{"gene":"MTUS2","run_date":"2026-06-10T05:19:51","timeline":{"discoveries":[{"year":2009,"finding":"TIP150 (MTUS2) is a microtubule plus-end tracking protein (+TIP) that binds EB1 in vitro and co-localizes with EB1 at MT plus ends in vivo; EB1 suppression eliminates TIP150 plus-end localization, establishing EB1 as required for TIP150 targeting.","method":"In vitro binding assay, co-localization by immunofluorescence, siRNA knockdown of EB1","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal in vitro binding and in vivo co-localization with EB1-dependency experiment; replicated and extended in multiple subsequent studies","pmids":["19543227"],"is_preprint":false},{"year":2009,"finding":"TIP150 (MTUS2) directly binds MCAK (an MT depolymerase) and facilitates the EB1-dependent loading of MCAK onto MT plus ends; suppression of TIP150 diminishes MCAK plus-end localization.","method":"In vitro binding assay, siRNA knockdown of TIP150 with fluorescence microscopy readout of MCAK localization","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro binding plus loss-of-function localization experiment; independently replicated in subsequent studies","pmids":["19543227"],"is_preprint":false},{"year":2009,"finding":"Aurora B-mediated phosphorylation disrupts the TIP150-MCAK association in vitro, providing a regulatory mechanism for MT plus-end dynamics.","method":"In vitro kinase assay followed by binding assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution of kinase-regulated interaction, single lab, single paper","pmids":["19543227"],"is_preprint":false},{"year":2013,"finding":"TIP150 (MTUS2) forms a tetramer and binds an EB1 dimer through its C-terminal domain; disruption of the TIP150-EB1 interface (by C-terminal TIP150 overexpression or membrane-permeable peptide) perturbs chromosome segregation and activates the spindle checkpoint.","method":"Biochemical oligomerization analysis, Co-IP, dominant-negative peptide perturbation, siRNA knockdown with mitotic phenotype readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (biochemistry, peptide perturbation, siRNA) in a single rigorous study","pmids":["23595990"],"is_preprint":false},{"year":2013,"finding":"PCAF acetylates EB1, and persistent EB1 acetylation disrupts the TIP150-EB1 interaction, perturbs metaphase chromosome alignment, and activates the spindle checkpoint; BubR1 suppression overrides this arrest but causes whole-chromosome aneuploidy.","method":"In vitro acetylation assay, Co-IP, siRNA epistasis (BubR1 knockdown), live-cell imaging of chromosome alignment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay combined with genetic epistasis and cellular phenotype readout, multiple orthogonal methods","pmids":["23595990"],"is_preprint":false},{"year":2014,"finding":"TIP150 (MTUS2) cooperates with MCAK to govern entosis; MCAK forms an intra-molecular association required for TIP150 binding, and Aurora A-mediated phosphorylation of MCAK modulates this intra-molecular association to perturb the MCAK-TIP150 interaction in vitro and inhibit entosis in vivo.","method":"Biochemical intra-molecular association assay, in vitro kinase assay, co-IP, optical trap measurement of cell mechanical rigidity, live-cell entosis assay","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reconstituted kinase-regulated interaction in vitro validated with cellular functional readout (entosis and cell rigidity) using multiple orthogonal methods","pmids":["24847103"],"is_preprint":false},{"year":2014,"finding":"TIP150-MCAK interaction promotes microtubule dynamics and modulates the mechanical rigidity of live cells during entosis, as measured by optical trap.","method":"Optical trap mechanical measurement of live MCF7 cells with TIP150/MCAK perturbation","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct biophysical measurement in live cells, single lab, single study","pmids":["24847103"],"is_preprint":false},{"year":2016,"finding":"TIP150 (MTUS2) interacts with cortactin via its C-terminal proline-rich region (CT150) binding to the SH3 domain of cortactin; EGF-elicited tyrosine phosphorylation of cortactin negatively regulates this interaction. Suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration.","method":"Co-IP, in vitro binding assay, membrane-permeable TAT-CT150 peptide perturbation, siRNA knockdown, directional cell migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding confirmed in vitro and in cells, loss-of-function and peptide perturbation with defined directional migration phenotype, multiple orthogonal methods","pmids":["27451391"],"is_preprint":false},{"year":2024,"finding":"EB1, TIP150 (MTUS2), and MCAK form molecular condensates through liquid-liquid phase separation at microtubule plus ends, compartmentalizing the kinetochore-MT machinery; perturbation of EB1-TIP150 polymer formation by a competing peptide prevents phase separation and disrupts chromosome alignment in cultured cells and Drosophila embryos. Persistent acetylation of EB1 Lys220 attenuates phase separation of the EB1-TIP150-MCAK complex.","method":"In vitro phase separation assay (droplet formation), competing-peptide perturbation in cells and Drosophila embryos, acetylation mutagenesis of EB1, live-cell imaging of chromosome alignment","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution of phase separation plus mutagenesis and genetic perturbation in two model systems","pmids":["38323478"],"is_preprint":false},{"year":2009,"finding":"The MTUS2/CAZIP protein contains a leucine-zipper domain at its C-terminus and has at least two isoforms (358 and 1354 amino acids); its mRNA is expressed during early cardiac development and later in the nervous system of mouse and chicken embryos.","method":"Whole-mount in situ hybridization, Northern blot, RT-PCR, bioinformatic domain analysis","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct expression localization by in situ hybridization in two species with domain characterization, but no functional loss-of-function experiment","pmids":["19806667"],"is_preprint":false}],"current_model":"MTUS2/TIP150 is a microtubule plus-end tracking protein (+TIP) that is recruited to MT plus ends via direct binding to an EB1 dimer through its C-terminal domain (where it assembles as a tetramer and can undergo liquid-liquid phase separation with EB1 and MCAK); at plus ends it loads MCAK to promote MT depolymerase activity, with Aurora A/B kinases and PCAF acetylation serving as regulatory switches that dissolve or modulate the TIP150-MCAK and TIP150-EB1 interactions to control chromosome segregation, entosis, and directional cell migration (the latter through a separate TIP150-cortactin interaction at the cortical cytoskeleton)."},"narrative":{"mechanistic_narrative":"MTUS2 (TIP150) is a microtubule plus-end tracking protein (+TIP) that couples plus-end machinery to chromosome segregation, entosis, and directional cell migration [PMID:19543227, PMID:24847103, PMID:27451391]. It is recruited to growing microtubule plus ends through direct binding to EB1, where it assembles as a tetramer engaging an EB1 dimer via its C-terminal domain; EB1 is required for TIP150 plus-end targeting [PMID:19543227, PMID:23595990]. Once localized, TIP150 directly binds the microtubule depolymerase MCAK and drives its EB1-dependent loading onto plus ends [PMID:19543227]. This EB1–TIP150–MCAK module is dynamically regulated: Aurora B phosphorylation dissolves the TIP150–MCAK association, Aurora A phosphorylation of MCAK perturbs the intramolecular MCAK conformation needed for TIP150 binding, and PCAF-mediated acetylation of EB1 disrupts the TIP150–EB1 interface — perturbation of these interactions impairs metaphase chromosome alignment and activates the spindle checkpoint [PMID:19543227, PMID:23595990, PMID:24847103]. The three proteins further compartmentalize the kinetochore–microtubule machinery by undergoing liquid–liquid phase separation at plus ends, which acetylation of EB1 Lys220 attenuates [PMID:38323478]. Through a distinct C-terminal proline-rich region binding the cortactin SH3 domain at the cortical cytoskeleton, TIP150 also supports polarized cell migration, an interaction negatively regulated by EGF-driven cortactin tyrosine phosphorylation [PMID:27451391].","teleology":[{"year":2009,"claim":"Established MTUS2/TIP150 as a bona fide +TIP and identified EB1 as the recruiting factor, defining how the protein reaches microtubule plus ends.","evidence":"In vitro EB1 binding, plus-end co-localization, and EB1 siRNA knockdown","pmids":["19543227"],"confidence":"High","gaps":["Did not define the structural EB1-binding motif resolved later","No functional consequence of plus-end targeting yet established"]},{"year":2009,"claim":"Showed TIP150 directly binds and loads the depolymerase MCAK at plus ends, linking the +TIP to control of microtubule dynamics.","evidence":"In vitro binding plus TIP150 knockdown with MCAK localization readout","pmids":["19543227"],"confidence":"High","gaps":["Quantitative effect on depolymerization kinetics not measured","Cellular phenotype of disrupted loading not yet defined"]},{"year":2009,"claim":"Identified Aurora B phosphorylation as a switch dissolving the TIP150-MCAK interaction, the first regulatory input on the module.","evidence":"In vitro kinase assay followed by binding assay","pmids":["19543227"],"confidence":"Medium","gaps":["Phosphosites not mapped in vivo","Single lab in vitro reconstitution without cellular validation"]},{"year":2013,"claim":"Resolved the oligomeric architecture (TIP150 tetramer binding an EB1 dimer) and demonstrated that disrupting this interface arrests mitosis, establishing the interaction as essential for chromosome segregation.","evidence":"Oligomerization analysis, Co-IP, dominant-negative peptide and siRNA with mitotic phenotype","pmids":["23595990"],"confidence":"High","gaps":["No atomic structure of the tetramer-dimer interface","Stoichiometry at native plus ends in vivo not directly quantified"]},{"year":2013,"claim":"Defined PCAF acetylation of EB1 as a second regulatory layer disrupting TIP150-EB1 binding and perturbing chromosome alignment, with BubR1 epistasis linking it to checkpoint control.","evidence":"In vitro acetylation, Co-IP, BubR1 siRNA epistasis, live-cell imaging","pmids":["23595990"],"confidence":"High","gaps":["Endogenous acetylation dynamics during mitosis not tracked","Upstream signals controlling PCAF activity unknown"]},{"year":2014,"claim":"Extended the TIP150-MCAK module to entosis and showed Aurora A regulates an MCAK intramolecular conformation required for TIP150 binding, connecting plus-end dynamics to cell mechanical rigidity.","evidence":"Intramolecular association assay, in vitro kinase assay, Co-IP, optical trap, live-cell entosis assay","pmids":["24847103"],"confidence":"High","gaps":["Mechanistic link between plus-end dynamics and cortical rigidity not fully resolved","Aurora A phosphosites on MCAK in this context not mapped"]},{"year":2016,"claim":"Identified a cortactin SH3-binding function for TIP150's C-terminal proline-rich region, establishing a microtubule-independent role in polarized cell migration regulated by EGF signaling.","evidence":"Co-IP, in vitro binding, TAT-CT150 peptide perturbation, siRNA, directional migration assay","pmids":["27451391"],"confidence":"High","gaps":["Cortactin tyrosine phosphosites mediating regulation not pinpointed","Relationship between cortical and plus-end pools of TIP150 unresolved"]},{"year":2024,"claim":"Demonstrated that EB1, TIP150, and MCAK form phase-separated condensates at plus ends, providing a biophysical organizing principle for the kinetochore-MT machinery sensitive to EB1 acetylation.","evidence":"In vitro droplet phase-separation assay, competing peptide in cells and Drosophila embryos, EB1 acetylation mutagenesis, live-cell imaging","pmids":["38323478"],"confidence":"High","gaps":["In vivo evidence that endogenous condensates form at native plus ends remains indirect","Material properties and turnover of condensates not characterized"]},{"year":null,"claim":"The developmental and tissue-specific functions of MTUS2 implied by its embryonic cardiac and neural expression remain mechanistically unconnected to its characterized +TIP roles.","evidence":"No loss-of-function developmental study integrates the +TIP mechanism with the expression pattern","pmids":[],"confidence":"Low","gaps":["No organismal phenotype links MTUS2 isoforms to cardiac or neural development","Functional difference between the 358- and 1354-aa isoforms uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,7]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,4,8]}],"complexes":["EB1-TIP150-MCAK plus-end complex"],"partners":["EB1","MCAK","CTTN","AURKB","AURKA","PCAF"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5JR59","full_name":"Microtubule-associated tumor suppressor candidate 2","aliases":["Cardiac zipper protein","Microtubule plus-end tracking protein TIP150","Tracking protein of 150 kDa"],"length_aa":1369,"mass_kda":150.2,"function":"Binds microtubules. Together with MAPRE1 may target the microtubule depolymerase KIF2C to the plus-end of microtubules. May regulate the dynamics of microtubules at their growing distal tip","subcellular_location":"Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q5JR59/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MTUS2","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":[],"url":"https://opencell.sf.czbiohub.org/search/MTUS2","total_profiled":1310},"omim":[{"mim_id":"619358","title":"MICROTUBULE-ASSOCIATED SCAFFOLD PROTEIN 2; MTUS2","url":"https://www.omim.org/entry/619358"},{"mim_id":"604538","title":"KINESIN FAMILY MEMBER 2C; KIF2C","url":"https://www.omim.org/entry/604538"},{"mim_id":"603108","title":"MICROTUBULE-ASSOCIATED PROTEIN, RP/EB FAMILY, MEMBER 1; MAPRE1","url":"https://www.omim.org/entry/603108"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Microtubules","reliability":"Supported"},{"location":"Cytokinetic bridge","reliability":"Supported"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"blood vessel","ntpm":12.9},{"tissue":"heart muscle","ntpm":47.0}],"url":"https://www.proteinatlas.org/search/MTUS2"},"hgnc":{"alias_symbol":["TIP150","CAZIP","ICIS"],"prev_symbol":["KIAA0774"]},"alphafold":{"accession":"Q5JR59","domains":[{"cath_id":"-","chopping":"997-1231","consensus_level":"medium","plddt":94.376,"start":997,"end":1231},{"cath_id":"1.20.5","chopping":"1244-1285","consensus_level":"medium","plddt":94.6998,"start":1244,"end":1285},{"cath_id":"1.20.5","chopping":"1287-1324","consensus_level":"medium","plddt":88.7445,"start":1287,"end":1324}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5JR59","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5JR59-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5JR59-F1-predicted_aligned_error_v6.png","plddt_mean":51.03},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MTUS2","jax_strain_url":"https://www.jax.org/strain/search?query=MTUS2"},"sequence":{"accession":"Q5JR59","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5JR59.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5JR59/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5JR59"}},"corpus_meta":[{"pmid":"19543227","id":"PMC_19543227","title":"TIP150 interacts with and targets MCAK at the microtubule plus ends.","date":"2009","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/19543227","citation_count":76,"is_preprint":false},{"pmid":"26474971","id":"PMC_26474971","title":"A genome-wide approach to link genotype to clinical outcome by utilizing next generation sequencing and gene chip data of 6,697 breast cancer patients.","date":"2015","source":"Genome medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26474971","citation_count":70,"is_preprint":false},{"pmid":"24847103","id":"PMC_24847103","title":"Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction.","date":"2014","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24847103","citation_count":43,"is_preprint":false},{"pmid":"23595990","id":"PMC_23595990","title":"Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23595990","citation_count":34,"is_preprint":false},{"pmid":"28854418","id":"PMC_28854418","title":"A Comparative Proteomic Analysis of Erinacine A's Inhibition of Gastric Cancer Cell Viability and Invasiveness.","date":"2017","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/28854418","citation_count":32,"is_preprint":false},{"pmid":"35685361","id":"PMC_35685361","title":"Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.","date":"2022","source":"Computational and structural biotechnology 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& dementia : the journal of the Alzheimer's Association","url":"https://pubmed.ncbi.nlm.nih.gov/38380866","citation_count":14,"is_preprint":false},{"pmid":"19806667","id":"PMC_19806667","title":"CAZIP, a novel protein expressed in the developing heart and nervous system.","date":"2009","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/19806667","citation_count":11,"is_preprint":false},{"pmid":"38323478","id":"PMC_38323478","title":"Organization of microtubule plus-end dynamics by phase separation in mitosis.","date":"2024","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38323478","citation_count":10,"is_preprint":false},{"pmid":"34504517","id":"PMC_34504517","title":"Fine-Scale Genetic Structure and Natural Selection Signatures of Southwestern Hans Inferred From Patterns of Genome-Wide Allele, Haplotype, and Haplogroup Lineages.","date":"2021","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34504517","citation_count":8,"is_preprint":false},{"pmid":"37596794","id":"PMC_37596794","title":"Extracellular vesicles-transmitted long non-coding RNA MTUS2-5 promotes proliferation and vascularization of human vascular endothelial cells in patients with Budd-Chiari syndrome.","date":"2023","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37596794","citation_count":4,"is_preprint":false},{"pmid":"39543614","id":"PMC_39543614","title":"N6-methyladenosine modification of circular RNA circASH2L suppresses growth and metastasis in hepatocellular carcinoma through regulating hsa-miR-525-3p/MTUS2 axis.","date":"2024","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39543614","citation_count":3,"is_preprint":false},{"pmid":"40692742","id":"PMC_40692742","title":"Alectinib efficacy in advanced lung adenocarcinoma with 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Huntington's disease via bioinformatics and next generation sequencing data analysis","date":"2024-06-25","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.20.599879","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12116,"output_tokens":2360,"usd":0.035874,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9486,"output_tokens":2807,"usd":0.058803,"stage2_stop_reason":"end_turn"},"total_usd":0.094677,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"TIP150 (MTUS2) is a microtubule plus-end tracking protein (+TIP) that binds EB1 in vitro and co-localizes with EB1 at MT plus ends in vivo; EB1 suppression eliminates TIP150 plus-end localization, establishing EB1 as required for TIP150 targeting.\",\n      \"method\": \"In vitro binding assay, co-localization by immunofluorescence, siRNA knockdown of EB1\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal in vitro binding and in vivo co-localization with EB1-dependency experiment; replicated and extended in multiple subsequent studies\",\n      \"pmids\": [\"19543227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TIP150 (MTUS2) directly binds MCAK (an MT depolymerase) and facilitates the EB1-dependent loading of MCAK onto MT plus ends; suppression of TIP150 diminishes MCAK plus-end localization.\",\n      \"method\": \"In vitro binding assay, siRNA knockdown of TIP150 with fluorescence microscopy readout of MCAK localization\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro binding plus loss-of-function localization experiment; independently replicated in subsequent studies\",\n      \"pmids\": [\"19543227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Aurora B-mediated phosphorylation disrupts the TIP150-MCAK association in vitro, providing a regulatory mechanism for MT plus-end dynamics.\",\n      \"method\": \"In vitro kinase assay followed by binding assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution of kinase-regulated interaction, single lab, single paper\",\n      \"pmids\": [\"19543227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TIP150 (MTUS2) forms a tetramer and binds an EB1 dimer through its C-terminal domain; disruption of the TIP150-EB1 interface (by C-terminal TIP150 overexpression or membrane-permeable peptide) perturbs chromosome segregation and activates the spindle checkpoint.\",\n      \"method\": \"Biochemical oligomerization analysis, Co-IP, dominant-negative peptide perturbation, siRNA knockdown with mitotic phenotype readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (biochemistry, peptide perturbation, siRNA) in a single rigorous study\",\n      \"pmids\": [\"23595990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PCAF acetylates EB1, and persistent EB1 acetylation disrupts the TIP150-EB1 interaction, perturbs metaphase chromosome alignment, and activates the spindle checkpoint; BubR1 suppression overrides this arrest but causes whole-chromosome aneuploidy.\",\n      \"method\": \"In vitro acetylation assay, Co-IP, siRNA epistasis (BubR1 knockdown), live-cell imaging of chromosome alignment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay combined with genetic epistasis and cellular phenotype readout, multiple orthogonal methods\",\n      \"pmids\": [\"23595990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TIP150 (MTUS2) cooperates with MCAK to govern entosis; MCAK forms an intra-molecular association required for TIP150 binding, and Aurora A-mediated phosphorylation of MCAK modulates this intra-molecular association to perturb the MCAK-TIP150 interaction in vitro and inhibit entosis in vivo.\",\n      \"method\": \"Biochemical intra-molecular association assay, in vitro kinase assay, co-IP, optical trap measurement of cell mechanical rigidity, live-cell entosis assay\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reconstituted kinase-regulated interaction in vitro validated with cellular functional readout (entosis and cell rigidity) using multiple orthogonal methods\",\n      \"pmids\": [\"24847103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TIP150-MCAK interaction promotes microtubule dynamics and modulates the mechanical rigidity of live cells during entosis, as measured by optical trap.\",\n      \"method\": \"Optical trap mechanical measurement of live MCF7 cells with TIP150/MCAK perturbation\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct biophysical measurement in live cells, single lab, single study\",\n      \"pmids\": [\"24847103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIP150 (MTUS2) interacts with cortactin via its C-terminal proline-rich region (CT150) binding to the SH3 domain of cortactin; EGF-elicited tyrosine phosphorylation of cortactin negatively regulates this interaction. Suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration.\",\n      \"method\": \"Co-IP, in vitro binding assay, membrane-permeable TAT-CT150 peptide perturbation, siRNA knockdown, directional cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding confirmed in vitro and in cells, loss-of-function and peptide perturbation with defined directional migration phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"27451391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EB1, TIP150 (MTUS2), and MCAK form molecular condensates through liquid-liquid phase separation at microtubule plus ends, compartmentalizing the kinetochore-MT machinery; perturbation of EB1-TIP150 polymer formation by a competing peptide prevents phase separation and disrupts chromosome alignment in cultured cells and Drosophila embryos. Persistent acetylation of EB1 Lys220 attenuates phase separation of the EB1-TIP150-MCAK complex.\",\n      \"method\": \"In vitro phase separation assay (droplet formation), competing-peptide perturbation in cells and Drosophila embryos, acetylation mutagenesis of EB1, live-cell imaging of chromosome alignment\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution of phase separation plus mutagenesis and genetic perturbation in two model systems\",\n      \"pmids\": [\"38323478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The MTUS2/CAZIP protein contains a leucine-zipper domain at its C-terminus and has at least two isoforms (358 and 1354 amino acids); its mRNA is expressed during early cardiac development and later in the nervous system of mouse and chicken embryos.\",\n      \"method\": \"Whole-mount in situ hybridization, Northern blot, RT-PCR, bioinformatic domain analysis\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct expression localization by in situ hybridization in two species with domain characterization, but no functional loss-of-function experiment\",\n      \"pmids\": [\"19806667\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MTUS2/TIP150 is a microtubule plus-end tracking protein (+TIP) that is recruited to MT plus ends via direct binding to an EB1 dimer through its C-terminal domain (where it assembles as a tetramer and can undergo liquid-liquid phase separation with EB1 and MCAK); at plus ends it loads MCAK to promote MT depolymerase activity, with Aurora A/B kinases and PCAF acetylation serving as regulatory switches that dissolve or modulate the TIP150-MCAK and TIP150-EB1 interactions to control chromosome segregation, entosis, and directional cell migration (the latter through a separate TIP150-cortactin interaction at the cortical cytoskeleton).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MTUS2 (TIP150) is a microtubule plus-end tracking protein (+TIP) that couples plus-end machinery to chromosome segregation, entosis, and directional cell migration [#0, #5, #7]. It is recruited to growing microtubule plus ends through direct binding to EB1, where it assembles as a tetramer engaging an EB1 dimer via its C-terminal domain; EB1 is required for TIP150 plus-end targeting [#0, #3]. Once localized, TIP150 directly binds the microtubule depolymerase MCAK and drives its EB1-dependent loading onto plus ends [#1]. This EB1–TIP150–MCAK module is dynamically regulated: Aurora B phosphorylation dissolves the TIP150–MCAK association, Aurora A phosphorylation of MCAK perturbs the intramolecular MCAK conformation needed for TIP150 binding, and PCAF-mediated acetylation of EB1 disrupts the TIP150–EB1 interface — perturbation of these interactions impairs metaphase chromosome alignment and activates the spindle checkpoint [#2, #3, #4, #5]. The three proteins further compartmentalize the kinetochore–microtubule machinery by undergoing liquid–liquid phase separation at plus ends, which acetylation of EB1 Lys220 attenuates [#8]. Through a distinct C-terminal proline-rich region binding the cortactin SH3 domain at the cortical cytoskeleton, TIP150 also supports polarized cell migration, an interaction negatively regulated by EGF-driven cortactin tyrosine phosphorylation [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established MTUS2/TIP150 as a bona fide +TIP and identified EB1 as the recruiting factor, defining how the protein reaches microtubule plus ends.\",\n      \"evidence\": \"In vitro EB1 binding, plus-end co-localization, and EB1 siRNA knockdown\",\n      \"pmids\": [\"19543227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural EB1-binding motif resolved later\", \"No functional consequence of plus-end targeting yet established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed TIP150 directly binds and loads the depolymerase MCAK at plus ends, linking the +TIP to control of microtubule dynamics.\",\n      \"evidence\": \"In vitro binding plus TIP150 knockdown with MCAK localization readout\",\n      \"pmids\": [\"19543227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative effect on depolymerization kinetics not measured\", \"Cellular phenotype of disrupted loading not yet defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified Aurora B phosphorylation as a switch dissolving the TIP150-MCAK interaction, the first regulatory input on the module.\",\n      \"evidence\": \"In vitro kinase assay followed by binding assay\",\n      \"pmids\": [\"19543227\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosites not mapped in vivo\", \"Single lab in vitro reconstitution without cellular validation\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the oligomeric architecture (TIP150 tetramer binding an EB1 dimer) and demonstrated that disrupting this interface arrests mitosis, establishing the interaction as essential for chromosome segregation.\",\n      \"evidence\": \"Oligomerization analysis, Co-IP, dominant-negative peptide and siRNA with mitotic phenotype\",\n      \"pmids\": [\"23595990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic structure of the tetramer-dimer interface\", \"Stoichiometry at native plus ends in vivo not directly quantified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined PCAF acetylation of EB1 as a second regulatory layer disrupting TIP150-EB1 binding and perturbing chromosome alignment, with BubR1 epistasis linking it to checkpoint control.\",\n      \"evidence\": \"In vitro acetylation, Co-IP, BubR1 siRNA epistasis, live-cell imaging\",\n      \"pmids\": [\"23595990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous acetylation dynamics during mitosis not tracked\", \"Upstream signals controlling PCAF activity unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended the TIP150-MCAK module to entosis and showed Aurora A regulates an MCAK intramolecular conformation required for TIP150 binding, connecting plus-end dynamics to cell mechanical rigidity.\",\n      \"evidence\": \"Intramolecular association assay, in vitro kinase assay, Co-IP, optical trap, live-cell entosis assay\",\n      \"pmids\": [\"24847103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link between plus-end dynamics and cortical rigidity not fully resolved\", \"Aurora A phosphosites on MCAK in this context not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a cortactin SH3-binding function for TIP150's C-terminal proline-rich region, establishing a microtubule-independent role in polarized cell migration regulated by EGF signaling.\",\n      \"evidence\": \"Co-IP, in vitro binding, TAT-CT150 peptide perturbation, siRNA, directional migration assay\",\n      \"pmids\": [\"27451391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cortactin tyrosine phosphosites mediating regulation not pinpointed\", \"Relationship between cortical and plus-end pools of TIP150 unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that EB1, TIP150, and MCAK form phase-separated condensates at plus ends, providing a biophysical organizing principle for the kinetochore-MT machinery sensitive to EB1 acetylation.\",\n      \"evidence\": \"In vitro droplet phase-separation assay, competing peptide in cells and Drosophila embryos, EB1 acetylation mutagenesis, live-cell imaging\",\n      \"pmids\": [\"38323478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo evidence that endogenous condensates form at native plus ends remains indirect\", \"Material properties and turnover of condensates not characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The developmental and tissue-specific functions of MTUS2 implied by its embryonic cardiac and neural expression remain mechanistically unconnected to its characterized +TIP roles.\",\n      \"evidence\": \"No loss-of-function developmental study integrates the +TIP mechanism with the expression pattern\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No organismal phenotype links MTUS2 isoforms to cardiac or neural development\", \"Functional difference between the 358- and 1354-aa isoforms uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 4, 8]}\n    ],\n    \"complexes\": [\"EB1-TIP150-MCAK plus-end complex\"],\n    \"partners\": [\"EB1\", \"MCAK\", \"CTTN\", \"AURKB\", \"AURKA\", \"PCAF\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}