{"gene":"MACF1","run_date":"2026-06-10T02:59:50","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; full-length MACF1 associates with both actin and microtubules in transfected cells, establishing it as a microtubule-actin cross-linking factor.","method":"In vitro binding assays, transient transfection, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro binding assays plus cell transfection with domain constructs; replicated in subsequent studies","pmids":["10601340"],"is_preprint":false},{"year":2001,"finding":"The C-terminus of MACF1 contains at least two microtubule-binding regions: a GAR domain (which binds and partially stabilizes MTs) and a GSR-repeat domain (which bundles MTs); together they synergize to bundle and stabilize MTs against nocodazole. The plakin domain M1 region does not bind MTs in the context of surrounding sequences.","method":"Transient transfection, in vitro MT-binding assays, nocodazole resistance assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — domain dissection with in vitro binding and cell-based assays, replicated across multiple constructs","pmids":["11112700"],"is_preprint":false},{"year":2003,"finding":"ACF7/MACF1 is required for microtubules to grow along polarized actin bundles and pause/tether at actin-rich cortical sites; in ACF7-null endodermal cells, microtubules still bind EB1 and CLIP170 but show altered dynamic instability and skewed trajectories. Rescue requires both ACF7's actin- and microtubule-binding domains, establishing ACF7 as an essential integrator of MT-actin dynamics for cellular polarization and coordinated migration.","method":"ACF7 null cell lines, live imaging, domain rescue experiments","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null with domain-specific rescue, multiple orthogonal readouts, published in Cell","pmids":["14636561"],"is_preprint":false},{"year":2006,"finding":"MACF1 participates in the Wnt signaling pathway: in the absence of Wnt, MACF1 associates with an Axin/β-catenin/GSK3β/APC complex. Upon Wnt stimulation, MACF1 facilitates translocation of the Axin complex to LRP6 at the cell membrane. siRNA knockdown of MACF1 decreases nuclear β-catenin and inhibits TCF/β-catenin-dependent transcriptional activation. MACF1-null embryos phenocopy Wnt-3 and LRP5/6 double-knockout embryos (primitive streak/mesoderm defects).","method":"Co-immunoprecipitation, siRNA knockdown, luciferase reporter assay, dominant-negative constructs, MACF1 knockout mice","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional knockdown with reporter assay, genetic knockout phenotype, multiple orthogonal methods","pmids":["16815997"],"is_preprint":false},{"year":2007,"finding":"The actin-binding domain (N-terminal ABD) of ACF7/MACF1 directly binds to the tetratricopeptide repeat (TPR) domains of rapsyn, as demonstrated by co-expression in fibroblasts, co-immunoprecipitation, surface plasmon resonance, and blot overlay assays.","method":"Co-immunoprecipitation, surface plasmon resonance, blot overlay, co-expression in fibroblasts","journal":"Neuroscience","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct binding confirmed by SPR and blot overlay plus Co-IP; multiple orthogonal methods in single study","pmids":["17222516"],"is_preprint":false},{"year":2008,"finding":"ACF7 harbors an intrinsic actin-regulated ATPase domain that is both functional and essential for targeting microtubules to focal adhesions (FAs), stabilizing FA-actin networks, and enabling directional epidermal cell migration. Binding domains for F-actin, MTs, and MT plus-end proteins alone are insufficient for these functions; the ATPase activity is indispensable.","method":"Conditional ACF7 knockout in skin epidermis, ATPase activity assays, domain rescue experiments, live imaging","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay plus conditional KO with domain-specific rescue; multiple orthogonal methods","pmids":["18854161"],"is_preprint":false},{"year":2009,"finding":"ACF7 (and its Drosophila orthologue Shot) regulates neuronal microtubule organisation (requiring both F-actin and MT-binding domains) and controls filopodia formation through a distinct mechanism requiring EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C.","method":"Neuronal culture knockdown/overexpression, domain deletion analysis, co-immunoprecipitation with eIF5C","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — parallel neuronal systems with domain analysis and binding partner identification; single lab","pmids":["19571116"],"is_preprint":false},{"year":2010,"finding":"ErbB2-Memo-mDia1 signaling inhibits GSK3, allowing ACF7 to localize to the plasma membrane and ruffles in a Memo-, GSK3-, and APC-dependent manner; ACF7 at the membrane is both required and sufficient for microtubule capture downstream of ErbB2 in migrating cells. This function does not require ACF7's ATPase activity.","method":"RNAi knockdown, live-cell imaging, overexpression rescue, epistasis analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis and rescue experiments with multiple constructs; single lab","pmids":["20937854"],"is_preprint":false},{"year":2011,"finding":"GSK3β directly phosphorylates ACF7 at sites within its microtubule-binding domain, and this phosphorylation uncouples ACF7 from microtubules. Wnt signaling (which inhibits GSK3β) promotes ACF7–MT coupling. Phosphorylation-refractory ACF7 rescues microtubule architecture but not polarized movement; phosphorylation must be dynamically regulated for directed stem-cell migration during wound repair.","method":"In vitro kinase assay, phospho-site mutagenesis, phospho-mimetic and -refractory ACF7 rescue in ACF7-null skin, live imaging of hair follicle stem cells","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with site mutagenesis and in vivo rescue; multiple orthogonal methods published in Cell","pmids":["21295697"],"is_preprint":false},{"year":2012,"finding":"ELMO proteins interact directly with ACF7 via a C-terminal polyproline segment in ELMO and the last spectrin repeat of ACF7. This ELMO–ACF7 interaction recruits ACF7 to the membrane to promote microtubule capture and stability, protrusion persistence, and requires the Rac-GEF DOCK180.","method":"Co-immunoprecipitation, domain mapping, live-cell protrusion assays, microtubule imaging","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct domain mapping plus functional microtubule and protrusion assays; single lab","pmids":["23184944"],"is_preprint":false},{"year":2004,"finding":"MACF1 C-terminal domain interacts with the N-terminal domain of the Golgi protein p230/Golgin-245, confirmed by yeast two-hybrid, co-immunoprecipitation, and in vitro binding. Expression of dominant-interfering fragments of either protein disrupts transport of a GPI-anchored protein from the TGN to the cell periphery without affecting transmembrane cargo trafficking.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, dominant-negative interference in HeLa cells","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus in vitro binding plus functional assay; single lab","pmids":["15265687"],"is_preprint":false},{"year":2014,"finding":"MACF1 regulates microtubule dynamics and mediates GSK-3 signaling in developing cortical pyramidal neurons; conditional MACF1 deletion disrupts leading-process/centrosome dynamics and neuronal positioning. MACF1 physically interacts with GSK-3 in neurons.","method":"Conditional knockout mice (in utero electroporation), co-immunoprecipitation, live imaging, microtubule stability assays","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with cellular phenotype plus Co-IP; single lab","pmids":["25224226"],"is_preprint":false},{"year":2016,"finding":"Src/FAK complex phosphorylates a key tyrosine residue in ACF7's calponin homology (CH) domain, which is essential for F-actin binding of ACF7, focal adhesion dynamics, and epidermal migration in vivo. Crystal structure of ACF7's N-terminal domain was resolved at sufficient resolution to identify the tyrosine residue.","method":"Crystal structure determination, phospho-site mutagenesis, conditional knockout mice, in vitro kinase assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vivo rescue; multiple orthogonal methods in single study","pmids":["27216888"],"is_preprint":false},{"year":2016,"finding":"ACF7/MACF1 specifically binds CAMSAP3 (Nezha) at minus ends of noncentrosomal microtubules and is required for apical localization of CAMSAP3-decorated MT minus ends in intestinal epithelial cells. Loss of ACF7 disrupts polarized epithelial cyst formation in 3D culture.","method":"Co-immunoprecipitation, knockout cell lines, 3D epithelial cyst assays, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP plus KO phenotype in multiple assays; single lab","pmids":["27802168"],"is_preprint":false},{"year":2016,"finding":"ACF7/MACF1 (at microtubule minus ends via CAMSAP3 interaction) coordinates noncentrosomal microtubule–actin crosstalk through retrograde flow to maintain MT orientation perpendicular to the cell edge and regulate focal adhesion size and cell migration in Caco2 cells.","method":"Co-immunoprecipitation, siRNA knockdown, live imaging, focal adhesion analysis","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus functional imaging; single lab","pmids":["27693509"],"is_preprint":false},{"year":2016,"finding":"MACF1 ablation in the developing retina abolishes ciliogenesis: basal bodies fail to dock to ciliary vesicles or migrate apically, randomizing photoreceptor polarity. MACF1 interacts with ciliary proteins MKKS and TALPID3.","method":"Conditional knockout mice, co-immunoprecipitation, electron microscopy, immunofluorescence","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined ciliogenesis phenotype plus Co-IP; single lab","pmids":["27783952"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of the hACF7 EF1-EF2-GAR microtubule-binding module resolved at 2.8 Å: the EF1-EF2 domain is an EFβ-scaffold with two Ca2+ ions; the GAR domain has a unique α/β sandwich fold coordinating Zn2+. A conserved basic patch distal to the Zn2+-binding site mediates MT binding; EF1-EF2 alone is insufficient for MT binding.","method":"X-ray crystallography (2.8 Å), mutagenesis of MT-binding residues, MT-binding assays","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure with mutational validation of MT-binding determinants","pmids":["28602822"],"is_preprint":false},{"year":2017,"finding":"MACF1 regulates GABAergic interneuron migration and positioning in the developing brain by controlling microtubule stability and leading process dynamics; conditional deletion reduces interneuron speed, alters migration mode, and shortens leading processes and dendrites.","method":"Conditional knockout mice (Dlx5/6-Cre, Nkx2.1-Cre), live imaging, microtubule stability assays","journal":"Cerebral cortex","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined migration phenotype and MT stability assay; single lab","pmids":["27756764"],"is_preprint":false},{"year":2017,"finding":"In zebrafish oocytes, Macf1a localizes to the Balbiani Body and functions via its actin-binding domain (ABD) to mediate Balbiani Body granule dissociation and nuclear positioning; the plectin repeat domain is dispensable. Disruption of actin (not MTs) phenocopies the macf1a mutant, indicating MACF1 links cortical actin to the Balbiani Body structure.","method":"CRISPR/Cas9 domain-specific deletion, live imaging, actin/MT depolymerization, localization studies","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — CRISPR domain knockouts with direct rescue analysis, live imaging, pharmacological epistasis","pmids":["28880872"],"is_preprint":false},{"year":2018,"finding":"The E3 ubiquitin ligase HectD1 promotes ACF7/MACF1 proteasome-mediated degradation. Depletion of HectD1 stabilizes ACF7 protein levels. ACF7 is required for maintenance of the EMT program and migration; HectD1-mediated degradation of ACF7 suppresses EMT and metastasis.","method":"shRNA screens, co-immunoprecipitation, ubiquitin assays, mouse metastasis models","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional shRNA screen plus Co-IP plus in vivo metastasis model; single lab","pmids":["29386124"],"is_preprint":false},{"year":2018,"finding":"De novo missense variants in the zinc-binding residues of MACF1's GAR domain cause a distinctive lissencephaly with brainstem malformation (absent anterior commissure, W-shaped brainstem). Cells from affected individuals show a higher proportion with short cilia, linking GAR domain integrity to ciliogenesis.","method":"Whole-exome/genome sequencing, ciliogenesis assay in patient cells","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — human genetics plus functional ciliogenesis assay in patient cells; single study","pmids":["30471716"],"is_preprint":false},{"year":2019,"finding":"MACF1 is concentrated at neuromuscular synapses where it binds Rapsyn (confirmed by co-purification and interaction assays) and organizes MT-associated proteins EB1 and MAP1b and the actin-associated protein Vinculin at the synapse. MACF1 deficiency impairs synaptic differentiation and neuromuscular transmission.","method":"Biochemical co-purification (AChR pull-down), proteomic screen, conditional knockout mice, electrophysiology","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomics screen followed by co-purification, KO mouse with electrophysiological phenotype; multiple orthogonal methods","pmids":["30842214"],"is_preprint":false},{"year":2019,"finding":"MACF1 knockdown in osteoclast precursors inhibits RANKL-induced osteoclastogenesis by attenuating phosphorylation of Akt and GSK3β, thereby inhibiting NFATc1 expression. An Akt activator rescues the inhibition, placing MACF1 upstream of Akt/GSK3β/NFATc1 in the osteoclast differentiation pathway. MACF1 knockdown also disrupts actin ring formation.","method":"shRNA lentiviral knockdown, western blot, pharmacological rescue with Akt activator, pit resorption assay","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — epistasis by pharmacological rescue plus knockdown phenotype; single lab","pmids":["31260729"],"is_preprint":false},{"year":2019,"finding":"MACF1 deficiency in osteoblast-specific conditional knockout mice (Osx-Cre) reduces bone mass and impairs osteoblast differentiation. Mechanistically, loss of MACF1 attenuates BMP2/Smad/Runx2 signaling in primary osteoblasts.","method":"Conditional knockout mice, micro-CT, primary osteoblast differentiation assays, western blot for Smad/Runx2 signaling","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with pathway analysis; single lab","pmids":["31709715"],"is_preprint":false},{"year":2020,"finding":"MACF1 directly interacts with SMAD7 and facilitates SMAD7 nuclear translocation, thereby activating downstream osteogenic pathways and promoting bone formation in mesenchymal stem cells.","method":"Co-immunoprecipitation, nuclear-cytoplasmic fractionation, conditional knockout mice","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct Co-IP plus fractionation plus KO model; single lab","pmids":["32143362"],"is_preprint":false},{"year":2020,"finding":"MACF1 promotes preosteoblast migration by mediating EB1 distribution on microtubules, reducing EB1 at focal adhesions, enhancing EB1 co-localization with activated Src, and diminishing EB1-APC co-localization, collectively driving focal adhesion turnover.","method":"MACF1 knockdown/overexpression in MC3T3-E1, immunofluorescence co-localization, migration assays","journal":"Biology open","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, immunofluorescence co-localization without direct binding evidence","pmids":["32139394"],"is_preprint":false},{"year":2021,"finding":"MACF1 is an essential regulator of myonuclei positioning in skeletal muscle; conditional muscle-specific MACF1 knockout causes aberrant extra-synaptic myonuclei positioning, altered microtubule network organization around nuclei, and NMJ fragmentation. In vitro, MACF1 controls MT dynamics, contributes to MT stabilization during myofiber maturation, and regulates MT density around myonuclei. MACF1 deficiency also leads to increased mitochondria biogenesis.","method":"Conditional knockout mice, Drosophila muscle genetics, cultured myotube KD, live imaging, electron microscopy","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in mammalian and Drosophila models with mechanistic live imaging; replicated across species and systems","pmids":["34448452"],"is_preprint":false},{"year":2021,"finding":"MACF1 promotes osteoblast differentiation by sequestering transcriptional repressors CDK12, MEAF6, TCF12, and E2F6 in the cytoplasm in a concentration-dependent manner, preventing their nuclear activity. MACF1 oppositely regulates TCF12 (repressor) and TCF7 (activator) localization.","method":"Co-immunoprecipitation, nuclear-cytoplasmic fractionation, transcriptomic analysis, MACF1 knockdown","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus fractionation plus transcriptomics; single lab","pmids":["33664480"],"is_preprint":false},{"year":2021,"finding":"MACF1 promotes osteoblastic cell migration through MAP1B: MACF1 decreases phosphorylation of MAP1B-T1265 by inhibiting GSK3β activity, and upregulates total MAP1B mRNA by strengthening TCF7 binding to the Map1b promoter (confirmed by ChIP-PCR and luciferase assay).","method":"siRNA knockdown, ChIP-PCR, luciferase reporter assay, LiCl/Wortmannin pharmacological epistasis, wound healing and transwell assays","journal":"Bone","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-PCR, luciferase reporter, pharmacological epistasis; single lab","pmids":["34700040"],"is_preprint":false},{"year":2021,"finding":"USP14 is identified as a deubiquitinase for ACF7/MACF1: USP14 physically interacts with ACF7 and stabilizes ACF7 protein levels by inhibiting its ubiquitin-proteasome-dependent degradation. USP14 knockdown reduces ACF7 levels and inhibits NSCLC cell migration.","method":"Proteomic approach, co-immunoprecipitation, overexpression/knockdown, ubiquitination assay, migration assay","journal":"Journal of biosciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus functional ubiquitination and migration assays; single lab","pmids":["33737492"],"is_preprint":false},{"year":2025,"finding":"Variants in the EF-hand domains of MACF1 (outside the GAR domain) result in brain malformation; experimental evidence shows that EF-hand/GAR module variants increase microtubule binding, suggesting a toxic gain-of-function mechanism. RNA sequencing and ChIP analyses of human fetal brain reveal five MACF1 isoforms with region-specific expression differing in exon 1 transcription start sites, explaining differential cortical phenotypes.","method":"Microtubule-binding assay with variant proteins, RNA sequencing, ChIP analysis of human fetal brain","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — direct MT-binding assay for variant proteins plus isoform-specific expression data; single study","pmids":["40925378"],"is_preprint":false}],"current_model":"MACF1/ACF7 is a giant spectraplakin scaffold (~600–700 kDa) that crosslinks F-actin and microtubules through distinct N-terminal (calponin homology/ABD) and C-terminal (GAR/GSR) domains; it guides MT plus ends along actin to focal adhesions via an intrinsic ATPase activity, is regulated by GSK3β phosphorylation of its MT-binding domain (downstream of Wnt signaling) and by Src/FAK phosphorylation of its actin-binding CH domain, interacts with Axin/β-catenin/APC complexes to facilitate Wnt/LRP6 signaling, directly binds Rapsyn at the neuromuscular junction to organize synaptic cytoskeletal scaffolds, interacts with CAMSAP3 to anchor noncentrosomal microtubule minus ends, is targeted for proteasomal degradation by the E3 ligase HectD1 and stabilized by the deubiquitinase USP14, and controls diverse processes—including neuronal migration, ciliogenesis, epithelial polarity, osteoblast differentiation, and myonuclei positioning—by integrating cytoskeletal dynamics with multiple signaling pathways."},"narrative":{"mechanistic_narrative":"MACF1/ACF7 is a giant spectraplakin scaffold that physically integrates the actin and microtubule cytoskeletons to drive cell polarization, directional migration, and tissue morphogenesis [PMID:10601340, PMID:14636561]. It binds F-actin through an N-terminal calponin-homology actin-binding domain and engages and stabilizes microtubules through a C-terminal module in which a GAR domain and adjacent GSR repeats cooperate to bundle and protect MTs, with structural work resolving an EF1-EF2/GAR unit that coordinates Ca2+ and a Zn2+-binding GAR fold required for MT binding [PMID:11112700, PMID:28602822]. Beyond passive crosslinking, ACF7 carries an intrinsic actin-regulated ATPase activity that is indispensable for guiding microtubule plus ends along actin to focal adhesions and enabling directional epidermal migration [PMID:18854161]. Its cytoskeletal coupling is dynamically gated by phosphorylation: GSK3β phosphorylates the MT-binding domain to uncouple ACF7 from microtubules, a step relieved by Wnt-mediated GSK3β inhibition, while Src/FAK phosphorylates a tyrosine in the CH domain to control actin binding and focal adhesion dynamics [PMID:21295697, PMID:27216888]. ACF7 also participates directly in Wnt signaling by escorting the Axin/β-catenin/GSK3β/APC complex to LRP6 at the membrane to promote β-catenin-dependent transcription [PMID:16815997], and anchors noncentrosomal microtubule minus ends through CAMSAP3 to establish epithelial apical-basal polarity [PMID:27802168, PMID:27693509]. Through these activities it organizes the cytoskeleton at the neuromuscular junction by binding Rapsyn and clustering EB1, MAP1b, and Vinculin [PMID:30842214], positions myonuclei and shapes the perinuclear MT network in skeletal muscle [PMID:34448452], and is required for basal body docking and ciliogenesis [PMID:27783952]. ACF7 protein levels are set by an opposing E3 ligase HectD1 (degradation) and deubiquitinase USP14 (stabilization), tuning its pro-migratory and EMT-promoting output [PMID:29386124, PMID:33737492]. De novo missense variants in the GAR and EF-hand microtubule-binding module cause a distinctive lissencephaly with brainstem malformation, with variant proteins showing increased microtubule binding consistent with a toxic gain-of-function mechanism [PMID:30471716, PMID:40925378].","teleology":[{"year":1999,"claim":"Established the foundational identity of MACF1 as a bona fide cytoskeletal crosslinker by showing it physically engages both actin and microtubules through separate terminal domains.","evidence":"In vitro binding assays and domain-construct transfection with immunofluorescence","pmids":["10601340"],"confidence":"High","gaps":["Did not resolve which C-terminal subdomains mediate MT binding versus stabilization","No in vivo functional consequence established"]},{"year":2001,"claim":"Dissected the C-terminal MT-binding apparatus, distinguishing a GAR domain that binds/stabilizes MTs from a GSR-repeat domain that bundles them, defining how MACF1 stabilizes the MT network.","evidence":"Domain dissection with in vitro MT-binding and nocodazole-resistance assays in transfected cells","pmids":["11112700"],"confidence":"High","gaps":["Atomic-resolution structure of these domains not yet determined","Regulation of these binding activities unaddressed"]},{"year":2003,"claim":"Showed via genetic null and domain rescue that MACF1 is required to guide MT growth along actin and tether MTs at cortical sites, establishing it as an essential integrator of MT-actin dynamics for polarized migration.","evidence":"ACF7-null endodermal cells, live imaging, domain-specific rescue","pmids":["14636561"],"confidence":"High","gaps":["Did not explain the energetic/enzymatic basis of MT guidance","Upstream regulators of cortical tethering unknown"]},{"year":2004,"claim":"Linked MACF1 to membrane trafficking by identifying a direct interaction with the Golgi protein p230/Golgin-245 required for GPI-anchored cargo transport from the TGN.","evidence":"Yeast two-hybrid, Co-IP, in vitro binding, dominant-negative interference in HeLa cells","pmids":["15265687"],"confidence":"Medium","gaps":["Single lab, no in vivo confirmation","Mechanistic link between cytoskeletal crosslinking and cargo selectivity unresolved"]},{"year":2006,"claim":"Placed MACF1 in the Wnt pathway as a factor that escorts the Axin/β-catenin/GSK3β/APC complex to LRP6, connecting cytoskeletal scaffolding to canonical Wnt transcriptional output.","evidence":"Reciprocal Co-IP, siRNA knockdown, luciferase reporter, knockout mice phenocopying Wnt-3/LRP5/6 mutants","pmids":["16815997"],"confidence":"High","gaps":["Direct binding partner within the Axin complex not pinpointed","Whether cytoskeletal binding is needed for complex translocation unresolved"]},{"year":2007,"claim":"Defined a direct neuromuscular interaction by mapping ACF7's actin-binding domain to the TPR domains of rapsyn, implicating MACF1 in synaptic scaffold organization.","evidence":"Co-IP, surface plasmon resonance, blot overlay, co-expression in fibroblasts","pmids":["17222516"],"confidence":"High","gaps":["Functional consequence at the NMJ not yet tested in this study","Stoichiometry and regulation of the interaction unknown"]},{"year":2008,"claim":"Revealed that MACF1 is an active enzyme, not merely a passive crosslinker: an intrinsic actin-regulated ATPase is indispensable for targeting MTs to focal adhesions and directional epidermal migration.","evidence":"Conditional epidermal KO, ATPase activity assays, domain rescue, live imaging","pmids":["18854161"],"confidence":"High","gaps":["Mechanistic coupling of ATP hydrolysis to MT movement not structurally resolved","Regulators of the ATPase cycle unidentified"]},{"year":2009,"claim":"Extended MACF1 function to neuronal MT organization and identified a filopodia-formation mechanism via EF-hand motifs and the translational regulator eIF5C, separating MT-organizing from filopodial roles.","evidence":"Neuronal knockdown/overexpression, domain deletion, Co-IP with eIF5C (Drosophila Shot ortholog)","pmids":["19571116"],"confidence":"Medium","gaps":["Single lab","Mechanism by which a translational regulator couples to MACF1 unclear"]},{"year":2010,"claim":"Identified an ATPase-independent membrane-recruitment route in which ErbB2-Memo-mDia1 signaling inhibits GSK3 to localize ACF7 to the membrane for MT capture during migration.","evidence":"RNAi, live-cell imaging, overexpression rescue, epistasis","pmids":["20937854"],"confidence":"Medium","gaps":["Single lab","Direct membrane anchor for ACF7 in this context not defined"]},{"year":2011,"claim":"Pinpointed GSK3β phosphorylation of ACF7's MT-binding domain as the switch that uncouples ACF7 from MTs, establishing dynamic phosphocycling as essential for directed stem-cell migration downstream of Wnt.","evidence":"In vitro kinase assay, phospho-site mutagenesis, phospho-mimetic/refractory rescue in ACF7-null skin, live imaging","pmids":["21295697"],"confidence":"High","gaps":["Phosphatase that reverses the modification unidentified","Spatial control of phosphorylation during migration not mapped"]},{"year":2012,"claim":"Defined an ELMO-ACF7 interaction (ELMO polyproline to ACF7 last spectrin repeat) that recruits ACF7 to the membrane for MT capture and protrusion persistence, requiring the Rac-GEF DOCK180.","evidence":"Co-IP, domain mapping, live-cell protrusion and MT imaging","pmids":["23184944"],"confidence":"Medium","gaps":["Single lab","Interplay with the ErbB2-Memo recruitment route not reconciled"]},{"year":2014,"claim":"Showed MACF1 controls cortical pyramidal neuron migration via MT dynamics and physical interaction with GSK-3, extending its migratory scaffold role to brain development.","evidence":"Conditional KO (in utero electroporation), Co-IP, live imaging, MT stability assays","pmids":["25224226"],"confidence":"Medium","gaps":["Single lab","Direct phosphorylation events in neurons not delineated"]},{"year":2016,"claim":"Identified the Src/FAK-phosphorylated CH-domain tyrosine, supported by a crystal structure of the N-terminal domain, as essential for ACF7 actin binding and focal adhesion dynamics, adding a second phospho-regulatory axis distinct from GSK3β.","evidence":"Crystal structure, phospho-site mutagenesis, conditional KO mice, in vitro kinase assay","pmids":["27216888"],"confidence":"High","gaps":["How CH-domain and MT-domain phospho-switches are coordinated in time unresolved","Phosphatase counterpart unknown"]},{"year":2016,"claim":"Established that ACF7 anchors noncentrosomal MT minus ends by binding CAMSAP3, coordinating MT-actin crosstalk via retrograde flow to set epithelial polarity, MT orientation, and focal adhesion size.","evidence":"Reciprocal Co-IP, KO cell lines, 3D cyst assays, live imaging, focal adhesion analysis","pmids":["27802168","27693509"],"confidence":"Medium","gaps":["Whether minus-end anchoring uses the same domains as plus-end guidance unresolved","Single lab for the mechanistic detail"]},{"year":2016,"claim":"Demonstrated MACF1 is required for ciliogenesis, mediating basal body docking and apical migration with interactions to MKKS and TALPID3, linking the scaffold to ciliary polarity.","evidence":"Conditional KO mice, Co-IP, electron microscopy, immunofluorescence","pmids":["27783952"],"confidence":"Medium","gaps":["Single lab","Direct binding interfaces with ciliary proteins not mapped"]},{"year":2017,"claim":"Resolved the atomic structure of the EF1-EF2-GAR MT-binding module, revealing Ca2+- and Zn2+-coordinating folds and a conserved basic patch as the MT-binding determinant, providing the structural basis for MT engagement.","evidence":"X-ray crystallography at 2.8 Å with mutagenesis of MT-binding residues","pmids":["28602822"],"confidence":"High","gaps":["Structure of the full-length protein and actin-binding module in complex not solved","How phosphorylation alters the structure not captured"]},{"year":2017,"claim":"Extended the neuronal migration role to GABAergic interneurons, showing MACF1 controls MT stability and leading process dynamics during cortical interneuron positioning.","evidence":"Conditional KO mice (Dlx5/6-Cre, Nkx2.1-Cre), live imaging, MT stability assays","pmids":["27756764"],"confidence":"Medium","gaps":["Single lab","Molecular partners specific to interneurons not identified"]},{"year":2017,"claim":"Showed in zebrafish oocytes that Macf1a links cortical actin (not MTs) to the Balbiani Body for granule dissociation and nuclear positioning, revealing an actin-dependent, MT-independent function.","evidence":"CRISPR domain-specific deletion, live imaging, actin/MT depolymerization, localization","pmids":["28880872"],"confidence":"High","gaps":["Direct Balbiani Body binding partner unidentified","Conservation of this role in mammals untested in this study"]},{"year":2018,"claim":"Identified HectD1 as the E3 ligase that targets ACF7 for proteasomal degradation, establishing ubiquitin-mediated control of ACF7 levels as a brake on EMT and metastasis.","evidence":"shRNA screens, Co-IP, ubiquitin assays, mouse metastasis models","pmids":["29386124"],"confidence":"Medium","gaps":["Single lab","Ubiquitination sites on ACF7 not mapped"]},{"year":2018,"claim":"Linked MACF1 to human disease, showing de novo GAR-domain zinc-binding variants cause lissencephaly with brainstem malformation and short cilia, tying the structurally defined MT-binding module to ciliogenesis defects.","evidence":"Whole-exome/genome sequencing, ciliogenesis assay in patient cells","pmids":["30471716"],"confidence":"Medium","gaps":["Single study","Whether variants act by loss or gain of function not resolved here"]},{"year":2019,"claim":"Showed MACF1 concentrates at neuromuscular synapses, binds Rapsyn, and organizes EB1, MAP1b, and Vinculin to support synaptic differentiation and transmission, defining its synaptic cytoskeletal scaffold role in vivo.","evidence":"AChR co-purification, proteomic screen, conditional KO mice, electrophysiology","pmids":["30842214"],"confidence":"High","gaps":["Hierarchy of recruitment among the synaptic partners unresolved","Phospho-regulation at the synapse not addressed"]},{"year":2019,"claim":"Expanded MACF1 into bone biology, showing it acts upstream of Akt/GSK3β/NFATc1 in osteoclastogenesis and of BMP2/Smad/Runx2 in osteoblast differentiation.","evidence":"shRNA knockdown, conditional KO mice, micro-CT, pharmacological rescue, western blot","pmids":["31260729","31709715"],"confidence":"Medium","gaps":["Single lab per finding","Direct molecular substrate of MACF1 in these pathways not defined"]},{"year":2020,"claim":"Identified a direct MACF1-SMAD7 interaction promoting SMAD7 nuclear translocation to activate osteogenic programs, adding a cytoplasm-to-nucleus shuttling role in mesenchymal stem cells.","evidence":"Co-IP, nuclear-cytoplasmic fractionation, conditional KO mice","pmids":["32143362"],"confidence":"Medium","gaps":["Single lab","How a cytoskeletal scaffold drives nuclear import mechanistically unclear"]},{"year":2021,"claim":"Established MACF1 as essential for myonuclei positioning, perinuclear MT organization, and NMJ integrity in skeletal muscle, conserved from Drosophila to mouse.","evidence":"Conditional KO mice, Drosophila genetics, myotube knockdown, live imaging, EM","pmids":["34448452"],"confidence":"High","gaps":["Link between MACF1 loss and increased mitochondrial biogenesis unexplained","Direct nuclear-anchoring partners not identified"]},{"year":2021,"claim":"Revealed a transcriptional-control mechanism in which MACF1 sequesters repressors (CDK12, MEAF6, TCF12, E2F6) in the cytoplasm and oppositely regulates TCF12/TCF7 localization to promote osteoblast differentiation.","evidence":"Co-IP, nuclear-cytoplasmic fractionation, transcriptomics, knockdown","pmids":["33664480"],"confidence":"Medium","gaps":["Single lab","Concentration-dependent sequestration mechanism not biochemically defined"]},{"year":2021,"claim":"Detailed how MACF1 promotes osteoblast migration via MAP1B: inhibiting GSK3β to reduce MAP1B-T1265 phosphorylation and strengthening TCF7 occupancy at the Map1b promoter to raise its transcription.","evidence":"siRNA, ChIP-PCR, luciferase reporter, pharmacological epistasis, migration assays","pmids":["34700040"],"confidence":"Medium","gaps":["Single lab","Whether MACF1 directly regulates TCF7 occupancy or acts indirectly unresolved"]},{"year":2021,"claim":"Identified USP14 as the deubiquitinase that stabilizes ACF7, defining the opposing arm to HectD1 in setting ACF7 protein levels and supporting cancer cell migration.","evidence":"Proteomics, Co-IP, ubiquitination assays, knockdown/overexpression, migration assays","pmids":["33737492"],"confidence":"Medium","gaps":["Single lab","Coordination between USP14 and HectD1 on ACF7 not directly tested"]},{"year":2025,"claim":"Refined the disease mechanism by showing EF-hand/GAR variants increase microtubule binding (toxic gain-of-function) and that region-specific MACF1 isoforms with distinct exon-1 start sites explain differential cortical phenotypes.","evidence":"MT-binding assays with variant proteins, RNA sequencing, ChIP of human fetal brain","pmids":["40925378"],"confidence":"Medium","gaps":["Single study","Cellular consequence of increased MT binding in neurons not demonstrated"]},{"year":null,"claim":"How MACF1's two phospho-switches (GSK3β on the MT domain, Src/FAK on the CH domain), its ATPase cycle, ubiquitin turnover, and its many context-specific partners are integrated within a single giant molecule to produce distinct outputs across tissues remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structure linking actin- and MT-binding modules","Phosphatases reversing the regulatory phosphorylations unidentified","Mechanistic basis for tissue-specific output selection unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,2,16]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,13,24,27]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,2,21]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2,16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,7,9]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[13,14,15]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[10]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[15,20]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,8,22,23]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[11,15,17,20,26]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[19,29]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[20,30]}],"complexes":["Axin/β-catenin/GSK3β/APC destruction complex","neuromuscular junction synaptic scaffold"],"partners":["CAMSAP3","RAPSN","GSK3B","AXIN1","ELMO","SMAD7","HECTD1","USP14"],"other_free_text":[]}},"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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Candidate Gene MACF1 is Associated with Autosomal Dominant Non-syndromic Hearing Loss in an Iranian Family.","date":"2025","source":"Archives of Iranian medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40001331","citation_count":2,"is_preprint":false},{"pmid":"40925378","id":"PMC_40925378","title":"A clinical and genotype-phenotype analysis of MACF1 variants.","date":"2025","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40925378","citation_count":1,"is_preprint":false},{"pmid":"41102854","id":"PMC_41102854","title":"Transcriptional activation of MACF1 by NR2F1 drives WNT-mediated focal adhesion and metastasis in lung adenocarcinoma.","date":"2025","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/41102854","citation_count":0,"is_preprint":false},{"pmid":"39555882","id":"PMC_39555882","title":"[Myonuclear domain settings by microtubules and MACF1].","date":"2024","source":"Medecine sciences : 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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":40822,"output_tokens":7228,"usd":0.115443,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16521,"output_tokens":7870,"usd":0.139678,"stage2_stop_reason":"end_turn"},"total_usd":0.255121,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","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; full-length MACF1 associates with both actin and microtubules in transfected cells, establishing it as a microtubule-actin cross-linking factor.\",\n      \"method\": \"In vitro binding assays, transient transfection, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro binding assays plus cell transfection with domain constructs; replicated in subsequent studies\",\n      \"pmids\": [\"10601340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-terminus of MACF1 contains at least two microtubule-binding regions: a GAR domain (which binds and partially stabilizes MTs) and a GSR-repeat domain (which bundles MTs); together they synergize to bundle and stabilize MTs against nocodazole. The plakin domain M1 region does not bind MTs in the context of surrounding sequences.\",\n      \"method\": \"Transient transfection, in vitro MT-binding assays, nocodazole resistance assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — domain dissection with in vitro binding and cell-based assays, replicated across multiple constructs\",\n      \"pmids\": [\"11112700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ACF7/MACF1 is required for microtubules to grow along polarized actin bundles and pause/tether at actin-rich cortical sites; in ACF7-null endodermal cells, microtubules still bind EB1 and CLIP170 but show altered dynamic instability and skewed trajectories. Rescue requires both ACF7's actin- and microtubule-binding domains, establishing ACF7 as an essential integrator of MT-actin dynamics for cellular polarization and coordinated migration.\",\n      \"method\": \"ACF7 null cell lines, live imaging, domain rescue experiments\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null with domain-specific rescue, multiple orthogonal readouts, published in Cell\",\n      \"pmids\": [\"14636561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MACF1 participates in the Wnt signaling pathway: in the absence of Wnt, MACF1 associates with an Axin/β-catenin/GSK3β/APC complex. Upon Wnt stimulation, MACF1 facilitates translocation of the Axin complex to LRP6 at the cell membrane. siRNA knockdown of MACF1 decreases nuclear β-catenin and inhibits TCF/β-catenin-dependent transcriptional activation. MACF1-null embryos phenocopy Wnt-3 and LRP5/6 double-knockout embryos (primitive streak/mesoderm defects).\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, luciferase reporter assay, dominant-negative constructs, MACF1 knockout mice\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional knockdown with reporter assay, genetic knockout phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"16815997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The actin-binding domain (N-terminal ABD) of ACF7/MACF1 directly binds to the tetratricopeptide repeat (TPR) domains of rapsyn, 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, co-expression in fibroblasts\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct binding confirmed by SPR and blot overlay plus Co-IP; multiple orthogonal methods in single study\",\n      \"pmids\": [\"17222516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ACF7 harbors an intrinsic actin-regulated ATPase domain that is both functional and essential for targeting microtubules to focal adhesions (FAs), stabilizing FA-actin networks, and enabling directional epidermal cell migration. Binding domains for F-actin, MTs, and MT plus-end proteins alone are insufficient for these functions; the ATPase activity is indispensable.\",\n      \"method\": \"Conditional ACF7 knockout in skin epidermis, ATPase activity assays, domain rescue experiments, live imaging\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay plus conditional KO with domain-specific rescue; multiple orthogonal methods\",\n      \"pmids\": [\"18854161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ACF7 (and its Drosophila orthologue Shot) regulates neuronal microtubule organisation (requiring both F-actin and MT-binding domains) and controls filopodia formation through a distinct mechanism requiring EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C.\",\n      \"method\": \"Neuronal culture knockdown/overexpression, domain deletion analysis, co-immunoprecipitation with eIF5C\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — parallel neuronal systems with domain analysis and binding partner identification; single lab\",\n      \"pmids\": [\"19571116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ErbB2-Memo-mDia1 signaling inhibits GSK3, allowing ACF7 to localize to the plasma membrane and ruffles in a Memo-, GSK3-, and APC-dependent manner; ACF7 at the membrane is both required and sufficient for microtubule capture downstream of ErbB2 in migrating cells. This function does not require ACF7's ATPase activity.\",\n      \"method\": \"RNAi knockdown, live-cell imaging, overexpression rescue, epistasis analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis and rescue experiments with multiple constructs; single lab\",\n      \"pmids\": [\"20937854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GSK3β directly phosphorylates ACF7 at sites within its microtubule-binding domain, and this phosphorylation uncouples ACF7 from microtubules. Wnt signaling (which inhibits GSK3β) promotes ACF7–MT coupling. Phosphorylation-refractory ACF7 rescues microtubule architecture but not polarized movement; phosphorylation must be dynamically regulated for directed stem-cell migration during wound repair.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, phospho-mimetic and -refractory ACF7 rescue in ACF7-null skin, live imaging of hair follicle stem cells\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with site mutagenesis and in vivo rescue; multiple orthogonal methods published in Cell\",\n      \"pmids\": [\"21295697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ELMO proteins interact directly with ACF7 via a C-terminal polyproline segment in ELMO and the last spectrin repeat of ACF7. This ELMO–ACF7 interaction recruits ACF7 to the membrane to promote microtubule capture and stability, protrusion persistence, and requires the Rac-GEF DOCK180.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, live-cell protrusion assays, microtubule imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct domain mapping plus functional microtubule and protrusion assays; single lab\",\n      \"pmids\": [\"23184944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MACF1 C-terminal domain interacts with the N-terminal domain of the Golgi protein p230/Golgin-245, confirmed by yeast two-hybrid, co-immunoprecipitation, and in vitro binding. Expression of dominant-interfering fragments of either protein disrupts transport of a GPI-anchored protein from the TGN to the cell periphery without affecting transmembrane cargo trafficking.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, dominant-negative interference in HeLa cells\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus in vitro binding plus functional assay; single lab\",\n      \"pmids\": [\"15265687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MACF1 regulates microtubule dynamics and mediates GSK-3 signaling in developing cortical pyramidal neurons; conditional MACF1 deletion disrupts leading-process/centrosome dynamics and neuronal positioning. MACF1 physically interacts with GSK-3 in neurons.\",\n      \"method\": \"Conditional knockout mice (in utero electroporation), co-immunoprecipitation, live imaging, microtubule stability assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with cellular phenotype plus Co-IP; single lab\",\n      \"pmids\": [\"25224226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Src/FAK complex phosphorylates a key tyrosine residue in ACF7's calponin homology (CH) domain, which is essential for F-actin binding of ACF7, focal adhesion dynamics, and epidermal migration in vivo. Crystal structure of ACF7's N-terminal domain was resolved at sufficient resolution to identify the tyrosine residue.\",\n      \"method\": \"Crystal structure determination, phospho-site mutagenesis, conditional knockout mice, in vitro kinase assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vivo rescue; multiple orthogonal methods in single study\",\n      \"pmids\": [\"27216888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ACF7/MACF1 specifically binds CAMSAP3 (Nezha) at minus ends of noncentrosomal microtubules and is required for apical localization of CAMSAP3-decorated MT minus ends in intestinal epithelial cells. Loss of ACF7 disrupts polarized epithelial cyst formation in 3D culture.\",\n      \"method\": \"Co-immunoprecipitation, knockout cell lines, 3D epithelial cyst assays, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP plus KO phenotype in multiple assays; single lab\",\n      \"pmids\": [\"27802168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ACF7/MACF1 (at microtubule minus ends via CAMSAP3 interaction) coordinates noncentrosomal microtubule–actin crosstalk through retrograde flow to maintain MT orientation perpendicular to the cell edge and regulate focal adhesion size and cell migration in Caco2 cells.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, live imaging, focal adhesion analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus functional imaging; single lab\",\n      \"pmids\": [\"27693509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MACF1 ablation in the developing retina abolishes ciliogenesis: basal bodies fail to dock to ciliary vesicles or migrate apically, randomizing photoreceptor polarity. MACF1 interacts with ciliary proteins MKKS and TALPID3.\",\n      \"method\": \"Conditional knockout mice, co-immunoprecipitation, electron microscopy, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined ciliogenesis phenotype plus Co-IP; single lab\",\n      \"pmids\": [\"27783952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of the hACF7 EF1-EF2-GAR microtubule-binding module resolved at 2.8 Å: the EF1-EF2 domain is an EFβ-scaffold with two Ca2+ ions; the GAR domain has a unique α/β sandwich fold coordinating Zn2+. A conserved basic patch distal to the Zn2+-binding site mediates MT binding; EF1-EF2 alone is insufficient for MT binding.\",\n      \"method\": \"X-ray crystallography (2.8 Å), mutagenesis of MT-binding residues, MT-binding assays\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure with mutational validation of MT-binding determinants\",\n      \"pmids\": [\"28602822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MACF1 regulates GABAergic interneuron migration and positioning in the developing brain by controlling microtubule stability and leading process dynamics; conditional deletion reduces interneuron speed, alters migration mode, and shortens leading processes and dendrites.\",\n      \"method\": \"Conditional knockout mice (Dlx5/6-Cre, Nkx2.1-Cre), live imaging, microtubule stability assays\",\n      \"journal\": \"Cerebral cortex\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined migration phenotype and MT stability assay; single lab\",\n      \"pmids\": [\"27756764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In zebrafish oocytes, Macf1a localizes to the Balbiani Body and functions via its actin-binding domain (ABD) to mediate Balbiani Body granule dissociation and nuclear positioning; the plectin repeat domain is dispensable. Disruption of actin (not MTs) phenocopies the macf1a mutant, indicating MACF1 links cortical actin to the Balbiani Body structure.\",\n      \"method\": \"CRISPR/Cas9 domain-specific deletion, live imaging, actin/MT depolymerization, localization studies\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — CRISPR domain knockouts with direct rescue analysis, live imaging, pharmacological epistasis\",\n      \"pmids\": [\"28880872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The E3 ubiquitin ligase HectD1 promotes ACF7/MACF1 proteasome-mediated degradation. Depletion of HectD1 stabilizes ACF7 protein levels. ACF7 is required for maintenance of the EMT program and migration; HectD1-mediated degradation of ACF7 suppresses EMT and metastasis.\",\n      \"method\": \"shRNA screens, co-immunoprecipitation, ubiquitin assays, mouse metastasis models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional shRNA screen plus Co-IP plus in vivo metastasis model; single lab\",\n      \"pmids\": [\"29386124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"De novo missense variants in the zinc-binding residues of MACF1's GAR domain cause a distinctive lissencephaly with brainstem malformation (absent anterior commissure, W-shaped brainstem). Cells from affected individuals show a higher proportion with short cilia, linking GAR domain integrity to ciliogenesis.\",\n      \"method\": \"Whole-exome/genome sequencing, ciliogenesis assay in patient cells\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — human genetics plus functional ciliogenesis assay in patient cells; single study\",\n      \"pmids\": [\"30471716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MACF1 is concentrated at neuromuscular synapses where it binds Rapsyn (confirmed by co-purification and interaction assays) and organizes MT-associated proteins EB1 and MAP1b and the actin-associated protein Vinculin at the synapse. MACF1 deficiency impairs synaptic differentiation and neuromuscular transmission.\",\n      \"method\": \"Biochemical co-purification (AChR pull-down), proteomic screen, conditional knockout mice, electrophysiology\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomics screen followed by co-purification, KO mouse with electrophysiological phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"30842214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MACF1 knockdown in osteoclast precursors inhibits RANKL-induced osteoclastogenesis by attenuating phosphorylation of Akt and GSK3β, thereby inhibiting NFATc1 expression. An Akt activator rescues the inhibition, placing MACF1 upstream of Akt/GSK3β/NFATc1 in the osteoclast differentiation pathway. MACF1 knockdown also disrupts actin ring formation.\",\n      \"method\": \"shRNA lentiviral knockdown, western blot, pharmacological rescue with Akt activator, pit resorption assay\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — epistasis by pharmacological rescue plus knockdown phenotype; single lab\",\n      \"pmids\": [\"31260729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MACF1 deficiency in osteoblast-specific conditional knockout mice (Osx-Cre) reduces bone mass and impairs osteoblast differentiation. Mechanistically, loss of MACF1 attenuates BMP2/Smad/Runx2 signaling in primary osteoblasts.\",\n      \"method\": \"Conditional knockout mice, micro-CT, primary osteoblast differentiation assays, western blot for Smad/Runx2 signaling\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with pathway analysis; single lab\",\n      \"pmids\": [\"31709715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MACF1 directly interacts with SMAD7 and facilitates SMAD7 nuclear translocation, thereby activating downstream osteogenic pathways and promoting bone formation in mesenchymal stem cells.\",\n      \"method\": \"Co-immunoprecipitation, nuclear-cytoplasmic fractionation, conditional knockout mice\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct Co-IP plus fractionation plus KO model; single lab\",\n      \"pmids\": [\"32143362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MACF1 promotes preosteoblast migration by mediating EB1 distribution on microtubules, reducing EB1 at focal adhesions, enhancing EB1 co-localization with activated Src, and diminishing EB1-APC co-localization, collectively driving focal adhesion turnover.\",\n      \"method\": \"MACF1 knockdown/overexpression in MC3T3-E1, immunofluorescence co-localization, migration assays\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, immunofluorescence co-localization without direct binding evidence\",\n      \"pmids\": [\"32139394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MACF1 is an essential regulator of myonuclei positioning in skeletal muscle; conditional muscle-specific MACF1 knockout causes aberrant extra-synaptic myonuclei positioning, altered microtubule network organization around nuclei, and NMJ fragmentation. In vitro, MACF1 controls MT dynamics, contributes to MT stabilization during myofiber maturation, and regulates MT density around myonuclei. MACF1 deficiency also leads to increased mitochondria biogenesis.\",\n      \"method\": \"Conditional knockout mice, Drosophila muscle genetics, cultured myotube KD, live imaging, electron microscopy\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in mammalian and Drosophila models with mechanistic live imaging; replicated across species and systems\",\n      \"pmids\": [\"34448452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MACF1 promotes osteoblast differentiation by sequestering transcriptional repressors CDK12, MEAF6, TCF12, and E2F6 in the cytoplasm in a concentration-dependent manner, preventing their nuclear activity. MACF1 oppositely regulates TCF12 (repressor) and TCF7 (activator) localization.\",\n      \"method\": \"Co-immunoprecipitation, nuclear-cytoplasmic fractionation, transcriptomic analysis, MACF1 knockdown\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus fractionation plus transcriptomics; single lab\",\n      \"pmids\": [\"33664480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MACF1 promotes osteoblastic cell migration through MAP1B: MACF1 decreases phosphorylation of MAP1B-T1265 by inhibiting GSK3β activity, and upregulates total MAP1B mRNA by strengthening TCF7 binding to the Map1b promoter (confirmed by ChIP-PCR and luciferase assay).\",\n      \"method\": \"siRNA knockdown, ChIP-PCR, luciferase reporter assay, LiCl/Wortmannin pharmacological epistasis, wound healing and transwell assays\",\n      \"journal\": \"Bone\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-PCR, luciferase reporter, pharmacological epistasis; single lab\",\n      \"pmids\": [\"34700040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP14 is identified as a deubiquitinase for ACF7/MACF1: USP14 physically interacts with ACF7 and stabilizes ACF7 protein levels by inhibiting its ubiquitin-proteasome-dependent degradation. USP14 knockdown reduces ACF7 levels and inhibits NSCLC cell migration.\",\n      \"method\": \"Proteomic approach, co-immunoprecipitation, overexpression/knockdown, ubiquitination assay, migration assay\",\n      \"journal\": \"Journal of biosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus functional ubiquitination and migration assays; single lab\",\n      \"pmids\": [\"33737492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Variants in the EF-hand domains of MACF1 (outside the GAR domain) result in brain malformation; experimental evidence shows that EF-hand/GAR module variants increase microtubule binding, suggesting a toxic gain-of-function mechanism. RNA sequencing and ChIP analyses of human fetal brain reveal five MACF1 isoforms with region-specific expression differing in exon 1 transcription start sites, explaining differential cortical phenotypes.\",\n      \"method\": \"Microtubule-binding assay with variant proteins, RNA sequencing, ChIP analysis of human fetal brain\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct MT-binding assay for variant proteins plus isoform-specific expression data; single study\",\n      \"pmids\": [\"40925378\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MACF1/ACF7 is a giant spectraplakin scaffold (~600–700 kDa) that crosslinks F-actin and microtubules through distinct N-terminal (calponin homology/ABD) and C-terminal (GAR/GSR) domains; it guides MT plus ends along actin to focal adhesions via an intrinsic ATPase activity, is regulated by GSK3β phosphorylation of its MT-binding domain (downstream of Wnt signaling) and by Src/FAK phosphorylation of its actin-binding CH domain, interacts with Axin/β-catenin/APC complexes to facilitate Wnt/LRP6 signaling, directly binds Rapsyn at the neuromuscular junction to organize synaptic cytoskeletal scaffolds, interacts with CAMSAP3 to anchor noncentrosomal microtubule minus ends, is targeted for proteasomal degradation by the E3 ligase HectD1 and stabilized by the deubiquitinase USP14, and controls diverse processes—including neuronal migration, ciliogenesis, epithelial polarity, osteoblast differentiation, and myonuclei positioning—by integrating cytoskeletal dynamics with multiple signaling pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MACF1/ACF7 is a giant spectraplakin scaffold that physically integrates the actin and microtubule cytoskeletons to drive cell polarization, directional migration, and tissue morphogenesis [#0, #2]. It binds F-actin through an N-terminal calponin-homology actin-binding domain and engages and stabilizes microtubules through a C-terminal module in which a GAR domain and adjacent GSR repeats cooperate to bundle and protect MTs, with structural work resolving an EF1-EF2/GAR unit that coordinates Ca2+ and a Zn2+-binding GAR fold required for MT binding [#1, #16]. Beyond passive crosslinking, ACF7 carries an intrinsic actin-regulated ATPase activity that is indispensable for guiding microtubule plus ends along actin to focal adhesions and enabling directional epidermal migration [#5]. Its cytoskeletal coupling is dynamically gated by phosphorylation: GSK3\\u03b2 phosphorylates the MT-binding domain to uncouple ACF7 from microtubules, a step relieved by Wnt-mediated GSK3\\u03b2 inhibition, while Src/FAK phosphorylates a tyrosine in the CH domain to control actin binding and focal adhesion dynamics [#8, #12]. ACF7 also participates directly in Wnt signaling by escorting the Axin/\\u03b2-catenin/GSK3\\u03b2/APC complex to LRP6 at the membrane to promote \\u03b2-catenin-dependent transcription [#3], and anchors noncentrosomal microtubule minus ends through CAMSAP3 to establish epithelial apical-basal polarity [#13, #14]. Through these activities it organizes the cytoskeleton at the neuromuscular junction by binding Rapsyn and clustering EB1, MAP1b, and Vinculin [#21], positions myonuclei and shapes the perinuclear MT network in skeletal muscle [#26], and is required for basal body docking and ciliogenesis [#15]. ACF7 protein levels are set by an opposing E3 ligase HectD1 (degradation) and deubiquitinase USP14 (stabilization), tuning its pro-migratory and EMT-promoting output [#19, #29]. De novo missense variants in the GAR and EF-hand microtubule-binding module cause a distinctive lissencephaly with brainstem malformation, with variant proteins showing increased microtubule binding consistent with a toxic gain-of-function mechanism [#20, #30].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the foundational identity of MACF1 as a bona fide cytoskeletal crosslinker by showing it physically engages both actin and microtubules through separate terminal domains.\",\n      \"evidence\": \"In vitro binding assays and domain-construct transfection with immunofluorescence\",\n      \"pmids\": [\"10601340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which C-terminal subdomains mediate MT binding versus stabilization\", \"No in vivo functional consequence established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Dissected the C-terminal MT-binding apparatus, distinguishing a GAR domain that binds/stabilizes MTs from a GSR-repeat domain that bundles them, defining how MACF1 stabilizes the MT network.\",\n      \"evidence\": \"Domain dissection with in vitro MT-binding and nocodazole-resistance assays in transfected cells\",\n      \"pmids\": [\"11112700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of these domains not yet determined\", \"Regulation of these binding activities unaddressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed via genetic null and domain rescue that MACF1 is required to guide MT growth along actin and tether MTs at cortical sites, establishing it as an essential integrator of MT-actin dynamics for polarized migration.\",\n      \"evidence\": \"ACF7-null endodermal cells, live imaging, domain-specific rescue\",\n      \"pmids\": [\"14636561\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain the energetic/enzymatic basis of MT guidance\", \"Upstream regulators of cortical tethering unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linked MACF1 to membrane trafficking by identifying a direct interaction with the Golgi protein p230/Golgin-245 required for GPI-anchored cargo transport from the TGN.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, in vitro binding, dominant-negative interference in HeLa cells\",\n      \"pmids\": [\"15265687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, no in vivo confirmation\", \"Mechanistic link between cytoskeletal crosslinking and cargo selectivity unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed MACF1 in the Wnt pathway as a factor that escorts the Axin/\\u03b2-catenin/GSK3\\u03b2/APC complex to LRP6, connecting cytoskeletal scaffolding to canonical Wnt transcriptional output.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA knockdown, luciferase reporter, knockout mice phenocopying Wnt-3/LRP5/6 mutants\",\n      \"pmids\": [\"16815997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding partner within the Axin complex not pinpointed\", \"Whether cytoskeletal binding is needed for complex translocation unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined a direct neuromuscular interaction by mapping ACF7's actin-binding domain to the TPR domains of rapsyn, implicating MACF1 in synaptic scaffold organization.\",\n      \"evidence\": \"Co-IP, surface plasmon resonance, blot overlay, co-expression in fibroblasts\",\n      \"pmids\": [\"17222516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence at the NMJ not yet tested in this study\", \"Stoichiometry and regulation of the interaction unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed that MACF1 is an active enzyme, not merely a passive crosslinker: an intrinsic actin-regulated ATPase is indispensable for targeting MTs to focal adhesions and directional epidermal migration.\",\n      \"evidence\": \"Conditional epidermal KO, ATPase activity assays, domain rescue, live imaging\",\n      \"pmids\": [\"18854161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic coupling of ATP hydrolysis to MT movement not structurally resolved\", \"Regulators of the ATPase cycle unidentified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended MACF1 function to neuronal MT organization and identified a filopodia-formation mechanism via EF-hand motifs and the translational regulator eIF5C, separating MT-organizing from filopodial roles.\",\n      \"evidence\": \"Neuronal knockdown/overexpression, domain deletion, Co-IP with eIF5C (Drosophila Shot ortholog)\",\n      \"pmids\": [\"19571116\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Mechanism by which a translational regulator couples to MACF1 unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified an ATPase-independent membrane-recruitment route in which ErbB2-Memo-mDia1 signaling inhibits GSK3 to localize ACF7 to the membrane for MT capture during migration.\",\n      \"evidence\": \"RNAi, live-cell imaging, overexpression rescue, epistasis\",\n      \"pmids\": [\"20937854\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct membrane anchor for ACF7 in this context not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Pinpointed GSK3\\u03b2 phosphorylation of ACF7's MT-binding domain as the switch that uncouples ACF7 from MTs, establishing dynamic phosphocycling as essential for directed stem-cell migration downstream of Wnt.\",\n      \"evidence\": \"In vitro kinase assay, phospho-site mutagenesis, phospho-mimetic/refractory rescue in ACF7-null skin, live imaging\",\n      \"pmids\": [\"21295697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase that reverses the modification unidentified\", \"Spatial control of phosphorylation during migration not mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined an ELMO-ACF7 interaction (ELMO polyproline to ACF7 last spectrin repeat) that recruits ACF7 to the membrane for MT capture and protrusion persistence, requiring the Rac-GEF DOCK180.\",\n      \"evidence\": \"Co-IP, domain mapping, live-cell protrusion and MT imaging\",\n      \"pmids\": [\"23184944\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Interplay with the ErbB2-Memo recruitment route not reconciled\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed MACF1 controls cortical pyramidal neuron migration via MT dynamics and physical interaction with GSK-3, extending its migratory scaffold role to brain development.\",\n      \"evidence\": \"Conditional KO (in utero electroporation), Co-IP, live imaging, MT stability assays\",\n      \"pmids\": [\"25224226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct phosphorylation events in neurons not delineated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified the Src/FAK-phosphorylated CH-domain tyrosine, supported by a crystal structure of the N-terminal domain, as essential for ACF7 actin binding and focal adhesion dynamics, adding a second phospho-regulatory axis distinct from GSK3\\u03b2.\",\n      \"evidence\": \"Crystal structure, phospho-site mutagenesis, conditional KO mice, in vitro kinase assay\",\n      \"pmids\": [\"27216888\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CH-domain and MT-domain phospho-switches are coordinated in time unresolved\", \"Phosphatase counterpart unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established that ACF7 anchors noncentrosomal MT minus ends by binding CAMSAP3, coordinating MT-actin crosstalk via retrograde flow to set epithelial polarity, MT orientation, and focal adhesion size.\",\n      \"evidence\": \"Reciprocal Co-IP, KO cell lines, 3D cyst assays, live imaging, focal adhesion analysis\",\n      \"pmids\": [\"27802168\", \"27693509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether minus-end anchoring uses the same domains as plus-end guidance unresolved\", \"Single lab for the mechanistic detail\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated MACF1 is required for ciliogenesis, mediating basal body docking and apical migration with interactions to MKKS and TALPID3, linking the scaffold to ciliary polarity.\",\n      \"evidence\": \"Conditional KO mice, Co-IP, electron microscopy, immunofluorescence\",\n      \"pmids\": [\"27783952\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct binding interfaces with ciliary proteins not mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the atomic structure of the EF1-EF2-GAR MT-binding module, revealing Ca2+- and Zn2+-coordinating folds and a conserved basic patch as the MT-binding determinant, providing the structural basis for MT engagement.\",\n      \"evidence\": \"X-ray crystallography at 2.8 \\u00c5 with mutagenesis of MT-binding residues\",\n      \"pmids\": [\"28602822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full-length protein and actin-binding module in complex not solved\", \"How phosphorylation alters the structure not captured\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended the neuronal migration role to GABAergic interneurons, showing MACF1 controls MT stability and leading process dynamics during cortical interneuron positioning.\",\n      \"evidence\": \"Conditional KO mice (Dlx5/6-Cre, Nkx2.1-Cre), live imaging, MT stability assays\",\n      \"pmids\": [\"27756764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Molecular partners specific to interneurons not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed in zebrafish oocytes that Macf1a links cortical actin (not MTs) to the Balbiani Body for granule dissociation and nuclear positioning, revealing an actin-dependent, MT-independent function.\",\n      \"evidence\": \"CRISPR domain-specific deletion, live imaging, actin/MT depolymerization, localization\",\n      \"pmids\": [\"28880872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Balbiani Body binding partner unidentified\", \"Conservation of this role in mammals untested in this study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified HectD1 as the E3 ligase that targets ACF7 for proteasomal degradation, establishing ubiquitin-mediated control of ACF7 levels as a brake on EMT and metastasis.\",\n      \"evidence\": \"shRNA screens, Co-IP, ubiquitin assays, mouse metastasis models\",\n      \"pmids\": [\"29386124\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Ubiquitination sites on ACF7 not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked MACF1 to human disease, showing de novo GAR-domain zinc-binding variants cause lissencephaly with brainstem malformation and short cilia, tying the structurally defined MT-binding module to ciliogenesis defects.\",\n      \"evidence\": \"Whole-exome/genome sequencing, ciliogenesis assay in patient cells\",\n      \"pmids\": [\"30471716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study\", \"Whether variants act by loss or gain of function not resolved here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed MACF1 concentrates at neuromuscular synapses, binds Rapsyn, and organizes EB1, MAP1b, and Vinculin to support synaptic differentiation and transmission, defining its synaptic cytoskeletal scaffold role in vivo.\",\n      \"evidence\": \"AChR co-purification, proteomic screen, conditional KO mice, electrophysiology\",\n      \"pmids\": [\"30842214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy of recruitment among the synaptic partners unresolved\", \"Phospho-regulation at the synapse not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanded MACF1 into bone biology, showing it acts upstream of Akt/GSK3\\u03b2/NFATc1 in osteoclastogenesis and of BMP2/Smad/Runx2 in osteoblast differentiation.\",\n      \"evidence\": \"shRNA knockdown, conditional KO mice, micro-CT, pharmacological rescue, western blot\",\n      \"pmids\": [\"31260729\", \"31709715\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab per finding\", \"Direct molecular substrate of MACF1 in these pathways not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified a direct MACF1-SMAD7 interaction promoting SMAD7 nuclear translocation to activate osteogenic programs, adding a cytoplasm-to-nucleus shuttling role in mesenchymal stem cells.\",\n      \"evidence\": \"Co-IP, nuclear-cytoplasmic fractionation, conditional KO mice\",\n      \"pmids\": [\"32143362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How a cytoskeletal scaffold drives nuclear import mechanistically unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established MACF1 as essential for myonuclei positioning, perinuclear MT organization, and NMJ integrity in skeletal muscle, conserved from Drosophila to mouse.\",\n      \"evidence\": \"Conditional KO mice, Drosophila genetics, myotube knockdown, live imaging, EM\",\n      \"pmids\": [\"34448452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link between MACF1 loss and increased mitochondrial biogenesis unexplained\", \"Direct nuclear-anchoring partners not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a transcriptional-control mechanism in which MACF1 sequesters repressors (CDK12, MEAF6, TCF12, E2F6) in the cytoplasm and oppositely regulates TCF12/TCF7 localization to promote osteoblast differentiation.\",\n      \"evidence\": \"Co-IP, nuclear-cytoplasmic fractionation, transcriptomics, knockdown\",\n      \"pmids\": [\"33664480\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Concentration-dependent sequestration mechanism not biochemically defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Detailed how MACF1 promotes osteoblast migration via MAP1B: inhibiting GSK3\\u03b2 to reduce MAP1B-T1265 phosphorylation and strengthening TCF7 occupancy at the Map1b promoter to raise its transcription.\",\n      \"evidence\": \"siRNA, ChIP-PCR, luciferase reporter, pharmacological epistasis, migration assays\",\n      \"pmids\": [\"34700040\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether MACF1 directly regulates TCF7 occupancy or acts indirectly unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified USP14 as the deubiquitinase that stabilizes ACF7, defining the opposing arm to HectD1 in setting ACF7 protein levels and supporting cancer cell migration.\",\n      \"evidence\": \"Proteomics, Co-IP, ubiquitination assays, knockdown/overexpression, migration assays\",\n      \"pmids\": [\"33737492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Coordination between USP14 and HectD1 on ACF7 not directly tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Refined the disease mechanism by showing EF-hand/GAR variants increase microtubule binding (toxic gain-of-function) and that region-specific MACF1 isoforms with distinct exon-1 start sites explain differential cortical phenotypes.\",\n      \"evidence\": \"MT-binding assays with variant proteins, RNA sequencing, ChIP of human fetal brain\",\n      \"pmids\": [\"40925378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study\", \"Cellular consequence of increased MT binding in neurons not demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MACF1's two phospho-switches (GSK3\\u03b2 on the MT domain, Src/FAK on the CH domain), its ATPase cycle, ubiquitin turnover, and its many context-specific partners are integrated within a single giant molecule to produce distinct outputs across tissues remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure linking actin- and MT-binding modules\", \"Phosphatases reversing the regulatory phosphorylations unidentified\", \"Mechanistic basis for tissue-specific output selection unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 2, 16]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 13, 24, 27]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2, 16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 7, 9]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [13, 14, 15]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [15, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 8, 22, 23]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [11, 15, 17, 20, 26]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [19, 29]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [20, 30]}\n    ],\n    \"complexes\": [\"Axin/\\u03b2-catenin/GSK3\\u03b2/APC destruction complex\", \"neuromuscular junction synaptic scaffold\"],\n    \"partners\": [\"CAMSAP3\", \"RAPSN\", \"GSK3B\", \"AXIN1\", \"ELMO\", \"SMAD7\", \"HECTD1\", \"USP14\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}