaling in developing neurons; MACF1 deletion causes aberrant neuronal positioning, abnormal leading process and centrosome dynamics, and reduced microtubule stability. MACF1 interacts with GSK-3 in developing neurons.\",\n      \"method\": \"Conditional knockout (floxed allele + in utero electroporation), co-immunoprecipitation (MACF1-GSK3), live imaging of neuron migration, MT stability assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with specific neuronal phenotype, Co-IP for GSK-3 interaction, microtubule dynamics measurements\",\n      \"pmids\": [\"25224226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Src/FAK complex phosphorylates a key tyrosine residue in the calponin homology (CH) domain of ACF7's N-terminal domain, and this phosphorylation is essential for F-actin binding of ACF7 and for focal adhesion dynamics and epidermal migration in vitro and in vivo. Crystal structure of the ACF7 NT domain was resolved to inform this mechanism.\",\n      \"method\": \"X-ray crystallography of ACF7 NT domain (2.x Å), phospho-mutant rescue, Src/FAK kinase assay, in vitro and in vivo (skin epidermis) migration assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro phosphorylation assay plus phospho-mutant functional rescue in vivo\",\n      \"pmids\": [\"27216888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ACF7 (MACF1) specifically binds CAMSAP3 and is required for apical localization of CAMSAP3-decorated microtubule minus ends in intestinal epithelial cells; loss of ACF7 impairs formation of polarized epithelial cysts and disrupts apico-basal polarity.\",\n      \"method\": \"Co-immunoprecipitation (ACF7-CAMSAP3), CRISPR/RNAi knockouts, 3D epithelial cyst assays, immunofluorescence of microtubule minus-end organization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with functional KO phenotype in 3D culture; replicated findings across CAMSAP family members provide specificity\",\n      \"pmids\": [\"27802168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In Caco2 epithelial cells, ACF7 interacts with CAMSAP3 (Nezha) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments; these minus-end MTs cooperate with actin retrograde flow to maintain MT orientation perpendicular to the cell edge and regulate focal adhesion size and cell migration.\",\n      \"method\": \"Co-immunoprecipitation, MT minus-end tracking, focal adhesion size quantification, migration assays, knockdown experiments\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional assays in multiple cellular readouts; independent replication of CAMSAP3-ACF7 interaction\",\n      \"pmids\": [\"27693509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MACF1 is required for ciliogenesis: MACF1 ablation in the developing retina abolishes ciliogenesis, prevents basal body docking to ciliary vesicles, and blocks apical migration; MACF1 interacts with ciliary proteins MKKS and TALPID3, suggesting it coordinates microtubule-to-actin trafficking required for ciliogenesis.\",\n      \"method\": \"Conditional knockout (retina-specific), co-immunoprecipitation (MACF1-MKKS, MACF1-TALPID3), electron microscopy, immunofluorescence of cilia and basal bodies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with specific ciliogenesis phenotype, Co-IP of ciliary protein interactions, EM analysis\",\n      \"pmids\": [\"27783952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ACF7 loss leads to aberrant microtubule organization, tight junction stabilization, and impaired wound closure in intestinal epithelium; ablation of ACF7 in mice inhibits intestinal wound healing and increases susceptibility to experimental colitis.\",\n      \"method\": \"Conditional intestinal KO, wound scratch assays, immunofluorescence of tight junction proteins and MTs, experimental colitis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with specific intestinal phenotype, multiple cellular and in vivo readouts\",\n      \"pmids\": [\"28541346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The 2.8 Å crystal structure of the hACF7 EF1-EF2-GAR MT-binding module revealed that the EF1-EF2 domain is an EFβ-scaffold with two Ca2+ ions and the GAR domain has a unique α/β sandwich fold coordinating Zn2+; the GAR domain alone is sufficient for MT binding via a conserved basic patch distal to the Zn2+-binding site, while EF1-EF2 alone is insufficient.\",\n      \"method\": \"X-ray crystallography (2.8 Å), domain deletion/mutagenesis, MT-binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure plus mutagenesis and MT-binding assays; comprehensive structure-function analysis\",\n      \"pmids\": [\"28602822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The E3 ubiquitin ligase HectD1 targets ACF7 for proteasome-mediated degradation; HectD1 depletion stabilizes ACF7, promoting EMT and cell migration. ACF7 is essential for maintenance of the EMT program.\",\n      \"method\": \"shRNA screens, ubiquitination assays, proteasome inhibitor rescue, mouse metastasis models, breast cancer patient biopsies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay identifying HectD1 as E3 ligase for ACF7, functional KD in multiple models, in vivo metastasis\",\n      \"pmids\": [\"29386124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MACF1 variants encoding zinc-binding residues in the GAR (microtubule-binding) domain cause a distinctive lissencephaly and brainstem malformation with absent pontine crossing fibers, and affected cells show increased proportions of short cilia.\",\n      \"method\": \"Whole-exome/genome sequencing (clinical), ciliogenesis assay in patient cells\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — clinical genetics plus functional ciliogenesis assay in patient cells; limited mechanistic follow-up\",\n      \"pmids\": [\"30471716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MACF1 is concentrated at neuromuscular synapses where it binds Rapsyn and serves as a synaptic organizer for MT-associated proteins EB1 and MAP1b and actin-associated protein Vinculin; MACF1 deficiency impairs synaptic differentiation and transmission in mice.\",\n      \"method\": \"Co-immunoprecipitation with AChRs/Rapsyn, mass spectrometry, conditional NMJ-specific analysis, electrophysiology\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP/MS identification, genetic KO with electrophysiological phenotype, multiple interaction partners validated\",\n      \"pmids\": [\"30842214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MACF1 regulates GABAergic interneuron migration and positioning in the developing mouse brain; conditional MACF1 deletion reduces interneuron numbers and disrupts positioning, speed, and mode of migration, associated with decreased microtubule stability in interneurons.\",\n      \"method\": \"Conditional KO (Dlx5/6-Cre and Nkx2.1-Cre), in vivo migration tracking, MT stability assays, morphometric analysis\",\n      \"journal\": \"Cerebral cortex\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent Cre drivers, specific neuronal migration phenotype, MT stability measurements\",\n      \"pmids\": [\"27756764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In zebrafish oocytes, Macf1a localizes to the Balbiani body and mediates its dissociation via its actin-binding domain (ABD); the ABD (not the plectin repeat domain) is required for linking cortical actin to the Balbiani body mRNP granule to drive its dissociation and nuclear positioning, establishing animal-vegetal polarity.\",\n      \"method\": \"CRISPR/Cas9 endogenous domain deletion, live imaging, pharmacological disruption of actin/MTs\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR domain deletion at endogenous locus, rigorous phenotypic rescue structure, genetic epistasis with cytoskeletal drugs\",\n      \"pmids\": [\"28880872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deficiency of MACF1 in osteoblasts attenuates BMP2/Smad/Runx2 signaling, inhibiting osteoblast differentiation and bone formation in vivo.\",\n      \"method\": \"Conditional KO (Osx-Cre), primary osteoblast differentiation assays, micro-CT, western blotting for Smad/Runx2 pathway\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — conditional KO with pathway analysis; pathway placement is indirect (no direct MACF1-SMAD interaction shown)\",\n      \"pmids\": [\"31709715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MACF1 directly interacts with SMAD7 and facilitates SMAD7 nuclear translocation to initiate downstream osteogenic pathways; mesenchymal-specific MACF1 deletion reduces nuclear SMAD7 and attenuates bone formation.\",\n      \"method\": \"Co-immunoprecipitation (MACF1-SMAD7 direct interaction), nuclear-cytoplasmic fractionation, conditional KO (Prrx1-Cre), bone histomorphometry\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP for direct interaction, functional KO; single lab, moderate evidence for the direct SMAD7 interaction\",\n      \"pmids\": [\"32143362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MACF1 is an essential regulator of myonuclei positioning in skeletal muscle: it controls microtubule dynamics and density around myonuclei, regulates myonuclei motion, and its conditional muscle-specific KO causes extra-synaptic myonuclei mispositioning, NMJ fragmentation, and increased mitochondrial biogenesis.\",\n      \"method\": \"Conditional muscle-specific KO (mouse), Drosophila muscle genetics, in vitro myotube microtubule dynamics, electron microscopy of triads, calcium release assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO replicated in two organisms, MT dynamics quantification, multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"34448452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MACF1 interacts with transcription factors TCF12 and E2F6 (repressors of osteoblast differentiation) and with CDK12 and MEAF6; MACF1 sequesters TCF12 and E2F6 in the cytoplasm in a concentration-dependent manner, preventing their nuclear repressor activity and thereby promoting osteoblast differentiation.\",\n      \"method\": \"Co-immunoprecipitation (MACF1 with TCF12, E2F6, CDK12, MEAF6), nuclear-cytoplasmic fractionation, transcriptome analysis, knockdown/overexpression\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP for direct interactions, nuclear-cytoplasmic fractionation shows localization dependency on MACF1 concentration; single lab\",\n      \"pmids\": [\"33664480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP14 is a deubiquitinating enzyme that directly associates with ACF7 and inhibits its proteasomal degradation by removing ubiquitin; USP14-mediated stabilization of ACF7 promotes NSCLC cell migration.\",\n      \"method\": \"Proteomic identification (Co-IP/MS), co-immunoprecipitation, ubiquitination assay, knockdown/overexpression of USP14, migration assay\",\n      \"journal\": \"Journal of biosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus functional ubiquitination assay; single lab with moderate follow-up\",\n      \"pmids\": [\"33737492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MACF1 promotes osteoblastic cell migration by stabilizing microtubules and increasing total MAP1B mRNA expression through TCF7 transcriptional activity, while also decreasing GSK3β-mediated phosphorylation of MAP1B at T1265, thereby regulating MAP1B activity downstream of the GSK3β pathway.\",\n      \"method\": \"siRNA knockdown, luciferase reporter assay, ChIP (TCF7 binding to Map1b promoter), in vitro and in vivo migration assays, GSK3β inhibitor (LiCl)\",\n      \"journal\": \"Bone\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ChIP plus functional assays; single lab, moderate mechanistic depth\",\n      \"pmids\": [\"34700040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"De novo heterozygous missense variants in the EF-hand domains of MACF1 (in addition to the GAR domain) cause brain malformation; experimental evidence shows that EF-hand/GAR module variants increase microtubule binding, indicating a toxic gain-of-function mechanism. Bi-allelic non-GAR domain variants (mainly Plakin domain) cause a distinct complex neurodevelopmental syndrome.\",\n      \"method\": \"Whole-exome/genome sequencing, MT co-sedimentation/binding assays for EF-hand mutants, RNA-seq and ChIP of human fetal brain tissue for isoform characterization\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — functional MT-binding assays for mutants plus isoform-specific RNA-seq and ChIP; domain-specific mechanism well supported\",\n      \"pmids\": [\"40925378\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MACF1 (ACF7) is a giant spectraplakin scaffold protein that crosslinks F-actin and microtubules through distinct N-terminal actin-binding (calponin homology) and C-terminal MT-binding (GAR/GSR) domains; it guides microtubule plus ends along actin filaments to focal adhesions using an intrinsic actin-regulated ATPase activity, is regulated by GSK3β phosphorylation (itself inhibited by Wnt) and Src/FAK phosphorylation at its CH domain, interacts with the Wnt destruction complex (Axin/β-catenin/GSK3β/APC) to facilitate LRP6 signaling, binds CAMSAP3 to anchor noncentrosomal microtubule minus ends, interacts with Rapsyn at the neuromuscular junction, is targeted for degradation by the E3 ligase HectD1 and stabilized by the deubiquitinase USP14, and is required for ciliogenesis, neuronal migration, epithelial polarity, and myonuclei positioning.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MACF1 (also called ACF7) is a giant spectraplakin that crosslinks the actin and microtubule cytoskeletons to coordinate cell polarity, migration, and differentiation across diverse tissues. Its N-terminal calponin homology domains bind F-actin (regulated by Src/FAK phosphorylation) while its C-terminal GAR and GSR domains bind and bundle microtubules (regulated by GSK3β phosphorylation downstream of Wnt); an intrinsic ATPase activity guides microtubule plus ends along actin filaments toward focal adhesions, and interaction with CAMSAP3 anchors noncentrosomal microtubule minus ends to establish epithelial apico-basal polarity [PMID:10601340, PMID:18854161, PMID:21295697, PMID:27802168]. MACF1 also functions as a scaffold in canonical Wnt signaling by associating with the Axin/β-catenin/GSK3β/APC destruction complex and mediating its Wnt-dependent translocation to LRP6, and it is required for ciliogenesis through interaction with ciliary proteins MKKS and TALPID3 [PMID:16815997, PMID:27783952]. De novo missense variants in the GAR or EF-hand domains cause lissencephaly and brain malformations through gain-of-function increased microtubule binding, while bi-allelic variants in the Plakin domain cause a distinct complex neurodevelopmental syndrome [PMID:30471716, PMID:40925378].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing that MACF1 is a bona fide actin–microtubule crosslinker resolved the fundamental question of whether a single molecule could simultaneously engage both cytoskeletal systems.\",\n      \"evidence\": \"In vitro binding assays and co-localization in transfected cells demonstrated separable N-terminal actin-binding and C-terminal MT-binding domains\",\n      \"pmids\": [\"10601340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length protein binding stoichiometry unknown\", \"No functional phenotype yet established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Dissecting the C-terminal MT-binding region into GAR (binding/stabilizing) and GSR (bundling) sub-domains revealed that MACF1 uses two distinct mechanisms to engage microtubules.\",\n      \"evidence\": \"Domain deletions tested by transfection and in vitro MT-binding/bundling assays\",\n      \"pmids\": [\"11112700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of GAR and GSR recognition of MTs unresolved\", \"Physiological relevance of MT bundling activity not tested in vivo\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Conditional knockout demonstrated that MACF1 is the essential molecule that guides MT plus-end growth along polarized actin to cortical sites, answering how cells coordinate the two cytoskeletons for polarized migration.\",\n      \"evidence\": \"Genetic KO in endodermal cells with live-cell imaging; rescue required both actin- and MT-binding domains\",\n      \"pmids\": [\"14636561\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of plus-end tracking not yet explained\", \"Upstream signals controlling MACF1 cortical localization unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of p230/Golgin-245 as a MACF1 binding partner linked the crosslinker to Golgi-to-surface transport of GPI-anchored proteins, revealing a cargo-selective trafficking role.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, in vitro binding, and GPI-AP trafficking assays\",\n      \"pmids\": [\"15265687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MACF1 physically accompanies vesicles or merely organizes tracks is unknown\", \"Specificity for GPI-APs over other cargoes not mechanistically explained\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that MACF1 scaffolds the Axin/β-catenin/GSK3β destruction complex and translocates it to LRP6 upon Wnt stimulation established an unexpected role for a cytoskeletal crosslinker in canonical Wnt signal transduction.\",\n      \"evidence\": \"Reciprocal Co-IP, TCF/β-catenin reporter assays, dominant-negative constructs, and MACF1-null mouse phenocopying Wnt3/LRP5/6 double KO\",\n      \"pmids\": [\"16815997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which MACF1 domain mediates Axin interaction not mapped\", \"Whether cytoskeletal crosslinking and Wnt scaffolding are mechanistically coupled is unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Direct binding of the MACF1 actin-binding domain to Rapsyn at the neuromuscular junction extended MACF1's role to synaptic organization, linking cytoskeletal scaffolding to receptor clustering.\",\n      \"evidence\": \"Co-IP, SPR, and blot overlay confirmed direct interaction with Rapsyn TPR domains\",\n      \"pmids\": [\"17222516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of MACF1-Rapsyn disruption at NMJ not yet shown genetically\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of an intrinsic actin-regulated ATPase activity in MACF1 revealed that crosslinking alone is insufficient — active energy-dependent motility is required to target MTs along F-actin to focal adhesions.\",\n      \"evidence\": \"In vitro ATPase assay, conditional epidermal KO, domain-specific rescue experiments, and live-cell focal adhesion dynamics imaging\",\n      \"pmids\": [\"18854161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATPase domain identity and structural basis not determined\", \"Whether ATPase drives processive movement or conformational cycling unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating separate requirements for actin/MT-binding (axonal MT organization) and EF-hand/eIF5C interaction (filopodia formation) in neurons revealed MACF1 executes mechanistically distinct functions within a single cell type.\",\n      \"evidence\": \"Domain deletions and Co-IP of eIF5C in neuronal culture systems; Drosophila Shot ortholog used for cross-species validation\",\n      \"pmids\": [\"19571116\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"eIF5C interaction confirmed only by single Co-IP\", \"Translational regulation link not mechanistically elaborated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placing MACF1 downstream of ErbB2→Memo→RhoA/mDia1→GSK3 inhibition established how receptor tyrosine kinase signaling recruits MACF1 to the membrane for MT capture during directed migration.\",\n      \"evidence\": \"Systematic shRNA knockdown of pathway components with MT capture as readout, rescue experiments\",\n      \"pmids\": [\"20937854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding between pathway components and MACF1 not demonstrated\", \"Whether this pathway operates in non-breast epithelial contexts unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Direct phosphorylation of MACF1's MT-binding domain by GSK3β, inhibited by Wnt, explained how dynamic phospho-cycling controls polarized MT guidance — constitutive engagement restores MT architecture but not directionality.\",\n      \"evidence\": \"In vitro kinase assay, phospho-site mapping, phospho-mutant rescue in ACF7-null keratinocytes, in vivo wound-repair model\",\n      \"pmids\": [\"21295697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Number and identity of all functionally relevant phospho-sites not exhaustively mapped\", \"How phospho-cycling is spatially restricted within the cell unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"ELMO/DOCK180-mediated recruitment of MACF1 to the membrane via a polyproline–spectrin repeat interaction identified an alternative, integrin-triggered pathway for MT capture distinct from the ErbB2 pathway.\",\n      \"evidence\": \"Co-IP with domain mapping, live-cell MT capture imaging, integrin-mediated spreading assays\",\n      \"pmids\": [\"23184944\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab finding; independent replication not reported\", \"Relationship between ELMO and ErbB2 recruitment pathways not clarified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Conditional KO in developing cortical neurons established that MACF1 is essential for radial neuronal migration through GSK3-regulated microtubule stabilization, extending the GSK3β–MACF1 axis to brain development.\",\n      \"evidence\": \"Conditional KO with in utero electroporation, Co-IP for GSK3 interaction, live neuron migration imaging\",\n      \"pmids\": [\"25224226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GSK3 phosphorylates the same sites in neurons as in keratinocytes not confirmed\", \"Contributions of other spectraplakins not excluded\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Multiple 2016 discoveries collectively resolved MACF1's structural basis and expanded its functional repertoire: crystal structures of the N-terminal and C-terminal domains revealed how Src/FAK phosphorylation activates actin binding and how the GAR domain coordinates Zn²⁺ for MT binding; CAMSAP3 interaction anchored noncentrosomal MT minus ends for epithelial polarity; and interaction with MKKS/TALPID3 established a requirement for ciliogenesis.\",\n      \"evidence\": \"X-ray crystallography of NT and EF1-EF2-GAR domains, phospho-mutant rescue in vivo, Co-IP of CAMSAP3 and ciliary proteins, CRISPR KO in epithelial and retinal systems\",\n      \"pmids\": [\"27216888\", \"28602822\", \"27802168\", \"27693509\", \"27783952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length MACF1 structure unavailable\", \"How MACF1 coordinates CAMSAP3 binding with plus-end tracking mechanistically unresolved\", \"Structural basis of MKKS/TALPID3 interaction unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Tissue-specific KO studies demonstrated MACF1's requirement for intestinal wound healing and GABAergic interneuron migration, broadening the phenotypic landscape and confirming MT stabilization as the common downstream mechanism.\",\n      \"evidence\": \"Conditional intestinal KO with colitis model; conditional brain KO with two independent Cre drivers for interneuron analysis\",\n      \"pmids\": [\"28541346\", \"27756764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether intestinal and neuronal phenotypes involve identical molecular pathways unknown\", \"Compensatory mechanisms in heterozygous animals not characterized\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"In zebrafish oocytes, MACF1's actin-binding domain was shown to link cortical actin to the Balbiani body for animal-vegetal axis establishment, extending MACF1's role to oocyte polarity.\",\n      \"evidence\": \"Endogenous CRISPR domain deletion and live imaging in zebrafish oocytes\",\n      \"pmids\": [\"28880872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mammalian oocytes use MACF1 for similar polarity establishment unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of HectD1 as the E3 ubiquitin ligase targeting MACF1 for proteasomal degradation revealed a post-translational control mechanism; MACF1 protein stability promotes EMT and migration.\",\n      \"evidence\": \"Ubiquitination assays, proteasome inhibitor rescue, shRNA screens, in vivo metastasis models\",\n      \"pmids\": [\"29386124\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific ubiquitination sites on MACF1 not mapped\", \"Whether HectD1 and USP14 compete at the same ubiquitin chains unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Clinical genetics linked de novo GAR-domain variants to lissencephaly with pontine malformation and shortened cilia, providing the first human disease association and supporting the ciliary function.\",\n      \"evidence\": \"Whole-exome/genome sequencing in affected families plus ciliogenesis assay in patient-derived cells\",\n      \"pmids\": [\"30471716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional rescue not performed\", \"Mechanism of cilia shortening by GAR variants not elucidated at molecular level\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"NMJ-specific analysis confirmed that MACF1 organizes a cytoskeletal scaffold at the synapse (recruiting EB1, MAP1b, Vinculin via Rapsyn), and its loss impairs synaptic transmission, closing the loop opened by the 2007 Rapsyn binding discovery.\",\n      \"evidence\": \"Co-IP/MS, conditional NMJ analysis, electrophysiology\",\n      \"pmids\": [\"30842214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MACF1 loss causes NMJ disease in humans unknown\", \"Relative contributions of MT vs actin scaffolding at NMJ not separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Multiple discoveries in 2021 established MACF1 as a regulator of myonuclei positioning in muscle, identified USP14 as a stabilizing deubiquitinase opposing HectD1, and revealed cytoplasmic sequestration of transcriptional repressors TCF12/E2F6 as a mechanism for promoting osteoblast differentiation.\",\n      \"evidence\": \"Conditional muscle KO in mouse and Drosophila; Co-IP/ubiquitination assays for USP14; Co-IP and nuclear-cytoplasmic fractionation for TCF12/E2F6\",\n      \"pmids\": [\"34448452\", \"33737492\", \"33664480\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"USP14 interaction confirmed in single lab only\", \"TCF12/E2F6 sequestration mechanism is correlative — no structural basis\", \"Whether myonuclei mispositioning causes functional myopathy not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanding the genotype-phenotype map revealed that EF-hand domain variants cause brain malformation through gain-of-function increased MT binding, while bi-allelic Plakin domain variants cause a distinct neurodevelopmental syndrome, establishing domain-specific pathomechanisms.\",\n      \"evidence\": \"WES/WGS, MT co-sedimentation assays for EF-hand mutants, RNA-seq and ChIP of human fetal brain for isoform characterization\",\n      \"pmids\": [\"40925378\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Animal model validation of EF-hand gain-of-function variants not reported\", \"Mechanism by which Plakin domain variants cause disease not functionally characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structural model of MACF1 is lacking, and it remains unknown how its multiple functional domains are spatially coordinated, how its ATPase activity is structurally organized, and how tissue-specific isoforms dictate distinct cellular functions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length cryo-EM or AlphaFold-validated structure\", \"ATPase domain identity and catalytic mechanism unresolved\", \"Isoform-specific functions in different tissues not systematically compared\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 2, 5, 13, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 2, 5, 14]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 10, 12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 26]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [15, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [11, 21, 22]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [9, 11, 21]}\n    ],\n    \"complexes\": [\n      \"Wnt destruction complex (Axin/β-catenin/GSK3β/APC)\"\n    ],\n    \"partners\": [\n      \"GSK3B\",\n      \"CAMSAP3\",\n      \"RAPSN\",\n      \"HECTD1\",\n      \"USP14\",\n      \"ELMO1\",\n      \"AXIN1\",\n      \"GOLGA4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}