{"gene":"TUBB6","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2019,"finding":"Overexpression of GFP-tubb6 (but not GFP-tubb5) in wild-type skeletal muscle fibers causes microtubule disorganization, and depletion of tubb6 (but not tubb5) in mdx muscle fibers normalizes microtubule architecture, demonstrating that tubb6 specifically disrupts the ordered microtubule grid when chronically elevated.","method":"GFP-tubb6 overexpression in isolated muscle fibers; shRNA knockdown of tubb6 vs tubb5 in mdx fibers; fluorescence microscopy of microtubule organization","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain-of-function and loss-of-function experiments with isotype-specific controls, replicated across mouse and human muscle models","pmids":["30535187"],"is_preprint":false},{"year":2019,"finding":"Tubb6 expression is selectively upregulated during muscle differentiation and regeneration (not in mature fibers), co-correlating with embryonic myosin heavy chain levels, positioning tubb6 as a regeneration-associated tubulin isotype.","method":"Immunofluorescence and western blot in mdx and cardiotoxin-injury mouse models; human DMD and myositis biopsies; correlation with embryonic myosin heavy chain (regeneration marker)","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple models and markers used in one study, but single lab","pmids":["30535187"],"is_preprint":false},{"year":2017,"finding":"A missense mutation in TUBB6 (p.Phe394Ser) causes autosomal dominant congenital cranial dysinnervation disorder; expression of the mutant protein in yeast impairs viability under microtubule-poison (benomyl) challenge, indicating the mutation compromises microtubule function.","method":"Yeast growth assay with benomyl; co-segregation analysis in five-generation pedigree","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast functional assay plus human genetic evidence, single lab","pmids":["29016863"],"is_preprint":false},{"year":2021,"finding":"TUBB6 controls microtubule dynamics in osteoclasts: CRISPR/Cas9 KO of Tubb6 reduces microtubule growth speed, increases microtubule growth lifetime, elevates acetylated α-tubulin, and produces smaller EB1-caps, indicating that TUBB6 promotes dynamic (less stable) microtubules.","method":"CRISPR/Cas9 knockout in osteoclast model; live-cell microtubule dynamics assay (EB1 tracking); immunofluorescence for acetylated tubulin","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple orthogonal readouts of microtubule dynamics in same study","pmids":["34869381"],"is_preprint":false},{"year":2021,"finding":"TUBB6 controls actin podosome dynamics in osteoclasts: Tubb6 KO increases individual podosome lifetime while destabilizing the podosome belt, linking TUBB6-regulated microtubule dynamics to actin cytoskeleton organization required for bone resorption.","method":"CRISPR/Cas9 knockout; live-cell imaging of podosome dynamics; bone resorption assay","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with direct imaging of podosome dynamics and functional bone resorption readout, single lab","pmids":["34869381"],"is_preprint":false},{"year":2021,"finding":"Proteomic analysis of microtubule-associated protein fractions revealed that ARHGAP10 (a negative regulator of CDC42) associates with microtubules, and this association is negatively regulated by TUBB6, suggesting a mechanism by which TUBB6 locally controls CDC42 activity and actin organization.","method":"Proteomic analysis of microtubule-associated protein-enriched fractions from Tubb6 KO vs wild-type osteoclasts","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — mass spectrometry-based enriched fraction analysis, single lab, functional inference supported by KO phenotype","pmids":["34869381"],"is_preprint":false},{"year":2025,"finding":"ARHGAP10 directly binds microtubules through its BAR-PH domain (requiring lysine residues K37, K41, K44 in the BAR domain), and TUBB6 negatively regulates this binding; ARHGAP10 KO impairs actin ring dynamics and osteoclast resorption activity, and complementation requires both microtubule binding and RHO-GTPase regulatory capacity.","method":"CRISPR/Cas9 KO; in vitro microtubule binding assay with BAR domain mutants; osteoclast resorption assay; complementation experiments","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro microtubule binding with mutagenesis is Tier 1, but preprint single lab","pmids":[],"is_preprint":true},{"year":2025,"finding":"Tubb6 mRNA is selectively expressed in Xenopus multiciliated cells (MCCs), and its protein localizes to ciliary axonemes; morpholino-mediated depletion of Tubb6 markedly reduces cilia number and length in MCCs causing defective ciliary motility, while mono-motile cilia in the gastrocoel roof plate are unaffected, revealing a cell-type-specific requirement for Tubb6 in motile cilia formation.","method":"Morpholino knockdown in Xenopus embryos; fluorescence imaging of cilia; in situ hybridization for mRNA localization; immunofluorescence for axonemal localization","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with direct protein localization and functional ciliary motility readout, peer-reviewed, Xenopus ortholog consistent with vertebrate TUBB6","pmids":["41074676"],"is_preprint":false},{"year":2025,"finding":"YBX1 binds the 3' UTR of TUBB6 mRNA and stabilizes it, functioning as an upstream post-transcriptional regulator that increases TUBB6 protein levels; TUBB6 knockdown in endothelial cells suppresses WNT signaling (specifically WNT3A and FZD8 expression) and reduces tip cell and proliferative cell marker expression, inhibiting sprouting angiogenesis.","method":"RNA binding assay (YBX1-TUBB6 3'UTR interaction); TUBB6 KO endothelial cell functional assays (migration, tube formation, flow cytometry); OIR and CNV in vivo mouse models with Tubb6 silencing; transcriptome analysis","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA binding assay plus KO with multiple functional readouts in vitro and in vivo, single lab","pmids":["40083923"],"is_preprint":false},{"year":2025,"finding":"NFKB1 binds the promoter region of TUBB6 and transcriptionally upregulates its expression in glioma cells; NFKB1 knockdown reduces TUBB6 expression, and TUBB6 knockdown suppresses glioma cell proliferation and promotes apoptosis via the Wnt/β-catenin signaling pathway.","method":"Promoter binding assay/ChIP-type analysis; NFKB1 knockdown with TUBB6 expression measurement; TUBB6 knockdown with cell proliferation, apoptosis, and Wnt/β-catenin pathway readouts in glioma cell lines","journal":"Discover oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcription factor-promoter binding plus knockdown with pathway readouts, single lab","pmids":["40169445"],"is_preprint":false},{"year":2025,"finding":"TUBB6 inhibition by antisense oligonucleotide (ASO) in a rat intracerebral hemorrhage model reduces hematoma volume, restores acetylated α-tubulin (microtubule stability marker), suppresses MAPK signaling, decreases pro-inflammatory cytokines, and reduces neuronal degeneration, demonstrating that elevated TUBB6 destabilizes microtubules and promotes neuroinflammation after hemorrhage.","method":"Antisense oligonucleotide inhibition in collagenase-injection ICH rat model; immunohistochemistry for acetylated α-tubulin; ELISA for cytokines; Western blot for MAPK pathway; Fluoro-Jade C staining for neurodegeneration; behavioral tests","journal":"Neuromolecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo ASO knockdown with multiple mechanistic readouts, single lab, no in vitro mechanistic validation","pmids":["39979490"],"is_preprint":false}],"current_model":"TUBB6 is a class V β-tubulin isotype that, when incorporated into microtubules, promotes dynamic (less stable) microtubule behavior; its chronic or context-inappropriate upregulation disrupts ordered microtubule networks in skeletal muscle and destabilizes microtubules after CNS injury, while in specialized cell types it is required for motile cilia axoneme assembly (multiciliated cells), actin-podosome belt organization and bone resorption in osteoclasts (partly via modulating ARHGAP10-CDC42 signaling at microtubules), and sprouting angiogenesis in endothelial cells (downstream of YBX1-mediated mRNA stabilization and upstream of WNT3A/FZD8 signaling), with its transcription in glioma driven by NFKB1 and its activity linked to Wnt/β-catenin pathway regulation."},"narrative":{"mechanistic_narrative":"TUBB6 is a β-tubulin isotype that, when incorporated into microtubules, biases them toward dynamic, less-stable behavior, and whose context-inappropriate elevation disorganizes microtubule networks [PMID:30535187, PMID:34869381]. Isotype-specific gain- and loss-of-function in skeletal muscle established that chronically elevated TUBB6 — but not TUBB5 — disrupts the ordered microtubule grid, and that TUBB6 is selectively induced during muscle regeneration rather than in mature fibers [PMID:30535187]. Live-cell imaging in osteoclasts directly demonstrated this destabilizing activity: loss of TUBB6 slows microtubule growth, prolongs growth lifetime, raises acetylated α-tubulin, and shortens EB1 caps, confirming that TUBB6 promotes dynamic microtubules [PMID:34869381]. Through these dynamic microtubules TUBB6 couples to the actin cytoskeleton, controlling podosome belt organization and bone resorption in part by negatively regulating the microtubule association of ARHGAP10, a CDC42 regulator that binds microtubules via its BAR-PH domain [PMID:34869381]. In specialized cells, TUBB6 is required for motile cilia axoneme assembly in multiciliated cells [PMID:41074676] and for sprouting angiogenesis, where it is stabilized post-transcriptionally by YBX1 binding its 3' UTR and acts upstream of WNT3A/FZD8 signaling [PMID:40083923]. A heterozygous missense mutation (p.Phe394Ser) causes autosomal dominant congenital cranial dysinnervation disorder and compromises microtubule function in a yeast challenge assay [PMID:29016863]. In disease contexts, TUBB6 is transcriptionally driven by NFKB1 in glioma and promotes proliferation through Wnt/β-catenin signaling [PMID:40169445], while its post-injury elevation destabilizes microtubules and drives neuroinflammation after intracerebral hemorrhage [PMID:39979490].","teleology":[{"year":2017,"claim":"Established the first human disease link for TUBB6 and provided initial evidence that its mutation impairs microtubule function.","evidence":"co-segregation in a five-generation pedigree plus a yeast benomyl growth assay of the mutant protein","pmids":["29016863"],"confidence":"Medium","gaps":["Mechanism by which p.Phe394Ser alters microtubule dynamics in human cells not defined","No structural model of the mutant tubulin","Tissue specificity of the cranial dysinnervation phenotype not mechanistically explained"]},{"year":2019,"claim":"Resolved whether TUBB6 is functionally distinct from other β-tubulins by showing isotype-specific disruption of ordered microtubule arrays and regeneration-associated induction.","evidence":"reciprocal GFP-tubb6 overexpression and shRNA knockdown versus tubb5 controls in mouse and human muscle fibers, with regeneration-marker correlation","pmids":["30535187"],"confidence":"High","gaps":["Molecular basis for isotype-specific destabilizing activity not identified","Whether TUBB6 disorganization is a cause or consequence of dystrophic pathology not separated","Upstream regulators of regeneration-associated induction not defined"]},{"year":2021,"claim":"Directly demonstrated that TUBB6 promotes dynamic microtubules and linked this activity to actin podosome organization and bone resorption.","evidence":"CRISPR/Cas9 KO in osteoclasts with EB1 tracking, acetylated tubulin readout, podosome live imaging, bone resorption assay, and proteomics of microtubule-associated fractions","pmids":["34869381"],"confidence":"High","gaps":["How TUBB6 negatively regulates ARHGAP10-microtubule association mechanistically unresolved at this stage","Direct binding partners of TUBB6 not identified","Single lab"]},{"year":2025,"claim":"Defined the molecular interface for the TUBB6–ARHGAP10–CDC42 axis by mapping direct microtubule binding to the ARHGAP10 BAR-PH domain.","evidence":"in vitro microtubule binding with BAR domain lysine mutants, osteoclast KO and complementation requiring both microtubule binding and RHO-GTPase regulation (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, single lab, not peer-reviewed","Direct physical contact between TUBB6 and ARHGAP10 versus indirect competition not distinguished","Quantitative effect of TUBB6 on ARHGAP10 affinity not measured"]},{"year":2025,"claim":"Extended TUBB6 function to motile cilia by showing a cell-type-specific requirement for axoneme formation in multiciliated cells.","evidence":"morpholino knockdown in Xenopus with cilia imaging, mRNA in situ hybridization, and axonemal immunolocalization","pmids":["41074676"],"confidence":"High","gaps":["Why mono-motile cilia are spared not explained","Whether human TUBB6 has the same ciliary role not tested","Molecular role within the axoneme undefined"]},{"year":2025,"claim":"Placed TUBB6 in a post-transcriptional and signaling network controlling sprouting angiogenesis.","evidence":"YBX1-TUBB6 3'UTR RNA binding assay, endothelial TUBB6 KO functional assays, OIR/CNV in vivo models, and transcriptome analysis showing WNT3A/FZD8 dependence","pmids":["40083923"],"confidence":"Medium","gaps":["How a tubulin isotype controls WNT3A/FZD8 expression mechanistically unclear","Direct versus indirect link to Wnt signaling not separated","Single lab"]},{"year":2025,"claim":"Connected TUBB6 transcriptional control to cancer by identifying NFKB1-driven expression and Wnt/β-catenin-dependent proliferation in glioma.","evidence":"promoter binding/ChIP-type analysis, NFKB1 and TUBB6 knockdown with proliferation, apoptosis, and Wnt/β-catenin pathway readouts in glioma cell lines","pmids":["40169445"],"confidence":"Medium","gaps":["Whether Wnt/β-catenin regulation is direct or via microtubule dynamics not resolved","In vivo tumor relevance not tested","Single lab"]},{"year":2025,"claim":"Demonstrated a pathogenic role for elevated TUBB6 in CNS injury through microtubule destabilization and neuroinflammation.","evidence":"antisense oligonucleotide inhibition in a rat intracerebral hemorrhage model with acetylated α-tubulin, MAPK, cytokine, neurodegeneration, and behavioral readouts","pmids":["39979490"],"confidence":"Medium","gaps":["No in vitro mechanistic validation linking TUBB6 to MAPK signaling","Cell type driving the effect not defined","Single lab"]},{"year":null,"claim":"The biophysical basis by which the TUBB6 isotype confers microtubule instability, and the direct molecular partners that translate this into actin, ciliary, Wnt, and inflammatory outputs, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural explanation for isotype-specific destabilization","No reconstituted system relating TUBB6 incorporation to dynamics quantitatively","Whether the diverse cell-type phenotypes share one molecular mechanism unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,3,7]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,7,8]}],"complexes":["microtubule"],"partners":["ARHGAP10","YBX1","NFKB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BUF5","full_name":"Tubulin beta-6 chain","aliases":["Tubulin beta class V"],"length_aa":446,"mass_kda":49.9,"function":"Tubulin is the major constituent of microtubules, a cylinder consisting of laterally associated linear protofilaments composed of alpha- and beta-tubulin heterodimers. Microtubules grow by the addition of GTP-tubulin dimers to the microtubule end, where a stabilizing cap forms. Below the cap, tubulin dimers are in GDP-bound state, owing to GTPase activity of alpha-tubulin","subcellular_location":"Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q9BUF5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TUBB6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"TUBA1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TUBB6","total_profiled":1310},"omim":[{"mim_id":"617732","title":"FACIAL PALSY, CONGENITAL, WITH PTOSIS AND VELOPHARYNGEAL DYSFUNCTION; FPVEPD","url":"https://www.omim.org/entry/617732"},{"mim_id":"615103","title":"TUBULIN, BETA-6; TUBB6","url":"https://www.omim.org/entry/615103"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"},{"location":"Flagellar centriole","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"},{"location":"End piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TUBB6"},"hgnc":{"alias_symbol":["MGC4083","HsT1601"],"prev_symbol":[]},"alphafold":{"accession":"Q9BUF5","domains":[{"cath_id":"3.40.50.1440","chopping":"2-258","consensus_level":"medium","plddt":93.6959,"start":2,"end":258},{"cath_id":"3.30.1330.20","chopping":"268-373","consensus_level":"medium","plddt":92.3363,"start":268,"end":373},{"cath_id":"1.10.287","chopping":"380-440","consensus_level":"medium","plddt":88.8356,"start":380,"end":440}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUF5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUF5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUF5-F1-predicted_aligned_error_v6.png","plddt_mean":92.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TUBB6","jax_strain_url":"https://www.jax.org/strain/search?query=TUBB6"},"sequence":{"accession":"Q9BUF5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BUF5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BUF5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUF5"}},"corpus_meta":[{"pmid":"30535187","id":"PMC_30535187","title":"Persistent upregulation of the β-tubulin tubb6, linked to muscle regeneration, is a source of microtubule disorganization in dystrophic muscle.","date":"2019","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30535187","citation_count":38,"is_preprint":false},{"pmid":"38030103","id":"PMC_38030103","title":"Human umbilical cord mesenchymal stem cell-derived exosomes provide neuroprotection in traumatic brain injury through the lncRNA TUBB6/Nrf2 pathway.","date":"2023","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/38030103","citation_count":28,"is_preprint":false},{"pmid":"29016863","id":"PMC_29016863","title":"A TUBB6 mutation is associated with autosomal dominant non-progressive congenital facial palsy, bilateral ptosis and velopharyngeal dysfunction.","date":"2017","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29016863","citation_count":21,"is_preprint":false},{"pmid":"34869381","id":"PMC_34869381","title":"The Beta-Tubulin Isotype TUBB6 Controls Microtubule and Actin Dynamics in Osteoclasts.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34869381","citation_count":21,"is_preprint":false},{"pmid":"40083923","id":"PMC_40083923","title":"YBX1-driven TUBB6 upregulation facilitates ocular angiogenesis via WNT3A-FZD8 pathway.","date":"2025","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/40083923","citation_count":2,"is_preprint":false},{"pmid":"39979490","id":"PMC_39979490","title":"Therapeutic Potential of TUBB6 Inhibition for Hematoma Reduction, Microtubule Stabilization, and Neurological Recovery in an In Vivo Model of Intracerebral Hemorrhage.","date":"2025","source":"Neuromolecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39979490","citation_count":2,"is_preprint":false},{"pmid":"39519211","id":"PMC_39519211","title":"Identification of Genes Associated with Familial Focal Segmental Glomerulosclerosis Through Transcriptomics and In Silico Analysis, Including RPL27, TUBB6, and PFDN5.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39519211","citation_count":1,"is_preprint":false},{"pmid":"40169445","id":"PMC_40169445","title":"Transcription factor NFKB1 mediates TUBB6 to promote the proliferation and suppress apoptosis in glioma via Wnt/β-catenin signaling pathway.","date":"2025","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40169445","citation_count":1,"is_preprint":false},{"pmid":"41074676","id":"PMC_41074676","title":"A Cell Type-Specific Role for Tubb6 in Ciliogenesis of Xenopus Epidermal Multiciliated Cells.","date":"2025","source":"Cytoskeleton (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/41074676","citation_count":0,"is_preprint":false},{"pmid":"40501596","id":"PMC_40501596","title":"A Cell Type-Specific Role for Tubb6 in Ciliogenesis of Xenopus Epidermal Multiciliated Cells.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40501596","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.31.657203","title":"A Cell Type-Specific Role for Tubb6 in Ciliogenesis of  <i>Xenopus</i>  Epidermal Multiciliated Cells","date":"2025-06-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.31.657203","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.12.659268","title":"ARHGAP10 is a novel microtubule-associated protein that regulates the resorption activity of osteoclasts","date":"2025-06-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.12.659268","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.24.661085","title":"Explainable machine learning models for glioma subtype classification and survival prediction","date":"2025-06-25","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.24.661085","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9069,"output_tokens":2924,"usd":0.035534,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10244,"output_tokens":3171,"usd":0.065247,"stage2_stop_reason":"end_turn"},"total_usd":0.100781,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"Overexpression of GFP-tubb6 (but not GFP-tubb5) in wild-type skeletal muscle fibers causes microtubule disorganization, and depletion of tubb6 (but not tubb5) in mdx muscle fibers normalizes microtubule architecture, demonstrating that tubb6 specifically disrupts the ordered microtubule grid when chronically elevated.\",\n      \"method\": \"GFP-tubb6 overexpression in isolated muscle fibers; shRNA knockdown of tubb6 vs tubb5 in mdx fibers; fluorescence microscopy of microtubule organization\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain-of-function and loss-of-function experiments with isotype-specific controls, replicated across mouse and human muscle models\",\n      \"pmids\": [\"30535187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Tubb6 expression is selectively upregulated during muscle differentiation and regeneration (not in mature fibers), co-correlating with embryonic myosin heavy chain levels, positioning tubb6 as a regeneration-associated tubulin isotype.\",\n      \"method\": \"Immunofluorescence and western blot in mdx and cardiotoxin-injury mouse models; human DMD and myositis biopsies; correlation with embryonic myosin heavy chain (regeneration marker)\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple models and markers used in one study, but single lab\",\n      \"pmids\": [\"30535187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A missense mutation in TUBB6 (p.Phe394Ser) causes autosomal dominant congenital cranial dysinnervation disorder; expression of the mutant protein in yeast impairs viability under microtubule-poison (benomyl) challenge, indicating the mutation compromises microtubule function.\",\n      \"method\": \"Yeast growth assay with benomyl; co-segregation analysis in five-generation pedigree\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast functional assay plus human genetic evidence, single lab\",\n      \"pmids\": [\"29016863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TUBB6 controls microtubule dynamics in osteoclasts: CRISPR/Cas9 KO of Tubb6 reduces microtubule growth speed, increases microtubule growth lifetime, elevates acetylated α-tubulin, and produces smaller EB1-caps, indicating that TUBB6 promotes dynamic (less stable) microtubules.\",\n      \"method\": \"CRISPR/Cas9 knockout in osteoclast model; live-cell microtubule dynamics assay (EB1 tracking); immunofluorescence for acetylated tubulin\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple orthogonal readouts of microtubule dynamics in same study\",\n      \"pmids\": [\"34869381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TUBB6 controls actin podosome dynamics in osteoclasts: Tubb6 KO increases individual podosome lifetime while destabilizing the podosome belt, linking TUBB6-regulated microtubule dynamics to actin cytoskeleton organization required for bone resorption.\",\n      \"method\": \"CRISPR/Cas9 knockout; live-cell imaging of podosome dynamics; bone resorption assay\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with direct imaging of podosome dynamics and functional bone resorption readout, single lab\",\n      \"pmids\": [\"34869381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Proteomic analysis of microtubule-associated protein fractions revealed that ARHGAP10 (a negative regulator of CDC42) associates with microtubules, and this association is negatively regulated by TUBB6, suggesting a mechanism by which TUBB6 locally controls CDC42 activity and actin organization.\",\n      \"method\": \"Proteomic analysis of microtubule-associated protein-enriched fractions from Tubb6 KO vs wild-type osteoclasts\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — mass spectrometry-based enriched fraction analysis, single lab, functional inference supported by KO phenotype\",\n      \"pmids\": [\"34869381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ARHGAP10 directly binds microtubules through its BAR-PH domain (requiring lysine residues K37, K41, K44 in the BAR domain), and TUBB6 negatively regulates this binding; ARHGAP10 KO impairs actin ring dynamics and osteoclast resorption activity, and complementation requires both microtubule binding and RHO-GTPase regulatory capacity.\",\n      \"method\": \"CRISPR/Cas9 KO; in vitro microtubule binding assay with BAR domain mutants; osteoclast resorption assay; complementation experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro microtubule binding with mutagenesis is Tier 1, but preprint single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Tubb6 mRNA is selectively expressed in Xenopus multiciliated cells (MCCs), and its protein localizes to ciliary axonemes; morpholino-mediated depletion of Tubb6 markedly reduces cilia number and length in MCCs causing defective ciliary motility, while mono-motile cilia in the gastrocoel roof plate are unaffected, revealing a cell-type-specific requirement for Tubb6 in motile cilia formation.\",\n      \"method\": \"Morpholino knockdown in Xenopus embryos; fluorescence imaging of cilia; in situ hybridization for mRNA localization; immunofluorescence for axonemal localization\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with direct protein localization and functional ciliary motility readout, peer-reviewed, Xenopus ortholog consistent with vertebrate TUBB6\",\n      \"pmids\": [\"41074676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"YBX1 binds the 3' UTR of TUBB6 mRNA and stabilizes it, functioning as an upstream post-transcriptional regulator that increases TUBB6 protein levels; TUBB6 knockdown in endothelial cells suppresses WNT signaling (specifically WNT3A and FZD8 expression) and reduces tip cell and proliferative cell marker expression, inhibiting sprouting angiogenesis.\",\n      \"method\": \"RNA binding assay (YBX1-TUBB6 3'UTR interaction); TUBB6 KO endothelial cell functional assays (migration, tube formation, flow cytometry); OIR and CNV in vivo mouse models with Tubb6 silencing; transcriptome analysis\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA binding assay plus KO with multiple functional readouts in vitro and in vivo, single lab\",\n      \"pmids\": [\"40083923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NFKB1 binds the promoter region of TUBB6 and transcriptionally upregulates its expression in glioma cells; NFKB1 knockdown reduces TUBB6 expression, and TUBB6 knockdown suppresses glioma cell proliferation and promotes apoptosis via the Wnt/β-catenin signaling pathway.\",\n      \"method\": \"Promoter binding assay/ChIP-type analysis; NFKB1 knockdown with TUBB6 expression measurement; TUBB6 knockdown with cell proliferation, apoptosis, and Wnt/β-catenin pathway readouts in glioma cell lines\",\n      \"journal\": \"Discover oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcription factor-promoter binding plus knockdown with pathway readouts, single lab\",\n      \"pmids\": [\"40169445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TUBB6 inhibition by antisense oligonucleotide (ASO) in a rat intracerebral hemorrhage model reduces hematoma volume, restores acetylated α-tubulin (microtubule stability marker), suppresses MAPK signaling, decreases pro-inflammatory cytokines, and reduces neuronal degeneration, demonstrating that elevated TUBB6 destabilizes microtubules and promotes neuroinflammation after hemorrhage.\",\n      \"method\": \"Antisense oligonucleotide inhibition in collagenase-injection ICH rat model; immunohistochemistry for acetylated α-tubulin; ELISA for cytokines; Western blot for MAPK pathway; Fluoro-Jade C staining for neurodegeneration; behavioral tests\",\n      \"journal\": \"Neuromolecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo ASO knockdown with multiple mechanistic readouts, single lab, no in vitro mechanistic validation\",\n      \"pmids\": [\"39979490\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TUBB6 is a class V β-tubulin isotype that, when incorporated into microtubules, promotes dynamic (less stable) microtubule behavior; its chronic or context-inappropriate upregulation disrupts ordered microtubule networks in skeletal muscle and destabilizes microtubules after CNS injury, while in specialized cell types it is required for motile cilia axoneme assembly (multiciliated cells), actin-podosome belt organization and bone resorption in osteoclasts (partly via modulating ARHGAP10-CDC42 signaling at microtubules), and sprouting angiogenesis in endothelial cells (downstream of YBX1-mediated mRNA stabilization and upstream of WNT3A/FZD8 signaling), with its transcription in glioma driven by NFKB1 and its activity linked to Wnt/β-catenin pathway regulation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TUBB6 is a β-tubulin isotype that, when incorporated into microtubules, biases them toward dynamic, less-stable behavior, and whose context-inappropriate elevation disorganizes microtubule networks [#0, #3]. Isotype-specific gain- and loss-of-function in skeletal muscle established that chronically elevated TUBB6 — but not TUBB5 — disrupts the ordered microtubule grid, and that TUBB6 is selectively induced during muscle regeneration rather than in mature fibers [#0, #1]. Live-cell imaging in osteoclasts directly demonstrated this destabilizing activity: loss of TUBB6 slows microtubule growth, prolongs growth lifetime, raises acetylated α-tubulin, and shortens EB1 caps, confirming that TUBB6 promotes dynamic microtubules [#3]. Through these dynamic microtubules TUBB6 couples to the actin cytoskeleton, controlling podosome belt organization and bone resorption in part by negatively regulating the microtubule association of ARHGAP10, a CDC42 regulator that binds microtubules via its BAR-PH domain [#4, #5, #6]. In specialized cells, TUBB6 is required for motile cilia axoneme assembly in multiciliated cells [#7] and for sprouting angiogenesis, where it is stabilized post-transcriptionally by YBX1 binding its 3' UTR and acts upstream of WNT3A/FZD8 signaling [#8]. A heterozygous missense mutation (p.Phe394Ser) causes autosomal dominant congenital cranial dysinnervation disorder and compromises microtubule function in a yeast challenge assay [#2]. In disease contexts, TUBB6 is transcriptionally driven by NFKB1 in glioma and promotes proliferation through Wnt/β-catenin signaling [#9], while its post-injury elevation destabilizes microtubules and drives neuroinflammation after intracerebral hemorrhage [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established the first human disease link for TUBB6 and provided initial evidence that its mutation impairs microtubule function.\",\n      \"evidence\": \"co-segregation in a five-generation pedigree plus a yeast benomyl growth assay of the mutant protein\",\n      \"pmids\": [\"29016863\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which p.Phe394Ser alters microtubule dynamics in human cells not defined\", \"No structural model of the mutant tubulin\", \"Tissue specificity of the cranial dysinnervation phenotype not mechanistically explained\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved whether TUBB6 is functionally distinct from other β-tubulins by showing isotype-specific disruption of ordered microtubule arrays and regeneration-associated induction.\",\n      \"evidence\": \"reciprocal GFP-tubb6 overexpression and shRNA knockdown versus tubb5 controls in mouse and human muscle fibers, with regeneration-marker correlation\",\n      \"pmids\": [\"30535187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for isotype-specific destabilizing activity not identified\", \"Whether TUBB6 disorganization is a cause or consequence of dystrophic pathology not separated\", \"Upstream regulators of regeneration-associated induction not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Directly demonstrated that TUBB6 promotes dynamic microtubules and linked this activity to actin podosome organization and bone resorption.\",\n      \"evidence\": \"CRISPR/Cas9 KO in osteoclasts with EB1 tracking, acetylated tubulin readout, podosome live imaging, bone resorption assay, and proteomics of microtubule-associated fractions\",\n      \"pmids\": [\"34869381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TUBB6 negatively regulates ARHGAP10-microtubule association mechanistically unresolved at this stage\", \"Direct binding partners of TUBB6 not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the molecular interface for the TUBB6–ARHGAP10–CDC42 axis by mapping direct microtubule binding to the ARHGAP10 BAR-PH domain.\",\n      \"evidence\": \"in vitro microtubule binding with BAR domain lysine mutants, osteoclast KO and complementation requiring both microtubule binding and RHO-GTPase regulation (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab, not peer-reviewed\", \"Direct physical contact between TUBB6 and ARHGAP10 versus indirect competition not distinguished\", \"Quantitative effect of TUBB6 on ARHGAP10 affinity not measured\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended TUBB6 function to motile cilia by showing a cell-type-specific requirement for axoneme formation in multiciliated cells.\",\n      \"evidence\": \"morpholino knockdown in Xenopus with cilia imaging, mRNA in situ hybridization, and axonemal immunolocalization\",\n      \"pmids\": [\"41074676\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why mono-motile cilia are spared not explained\", \"Whether human TUBB6 has the same ciliary role not tested\", \"Molecular role within the axoneme undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed TUBB6 in a post-transcriptional and signaling network controlling sprouting angiogenesis.\",\n      \"evidence\": \"YBX1-TUBB6 3'UTR RNA binding assay, endothelial TUBB6 KO functional assays, OIR/CNV in vivo models, and transcriptome analysis showing WNT3A/FZD8 dependence\",\n      \"pmids\": [\"40083923\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a tubulin isotype controls WNT3A/FZD8 expression mechanistically unclear\", \"Direct versus indirect link to Wnt signaling not separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected TUBB6 transcriptional control to cancer by identifying NFKB1-driven expression and Wnt/β-catenin-dependent proliferation in glioma.\",\n      \"evidence\": \"promoter binding/ChIP-type analysis, NFKB1 and TUBB6 knockdown with proliferation, apoptosis, and Wnt/β-catenin pathway readouts in glioma cell lines\",\n      \"pmids\": [\"40169445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Wnt/β-catenin regulation is direct or via microtubule dynamics not resolved\", \"In vivo tumor relevance not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated a pathogenic role for elevated TUBB6 in CNS injury through microtubule destabilization and neuroinflammation.\",\n      \"evidence\": \"antisense oligonucleotide inhibition in a rat intracerebral hemorrhage model with acetylated α-tubulin, MAPK, cytokine, neurodegeneration, and behavioral readouts\",\n      \"pmids\": [\"39979490\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro mechanistic validation linking TUBB6 to MAPK signaling\", \"Cell type driving the effect not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The biophysical basis by which the TUBB6 isotype confers microtubule instability, and the direct molecular partners that translate this into actin, ciliary, Wnt, and inflammatory outputs, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural explanation for isotype-specific destabilization\", \"No reconstituted system relating TUBB6 incorporation to dynamics quantitatively\", \"Whether the diverse cell-type phenotypes share one molecular mechanism unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 3, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 7, 8]}\n    ],\n    \"complexes\": [\"microtubule\"],\n    \"partners\": [\"ARHGAP10\", \"YBX1\", \"NFKB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}