{"gene":"MIS12","run_date":"2026-04-28T18:30:28","timeline":{"discoveries":[{"year":2003,"finding":"hMis12 localizes to the kinetochore region and is required for equal chromosome segregation; RNAi depletion causes misaligned metaphase chromosomes, lagging anaphase chromosomes, and extended metaphase spindle length, while kinetochore localization of hMis12 is independent of CENP-A loading pathway.","method":"RNAi depletion in HeLa cells, immunofluorescence, live imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — clean loss-of-function with specific phenotypic readouts, replicated across multiple kinetochore proteins, foundational paper with 201 citations","pmids":["12515822"],"is_preprint":false},{"year":2004,"finding":"Human Mis12 forms a core complex with nine binding partners including HEC1, Zwint-1, c20orf172, DC8, PMF1, and KIAA1570, and also stably associates with centromeric heterochromatin proteins HP1α and HP1γ; double RNAi of HP1 abolishes kinetochore localization of hMis12 and DC8, indicating HP1 anchors the Mis12 complex at centromeric heterochromatin.","method":"Co-immunoprecipitation, mass spectrometry, RNAi in HeLa cells, immunofluorescence","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP/MS interactome plus functional RNAi epistasis, highly cited foundational paper","pmids":["15502821"],"is_preprint":false},{"year":2006,"finding":"A stable four-subunit human Mis12 complex (hMis12, hDsn1, hNnf1/PMF1, hNsl1) is required for kinetochore assembly; depletion reduces outer kinetochore Ndc80/HEC1 localization, diminishes CENP-A, CENP-H, BubR1, and CENP-E levels at kinetochores, causes chromosome misalignment and reduced centromere stretch.","method":"Bacterial co-expression, mitotic extract fractionation, RNAi in human and chicken DT40 cells, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical reconstitution of complex plus clean KO/KD with multiple defined phenotypic readouts","pmids":["16585270"],"is_preprint":false},{"year":2010,"finding":"The human MIS12 complex has an elongated structure (~22 nm long axis) and the NSL1 subunit acts as a scaffold that supports interactions of the MIS12 complex with both the NDC80 and KNL1 complexes within the KMN network.","method":"Biochemical analysis, cross-linking mass spectrometry, negative-stain electron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — structural EM combined with biochemical cross-linking, replicated in budding yeast system","pmids":["20819937"],"is_preprint":false},{"year":2010,"finding":"Human Hsp90-Sgt1 chaperone complex interacts with and stabilizes the Mis12 complex; inhibition of Hsp90 or Sgt1 destabilizes Mis12 complex and delays chromosome alignment by impairing kinetochore microtubule-binding site formation. Co-inhibition of Sgt1 and Skp1 increases Mis12 at kinetochores, suggesting a balance of Mis12 assembly and turnover is required.","method":"Co-immunoprecipitation, Hsp90/Sgt1 inhibition, siRNA depletion, immunofluorescence in human cells","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional depletion with defined cellular phenotype, single lab","pmids":["20404110"],"is_preprint":false},{"year":2011,"finding":"A conserved N-terminal motif of CENP-C directly binds the Mis12 complex with high affinity, linking the inner kinetochore (CCAN) to the outer kinetochore (KMN network); expression of the isolated CENP-C N-terminal segment in HeLa cells prevents outer kinetochore assembly and causes chromosome missegregation and spindle assembly checkpoint impairment.","method":"In vitro binding assays, HeLa cell expression of dominant-negative CENP-C fragment, immunofluorescence, checkpoint assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 — direct binding assay plus dominant-negative functional validation, highly cited","pmids":["21353556"],"is_preprint":false},{"year":2014,"finding":"RWD domains of Knl1 bind the Mis12 complex and mediate kinetochore targeting of Knl1; the KMN network 3D EM structure shows that RWD domain-containing proteins Spc24, Spc25, and Knl1 all use the Mis12 complex as a central interaction hub for outer kinetochore topology.","method":"Biochemical interaction assays, 3D electron microscopy structure of KMN network","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — structural EM combined with biochemical reconstitution and functional validation in cells","pmids":["24530301"],"is_preprint":false},{"year":2015,"finding":"The budding yeast MIND (Mis12) complex enhances microtubule-binding affinity of a single Ndc80 complex by fourfold in a single-molecule assay; MIND does not bind microtubules itself but binds Ndc80 far from the microtubule-binding domain to allosterically activate microtubule interaction, and this activation is redundant with a Ndc80 mutation that may alter its folded conformation.","method":"Single-molecule microtubule-binding assays, biochemical binding studies, yeast genetics","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — quantitative single-molecule reconstitution assay demonstrating allosteric activation mechanism","pmids":["26430240"],"is_preprint":false},{"year":2016,"finding":"Crystal structures of human MIS12 complex bound to a CENP-C fragment reveal the structural basis for MIS12C's bridging function between the KMN network and chromosome-proximal kinetochore; Aurora B kinase phosphorylates Dsn1 within MIS12C to regulate the CENP-C interaction and strengthen inner-outer kinetochore connectivity.","method":"X-ray crystallography, in vitro kinase assays, mutagenesis, cell-based functional assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with biochemical and functional validation, >130 citations","pmids":["27881301"],"is_preprint":false},{"year":2016,"finding":"Cep57 localizes to kinetochores and directly binds Mis12 (a KMN network component) as well as Mad1; depletion of Cep57 reduces kinetochore localization of Mad1-Mad2 and impairs spindle assembly checkpoint signaling, while Cep57's microtubule-binding activity mediates timely removal of Mad1 from kinetochores.","method":"Co-immunoprecipitation, siRNA depletion, immunofluorescence, checkpoint signaling assays in human cells","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus RNAi functional phenotype with defined SAC readout, single lab","pmids":["26743940"],"is_preprint":false},{"year":2018,"finding":"In living human interphase cells, hMis12, Nsl1, Dsn1, and Nnf1 form a complex in the nucleoplasm outside centromeres, and at least hMis12 in this soluble pool associates with the CENP-C/H/I/K/M/T/W/N/L complex, as measured by fluorescence cross-correlation spectroscopy.","method":"Fluorescence cross-correlation spectroscopy (FCCS) in living human cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — direct live-cell protein interaction measurement with quantitative dissociation constants, single lab","pmids":["29509805"],"is_preprint":false},{"year":2020,"finding":"METTL3-mediated m6A methylation of MIS12 mRNA stabilizes the transcript through recognition by the m6A reader IGF2BP2; loss of m6A modifications accelerates MIS12 mRNA turnover and reduces MIS12 protein, leading to accelerated human mesenchymal stem cell senescence.","method":"m6A transcriptome profiling, METTL3 knockout/overexpression, IGF2BP2 reader assay, mRNA stability assay in human cells","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (m6A mapping, KO, overexpression, reader identification, mRNA stability), strong mechanistic chain","pmids":["33035345"],"is_preprint":false},{"year":2020,"finding":"In mouse oocytes, Mis12 localizes to the cytoplasm and spindle poles (not kinetochores) and is required for G2/M transition by regulating cyclin B1 accumulation through a Cdc14B-APC/CCdh1 pathway; depletion impairs GVBD, rescued by cyclin B1 overexpression or Cdc14B/Cdh1 depletion, while kinetochore functions appear dispensable for meiotic progression.","method":"Morpholino/siRNA depletion in mouse oocytes, rescue experiments with cyclin B1 overexpression and Cdc14B/Cdh1 depletion, immunofluorescence","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis rescue experiments with multiple genetic interventions establishing pathway position, single lab","pmids":["32341029"],"is_preprint":false},{"year":2024,"finding":"FTO stabilizes MIS12 protein in vascular smooth muscle cells through a proteasome-mediated pathway; FTO upregulation prevents ox-LDL-induced VSMC senescence and cell cycle arrest by maintaining MIS12 protein levels.","method":"FTO overexpression/knockdown in primary VSMCs, proteasome inhibitor experiments, Western blotting, SA-β-gal assay","journal":"Journal of inflammation research","confidence":"Low","confidence_rationale":"Tier 3 — single lab, mechanism of FTO-MIS12 interaction not directly established at molecular level","pmids":["38523689"],"is_preprint":false},{"year":2024,"finding":"CENP-C binding to the Mis12 complex facilitates centromeric recruitment of Aurora B, and Aurora B in turn reinforces the CENP-C-Mis12C interaction, creating a positive regulatory loop that ensures kinetochore-microtubule error correction and chromosome biorientation.","method":"CENP-C mutant cell lines lacking Mis12C-binding region, Aurora B localization assays, chromosome segregation analysis in human RPE-1 and mouse cells","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 — genetic mutant analysis with functional phenotype readouts and Aurora B localization measurements establishing regulatory loop","pmids":["39433344"],"is_preprint":false},{"year":2024,"finding":"CENP-T interacts with the Mis12 complex through three binding surfaces; this interaction is cooperatively regulated by dual phosphorylation of Dsn1 (Mis12C component) and CENP-T, ensuring robust Mis12C recruitment to CENP-T and proper mitotic progression.","method":"AlphaFold2 structure prediction, biochemical binding assays, mutagenesis, DT40 cells lacking CENP-C-Mis12C interaction, cell biological analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — structural prediction combined with biochemical validation and cell biology in a separation-of-function cell system","pmids":["39628583"],"is_preprint":false},{"year":2025,"finding":"MIS12 is phosphorylated at Ser177 by NEK2A from prophase to prometaphase, which expands the fibrous corona (outer kinetochore projection) to facilitate microtubule attachment; PP1 dephosphorylates Ser177 upon chromosome alignment to promote kinetochore compaction and end-on attachment conversion.","method":"Phosphosite mapping, in vitro kinase assay with NEK2A, phospho-specific antibody, PP1 functional assay, super-resolution imaging of fibrous corona in human cells","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 — identification of writer (NEK2A) and eraser (PP1) with in vitro kinase assay, mutagenesis, and structural phenotype (fibrous corona expansion)","pmids":["40560426"],"is_preprint":false},{"year":2025,"finding":"In budding yeast, α-helical C-terminal motifs of Mtw1 complex subunits Dsn1, Mtw1, and Nnf1 bind Knl1c and Ndc80c; an N-terminal auto-inhibitory segment of Dsn1 occludes binding sites for inner kinetochore subunits CENP-C/Mif2 and CENP-U/Ame1, and Aurora B/Ipl1 phosphorylation of this segment releases auto-inhibition to strengthen inner-outer kinetochore connections.","method":"Cryo-EM structure determination, biochemical binding assays, genetic experiments in S. cerevisiae","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — cryo-EM structure with biochemical and genetic validation, but preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"MIS12 is the central subunit of the tetrameric MIS12 complex (with DSN1, NSL1, PMF1), which forms an elongated ~22 nm scaffold at the outer kinetochore that directly bridges the inner kinetochore (by binding CENP-C through its N-terminal motif, regulated by Aurora B phosphorylation of DSN1 to relieve auto-inhibition) to the outer KMN network (by serving as a scaffold for NDC80 and KNL1 complexes via NSL1 and RWD domain interactions), allosterically enhancing Ndc80 microtubule-binding affinity; dynamic phosphorylation of MIS12-Ser177 by NEK2A expands the fibrous corona for initial microtubule capture, while PP1-mediated dephosphorylation drives compaction for stable end-on attachment, and at the mRNA level, METTL3-mediated m6A modification stabilizes MIS12 transcript via the IGF2BP2 reader."},"narrative":{"teleology":[{"year":2003,"claim":"Establishing that MIS12 is a kinetochore protein required for chromosome alignment answered the fundamental question of where and why this gene acts during mitosis.","evidence":"RNAi depletion in HeLa cells with immunofluorescence and live imaging showing misalignment and lagging chromosomes","pmids":["12515822"],"confidence":"High","gaps":["Binding partners and complex composition unknown","Mechanism of kinetochore targeting not determined"]},{"year":2004,"claim":"Identification of a multi-subunit MIS12 interactome (including HEC1, Zwint-1, PMF1, HP1α/γ) revealed that MIS12 operates as a hub connecting centromeric heterochromatin to outer kinetochore components.","evidence":"Co-immunoprecipitation and mass spectrometry in HeLa cells, HP1 double RNAi abolishing MIS12 kinetochore localization","pmids":["15502821"],"confidence":"High","gaps":["Stoichiometry and minimal complex composition not resolved","Direct versus bridged interactions not distinguished"]},{"year":2006,"claim":"Reconstitution of a defined four-subunit MIS12 complex (MIS12, DSN1, PMF1, NSL1) established the minimal functional unit and showed it is required for recruitment of outer kinetochore components including NDC80/HEC1.","evidence":"Bacterial co-expression, mitotic extract fractionation, RNAi in human and chicken DT40 cells","pmids":["16585270"],"confidence":"High","gaps":["Structural architecture of the complex unknown","How MIS12 complex connects to inner kinetochore not resolved"]},{"year":2010,"claim":"Structural and biochemical dissection revealed the elongated ~22 nm shape of the MIS12 complex and identified NSL1 as the scaffold bridging NDC80 and KNL1 complexes, defining MIS12 complex topology within the KMN network.","evidence":"Negative-stain EM, cross-linking mass spectrometry, biochemical reconstitution","pmids":["20819937"],"confidence":"High","gaps":["Atomic-resolution structure lacking","CENP-C binding interface not mapped"]},{"year":2011,"claim":"Discovery that CENP-C directly binds the MIS12 complex through a conserved N-terminal motif answered how the inner kinetochore physically connects to the outer KMN network.","evidence":"In vitro binding assays and dominant-negative CENP-C fragment expression in HeLa cells","pmids":["21353556"],"confidence":"High","gaps":["Structural basis of the CENP-C–MIS12 interface unresolved","Regulatory mechanism controlling this interaction unknown"]},{"year":2014,"claim":"Demonstration that RWD domains of KNL1 bind the MIS12 complex, together with 3D EM of the KMN network, established MIS12 as the central interaction hub organizing outer kinetochore topology.","evidence":"Biochemical interaction assays and 3D EM reconstruction of the KMN network","pmids":["24530301"],"confidence":"High","gaps":["Allosteric consequences of hub assembly on microtubule binding not yet tested"]},{"year":2015,"claim":"Single-molecule reconstitution showed the MIS12 (MIND) complex allosterically enhances Ndc80 microtubule-binding affinity fourfold without contacting microtubules itself, revealing a non-obvious activation mechanism.","evidence":"Single-molecule microtubule-binding assays with purified budding yeast complexes","pmids":["26430240"],"confidence":"High","gaps":["Structural basis of allosteric activation not determined","Whether human MIS12 complex has identical quantitative effect untested"]},{"year":2016,"claim":"Crystal structures of the human MIS12 complex bound to CENP-C revealed atomic-level architecture and showed Aurora B phosphorylation of DSN1 regulates the CENP-C interaction, providing the structural and regulatory logic of inner–outer kinetochore coupling.","evidence":"X-ray crystallography, in vitro kinase assays, mutagenesis, and cell-based assays","pmids":["27881301"],"confidence":"High","gaps":["Full-length complex structure with all binding partners not obtained","How phosphorylation dynamics are temporally controlled in vivo unclear"]},{"year":2020,"claim":"Epitranscriptomic regulation of MIS12 was established: METTL3-deposited m6A marks on MIS12 mRNA are read by IGF2BP2 to stabilize the transcript, linking RNA modification to kinetochore protein abundance and cellular senescence.","evidence":"m6A profiling, METTL3 KO/overexpression, IGF2BP2 reader identification, mRNA stability assays in human mesenchymal stem cells","pmids":["33035345"],"confidence":"High","gaps":["Which specific m6A sites on MIS12 mRNA are functionally critical not mapped","Whether this regulation operates in mitotic cells beyond stem cell senescence unknown"]},{"year":2024,"claim":"A positive feedback loop was uncovered in which CENP-C binding to MIS12 complex recruits Aurora B, and Aurora B reinforces the CENP-C–MIS12 interaction, ensuring error correction and biorientation; simultaneously, CENP-T was shown to engage MIS12 complex through three phospho-regulated binding surfaces, establishing dual inner-kinetochore receptor logic.","evidence":"CENP-C mutant cell lines with Aurora B localization assays; AlphaFold2 predictions validated by biochemical binding and DT40 genetic analysis","pmids":["39433344","39628583"],"confidence":"Medium","gaps":["Quantitative contribution of CENP-T versus CENP-C pathways to MIS12 recruitment in different organisms not resolved","How Aurora B feedback is terminated upon biorientation unclear"]},{"year":2025,"claim":"Phosphorylation of MIS12-Ser177 by NEK2A was shown to expand the fibrous corona for initial microtubule capture, with PP1 dephosphorylation compacting the kinetochore for stable end-on attachment—establishing MIS12 as a direct regulatory target controlling kinetochore structural dynamics.","evidence":"Phosphosite mapping, in vitro NEK2A kinase assay, phospho-specific antibody, PP1 functional assay, super-resolution imaging in human cells","pmids":["40560426"],"confidence":"High","gaps":["Structural mechanism by which Ser177 phosphorylation causes corona expansion not determined","Whether other MIS12 phosphosites cooperate with Ser177 untested"]},{"year":null,"claim":"A complete atomic model of the MIS12 complex simultaneously engaged with CENP-C, CENP-T, NDC80, and KNL1 in the context of the full kinetochore is still lacking, and the interplay between DSN1 auto-inhibition relief, MIS12-Ser177 phosphorylation, and Aurora B feedback in dictating kinetochore maturation timing remains to be dissected.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full reconstituted kinetochore structure with all MIS12 complex interfaces resolved","Temporal coordination of NEK2A, Aurora B, and PP1 phospho-regulation on MIS12 complex not integrated","In vivo stoichiometry of CENP-C vs CENP-T pathways for MIS12 recruitment unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3,6,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,16]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,2,5,8]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,2,16]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[1,8]}],"complexes":["MIS12 complex (MIS12/DSN1/NSL1/PMF1)","KMN network"],"partners":["DSN1","NSL1","PMF1","CENP-C","CENP-T","KNL1","NEK2A","IGF2BP2"],"other_free_text":[]},"mechanistic_narrative":"MIS12 is the central organizing subunit of the tetrameric MIS12 complex (with DSN1, NSL1/KIAA1570, PMF1/NNF1), which serves as the principal structural bridge between the inner centromere-proximal kinetochore and the outer microtubule-binding KMN network during chromosome segregation. The ~22 nm elongated MIS12 complex binds CENP-C and CENP-T at its inner face—connections regulated by Aurora B phosphorylation of DSN1 to relieve an auto-inhibitory conformation—and uses NSL1 as a scaffold to recruit the NDC80 and KNL1 complexes via RWD-domain interactions, allosterically enhancing Ndc80 microtubule-binding affinity without contacting microtubules itself [PMID:27881301, PMID:20819937, PMID:26430240, PMID:24530301]. Dynamic phosphorylation of MIS12-Ser177 by NEK2A expands the fibrous corona for initial microtubule capture in prometaphase, while PP1-mediated dephosphorylation compacts the kinetochore for stable end-on attachment [PMID:40560426]. At the transcript level, METTL3-mediated m6A modification stabilizes MIS12 mRNA through the IGF2BP2 reader, linking epitranscriptomic regulation to MIS12 protein abundance and cellular senescence [PMID:33035345]."},"prefetch_data":{"uniprot":{"accession":"Q9H081","full_name":"Protein MIS12 homolog","aliases":[],"length_aa":205,"mass_kda":24.1,"function":"Part of the MIS12 complex which is required for normal chromosome alignment and segregation and for kinetochore formation during mitosis (PubMed:12515822, PubMed:15502821, PubMed:16585270). Essential for proper kinetochore microtubule attachments (PubMed:23891108)","subcellular_location":"Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q9H081/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MIS12","classification":"Common Essential","n_dependent_lines":1134,"n_total_lines":1208,"dependency_fraction":0.9387417218543046},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000167842","cell_line_id":"CID000469","localizations":[{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"DSN1","stoichiometry":10.0},{"gene":"ARPC1A","stoichiometry":10.0},{"gene":"PMF1-BGLAP;PMF1","stoichiometry":10.0},{"gene":"SPC25","stoichiometry":10.0},{"gene":"YWHAG","stoichiometry":10.0},{"gene":"CASC5","stoichiometry":10.0},{"gene":"NSL1","stoichiometry":10.0},{"gene":"ZWINT","stoichiometry":4.0},{"gene":"SPC24","stoichiometry":4.0},{"gene":"NDC80","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000469","total_profiled":1310},"omim":[{"mim_id":"609178","title":"MIS12 KINETOCHORE COMPLEX COMPONENT; MIS12","url":"https://www.omim.org/entry/609178"},{"mim_id":"609177","title":"ZW10 INTERACTING KINETOCHORE PROTEIN; ZWINT","url":"https://www.omim.org/entry/609177"},{"mim_id":"609176","title":"POLYAMINE-MODULATED FACTOR 1; PMF1","url":"https://www.omim.org/entry/609176"},{"mim_id":"609175","title":"DSN1, MIS12 KINETOCHORE COMPLEX COMPONENT; DSN1","url":"https://www.omim.org/entry/609175"},{"mim_id":"609174","title":"NSL1, MIS12 KINETOCHORE COMPLEX COMPONENT; NSL1","url":"https://www.omim.org/entry/609174"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MIS12"},"hgnc":{"alias_symbol":["MGC2488","hMIS12","KNTC2AP","MTW1"],"prev_symbol":[]},"alphafold":{"accession":"Q9H081","domains":[{"cath_id":"-","chopping":"6-97","consensus_level":"high","plddt":91.425,"start":6,"end":97},{"cath_id":"4.10.91","chopping":"149-205","consensus_level":"medium","plddt":81.1791,"start":149,"end":205}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H081","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H081-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H081-F1-predicted_aligned_error_v6.png","plddt_mean":87.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MIS12","jax_strain_url":"https://www.jax.org/strain/search?query=MIS12"},"sequence":{"accession":"Q9H081","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H081.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H081/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H081"}},"corpus_meta":[{"pmid":"15502821","id":"PMC_15502821","title":"A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1.","date":"2004","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15502821","citation_count":222,"is_preprint":false},{"pmid":"21353556","id":"PMC_21353556","title":"Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore.","date":"2011","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/21353556","citation_count":219,"is_preprint":false},{"pmid":"12515822","id":"PMC_12515822","title":"Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.","date":"2003","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/12515822","citation_count":201,"is_preprint":false},{"pmid":"20819937","id":"PMC_20819937","title":"The MIS12 complex is a protein interaction hub for outer kinetochore assembly.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20819937","citation_count":190,"is_preprint":false},{"pmid":"16585270","id":"PMC_16585270","title":"The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation.","date":"2006","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16585270","citation_count":178,"is_preprint":false},{"pmid":"10398680","id":"PMC_10398680","title":"Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation.","date":"1999","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10398680","citation_count":178,"is_preprint":false},{"pmid":"33035345","id":"PMC_33035345","title":"METTL3 counteracts premature aging via m6A-dependent stabilization of MIS12 mRNA.","date":"2020","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/33035345","citation_count":154,"is_preprint":false},{"pmid":"27881301","id":"PMC_27881301","title":"Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores.","date":"2016","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/27881301","citation_count":131,"is_preprint":false},{"pmid":"24530301","id":"PMC_24530301","title":"Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24530301","citation_count":107,"is_preprint":false},{"pmid":"14602074","id":"PMC_14602074","title":"An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase.","date":"2003","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/14602074","citation_count":96,"is_preprint":false},{"pmid":"21075115","id":"PMC_21075115","title":"Molecular architecture and connectivity of the budding yeast Mtw1 kinetochore complex.","date":"2010","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21075115","citation_count":51,"is_preprint":false},{"pmid":"20404110","id":"PMC_20404110","title":"Hsp90-Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore-microtubule binding sites.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20404110","citation_count":49,"is_preprint":false},{"pmid":"16331414","id":"PMC_16331414","title":"Characterization of a Mis12 homologue in Arabidopsis thaliana.","date":"2005","source":"Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology","url":"https://pubmed.ncbi.nlm.nih.gov/16331414","citation_count":37,"is_preprint":false},{"pmid":"26430240","id":"PMC_26430240","title":"Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26430240","citation_count":35,"is_preprint":false},{"pmid":"26743940","id":"PMC_26743940","title":"Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1-Mad2.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26743940","citation_count":27,"is_preprint":false},{"pmid":"21276093","id":"PMC_21276093","title":"CaMtw1, a member of the evolutionarily conserved Mis12 kinetochore protein family, is required for efficient inner kinetochore assembly in the pathogenic yeast Candida albicans.","date":"2011","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/21276093","citation_count":27,"is_preprint":false},{"pmid":"20980244","id":"PMC_20980244","title":"Drosophila Mis12 complex acts as a single functional unit essential for anaphase chromosome movement and a robust spindle assembly checkpoint.","date":"2010","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20980244","citation_count":24,"is_preprint":false},{"pmid":"31611218","id":"PMC_31611218","title":"Antigen-driven selection of antibodies against SSA, SSB and the centromere 'complex', including a novel antigen, MIS12 complex, in human salivary glands.","date":"2019","source":"Annals of the rheumatic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/31611218","citation_count":20,"is_preprint":false},{"pmid":"32341029","id":"PMC_32341029","title":"Mis12 controls cyclin B1 stabilization via Cdc14B-mediated APC/CCdh1 regulation during meiotic G2/M transition in mouse oocytes.","date":"2020","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/32341029","citation_count":15,"is_preprint":false},{"pmid":"29509805","id":"PMC_29509805","title":"CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29509805","citation_count":11,"is_preprint":false},{"pmid":"32997987","id":"PMC_32997987","title":"C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32997987","citation_count":6,"is_preprint":false},{"pmid":"38523689","id":"PMC_38523689","title":"FTO Stabilizes MIS12 to Inhibit Vascular Smooth Muscle Cell Senescence in Atherosclerotic Plaque.","date":"2024","source":"Journal of inflammation research","url":"https://pubmed.ncbi.nlm.nih.gov/38523689","citation_count":6,"is_preprint":false},{"pmid":"19697146","id":"PMC_19697146","title":"Characterization of the two centromeric proteins CENP-C and MIS12 in Nicotiana species.","date":"2009","source":"Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology","url":"https://pubmed.ncbi.nlm.nih.gov/19697146","citation_count":5,"is_preprint":false},{"pmid":"27814517","id":"PMC_27814517","title":"MIS12/MIND Control at the Kinetochore.","date":"2016","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/27814517","citation_count":5,"is_preprint":false},{"pmid":"39433344","id":"PMC_39433344","title":"CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation.","date":"2024","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/39433344","citation_count":3,"is_preprint":false},{"pmid":"31222108","id":"PMC_31222108","title":"Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31222108","citation_count":2,"is_preprint":false},{"pmid":"39628583","id":"PMC_39628583","title":"Molecular details and phosphoregulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/39628583","citation_count":1,"is_preprint":false},{"pmid":"40560426","id":"PMC_40560426","title":"Dynamic phosphorylation of MIS12 ensures accurate kinetochore-microtubule attachment by expanding the fibrous corona.","date":"2025","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/40560426","citation_count":0,"is_preprint":false},{"pmid":"41787525","id":"PMC_41787525","title":"Screening, validation, and transcriptional regulation analysis of oxidative stress-related biomarkers in gestational diabetes mellitus: SH3BP5, ITGAM, PRRG1, and MIS12.","date":"2026","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/41787525","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.28.691167","title":"Unconventional components complement the cryptic kinetochore of the ciliate  <i>Tetrahymena thermophila</i>","date":"2025-12-01","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.28.691167","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.06.20.599825","title":"Molecular details and phospho-regulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells","date":"2024-06-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.20.599825","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.03.657598","title":"Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex","date":"2025-06-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.03.657598","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15511,"output_tokens":4347,"usd":0.055869},"stage2":{"model":"claude-opus-4-6","input_tokens":7807,"output_tokens":2835,"usd":0.164865},"total_usd":0.220734,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"hMis12 localizes to the kinetochore region and is required for equal chromosome segregation; RNAi depletion causes misaligned metaphase chromosomes, lagging anaphase chromosomes, and extended metaphase spindle length, while kinetochore localization of hMis12 is independent of CENP-A loading pathway.\",\n      \"method\": \"RNAi depletion in HeLa cells, immunofluorescence, live imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean loss-of-function with specific phenotypic readouts, replicated across multiple kinetochore proteins, foundational paper with 201 citations\",\n      \"pmids\": [\"12515822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Mis12 forms a core complex with nine binding partners including HEC1, Zwint-1, c20orf172, DC8, PMF1, and KIAA1570, and also stably associates with centromeric heterochromatin proteins HP1α and HP1γ; double RNAi of HP1 abolishes kinetochore localization of hMis12 and DC8, indicating HP1 anchors the Mis12 complex at centromeric heterochromatin.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, RNAi in HeLa cells, immunofluorescence\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP/MS interactome plus functional RNAi epistasis, highly cited foundational paper\",\n      \"pmids\": [\"15502821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"A stable four-subunit human Mis12 complex (hMis12, hDsn1, hNnf1/PMF1, hNsl1) is required for kinetochore assembly; depletion reduces outer kinetochore Ndc80/HEC1 localization, diminishes CENP-A, CENP-H, BubR1, and CENP-E levels at kinetochores, causes chromosome misalignment and reduced centromere stretch.\",\n      \"method\": \"Bacterial co-expression, mitotic extract fractionation, RNAi in human and chicken DT40 cells, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical reconstitution of complex plus clean KO/KD with multiple defined phenotypic readouts\",\n      \"pmids\": [\"16585270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The human MIS12 complex has an elongated structure (~22 nm long axis) and the NSL1 subunit acts as a scaffold that supports interactions of the MIS12 complex with both the NDC80 and KNL1 complexes within the KMN network.\",\n      \"method\": \"Biochemical analysis, cross-linking mass spectrometry, negative-stain electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural EM combined with biochemical cross-linking, replicated in budding yeast system\",\n      \"pmids\": [\"20819937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human Hsp90-Sgt1 chaperone complex interacts with and stabilizes the Mis12 complex; inhibition of Hsp90 or Sgt1 destabilizes Mis12 complex and delays chromosome alignment by impairing kinetochore microtubule-binding site formation. Co-inhibition of Sgt1 and Skp1 increases Mis12 at kinetochores, suggesting a balance of Mis12 assembly and turnover is required.\",\n      \"method\": \"Co-immunoprecipitation, Hsp90/Sgt1 inhibition, siRNA depletion, immunofluorescence in human cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional depletion with defined cellular phenotype, single lab\",\n      \"pmids\": [\"20404110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A conserved N-terminal motif of CENP-C directly binds the Mis12 complex with high affinity, linking the inner kinetochore (CCAN) to the outer kinetochore (KMN network); expression of the isolated CENP-C N-terminal segment in HeLa cells prevents outer kinetochore assembly and causes chromosome missegregation and spindle assembly checkpoint impairment.\",\n      \"method\": \"In vitro binding assays, HeLa cell expression of dominant-negative CENP-C fragment, immunofluorescence, checkpoint assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct binding assay plus dominant-negative functional validation, highly cited\",\n      \"pmids\": [\"21353556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RWD domains of Knl1 bind the Mis12 complex and mediate kinetochore targeting of Knl1; the KMN network 3D EM structure shows that RWD domain-containing proteins Spc24, Spc25, and Knl1 all use the Mis12 complex as a central interaction hub for outer kinetochore topology.\",\n      \"method\": \"Biochemical interaction assays, 3D electron microscopy structure of KMN network\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural EM combined with biochemical reconstitution and functional validation in cells\",\n      \"pmids\": [\"24530301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The budding yeast MIND (Mis12) complex enhances microtubule-binding affinity of a single Ndc80 complex by fourfold in a single-molecule assay; MIND does not bind microtubules itself but binds Ndc80 far from the microtubule-binding domain to allosterically activate microtubule interaction, and this activation is redundant with a Ndc80 mutation that may alter its folded conformation.\",\n      \"method\": \"Single-molecule microtubule-binding assays, biochemical binding studies, yeast genetics\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative single-molecule reconstitution assay demonstrating allosteric activation mechanism\",\n      \"pmids\": [\"26430240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structures of human MIS12 complex bound to a CENP-C fragment reveal the structural basis for MIS12C's bridging function between the KMN network and chromosome-proximal kinetochore; Aurora B kinase phosphorylates Dsn1 within MIS12C to regulate the CENP-C interaction and strengthen inner-outer kinetochore connectivity.\",\n      \"method\": \"X-ray crystallography, in vitro kinase assays, mutagenesis, cell-based functional assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with biochemical and functional validation, >130 citations\",\n      \"pmids\": [\"27881301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cep57 localizes to kinetochores and directly binds Mis12 (a KMN network component) as well as Mad1; depletion of Cep57 reduces kinetochore localization of Mad1-Mad2 and impairs spindle assembly checkpoint signaling, while Cep57's microtubule-binding activity mediates timely removal of Mad1 from kinetochores.\",\n      \"method\": \"Co-immunoprecipitation, siRNA depletion, immunofluorescence, checkpoint signaling assays in human cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus RNAi functional phenotype with defined SAC readout, single lab\",\n      \"pmids\": [\"26743940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In living human interphase cells, hMis12, Nsl1, Dsn1, and Nnf1 form a complex in the nucleoplasm outside centromeres, and at least hMis12 in this soluble pool associates with the CENP-C/H/I/K/M/T/W/N/L complex, as measured by fluorescence cross-correlation spectroscopy.\",\n      \"method\": \"Fluorescence cross-correlation spectroscopy (FCCS) in living human cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell protein interaction measurement with quantitative dissociation constants, single lab\",\n      \"pmids\": [\"29509805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"METTL3-mediated m6A methylation of MIS12 mRNA stabilizes the transcript through recognition by the m6A reader IGF2BP2; loss of m6A modifications accelerates MIS12 mRNA turnover and reduces MIS12 protein, leading to accelerated human mesenchymal stem cell senescence.\",\n      \"method\": \"m6A transcriptome profiling, METTL3 knockout/overexpression, IGF2BP2 reader assay, mRNA stability assay in human cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (m6A mapping, KO, overexpression, reader identification, mRNA stability), strong mechanistic chain\",\n      \"pmids\": [\"33035345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mouse oocytes, Mis12 localizes to the cytoplasm and spindle poles (not kinetochores) and is required for G2/M transition by regulating cyclin B1 accumulation through a Cdc14B-APC/CCdh1 pathway; depletion impairs GVBD, rescued by cyclin B1 overexpression or Cdc14B/Cdh1 depletion, while kinetochore functions appear dispensable for meiotic progression.\",\n      \"method\": \"Morpholino/siRNA depletion in mouse oocytes, rescue experiments with cyclin B1 overexpression and Cdc14B/Cdh1 depletion, immunofluorescence\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis rescue experiments with multiple genetic interventions establishing pathway position, single lab\",\n      \"pmids\": [\"32341029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FTO stabilizes MIS12 protein in vascular smooth muscle cells through a proteasome-mediated pathway; FTO upregulation prevents ox-LDL-induced VSMC senescence and cell cycle arrest by maintaining MIS12 protein levels.\",\n      \"method\": \"FTO overexpression/knockdown in primary VSMCs, proteasome inhibitor experiments, Western blotting, SA-β-gal assay\",\n      \"journal\": \"Journal of inflammation research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, mechanism of FTO-MIS12 interaction not directly established at molecular level\",\n      \"pmids\": [\"38523689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CENP-C binding to the Mis12 complex facilitates centromeric recruitment of Aurora B, and Aurora B in turn reinforces the CENP-C-Mis12C interaction, creating a positive regulatory loop that ensures kinetochore-microtubule error correction and chromosome biorientation.\",\n      \"method\": \"CENP-C mutant cell lines lacking Mis12C-binding region, Aurora B localization assays, chromosome segregation analysis in human RPE-1 and mouse cells\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic mutant analysis with functional phenotype readouts and Aurora B localization measurements establishing regulatory loop\",\n      \"pmids\": [\"39433344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CENP-T interacts with the Mis12 complex through three binding surfaces; this interaction is cooperatively regulated by dual phosphorylation of Dsn1 (Mis12C component) and CENP-T, ensuring robust Mis12C recruitment to CENP-T and proper mitotic progression.\",\n      \"method\": \"AlphaFold2 structure prediction, biochemical binding assays, mutagenesis, DT40 cells lacking CENP-C-Mis12C interaction, cell biological analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structural prediction combined with biochemical validation and cell biology in a separation-of-function cell system\",\n      \"pmids\": [\"39628583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MIS12 is phosphorylated at Ser177 by NEK2A from prophase to prometaphase, which expands the fibrous corona (outer kinetochore projection) to facilitate microtubule attachment; PP1 dephosphorylates Ser177 upon chromosome alignment to promote kinetochore compaction and end-on attachment conversion.\",\n      \"method\": \"Phosphosite mapping, in vitro kinase assay with NEK2A, phospho-specific antibody, PP1 functional assay, super-resolution imaging of fibrous corona in human cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — identification of writer (NEK2A) and eraser (PP1) with in vitro kinase assay, mutagenesis, and structural phenotype (fibrous corona expansion)\",\n      \"pmids\": [\"40560426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In budding yeast, α-helical C-terminal motifs of Mtw1 complex subunits Dsn1, Mtw1, and Nnf1 bind Knl1c and Ndc80c; an N-terminal auto-inhibitory segment of Dsn1 occludes binding sites for inner kinetochore subunits CENP-C/Mif2 and CENP-U/Ame1, and Aurora B/Ipl1 phosphorylation of this segment releases auto-inhibition to strengthen inner-outer kinetochore connections.\",\n      \"method\": \"Cryo-EM structure determination, biochemical binding assays, genetic experiments in S. cerevisiae\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with biochemical and genetic validation, but preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MIS12 is the central subunit of the tetrameric MIS12 complex (with DSN1, NSL1, PMF1), which forms an elongated ~22 nm scaffold at the outer kinetochore that directly bridges the inner kinetochore (by binding CENP-C through its N-terminal motif, regulated by Aurora B phosphorylation of DSN1 to relieve auto-inhibition) to the outer KMN network (by serving as a scaffold for NDC80 and KNL1 complexes via NSL1 and RWD domain interactions), allosterically enhancing Ndc80 microtubule-binding affinity; dynamic phosphorylation of MIS12-Ser177 by NEK2A expands the fibrous corona for initial microtubule capture, while PP1-mediated dephosphorylation drives compaction for stable end-on attachment, and at the mRNA level, METTL3-mediated m6A modification stabilizes MIS12 transcript via the IGF2BP2 reader.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MIS12 is the central organizing subunit of the tetrameric MIS12 complex (with DSN1, NSL1/KIAA1570, PMF1/NNF1), which serves as the principal structural bridge between the inner centromere-proximal kinetochore and the outer microtubule-binding KMN network during chromosome segregation. The ~22 nm elongated MIS12 complex binds CENP-C and CENP-T at its inner face—connections regulated by Aurora B phosphorylation of DSN1 to relieve an auto-inhibitory conformation—and uses NSL1 as a scaffold to recruit the NDC80 and KNL1 complexes via RWD-domain interactions, allosterically enhancing Ndc80 microtubule-binding affinity without contacting microtubules itself [PMID:27881301, PMID:20819937, PMID:26430240, PMID:24530301]. Dynamic phosphorylation of MIS12-Ser177 by NEK2A expands the fibrous corona for initial microtubule capture in prometaphase, while PP1-mediated dephosphorylation compacts the kinetochore for stable end-on attachment [PMID:40560426]. At the transcript level, METTL3-mediated m6A modification stabilizes MIS12 mRNA through the IGF2BP2 reader, linking epitranscriptomic regulation to MIS12 protein abundance and cellular senescence [PMID:33035345].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that MIS12 is a kinetochore protein required for chromosome alignment answered the fundamental question of where and why this gene acts during mitosis.\",\n      \"evidence\": \"RNAi depletion in HeLa cells with immunofluorescence and live imaging showing misalignment and lagging chromosomes\",\n      \"pmids\": [\"12515822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding partners and complex composition unknown\", \"Mechanism of kinetochore targeting not determined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of a multi-subunit MIS12 interactome (including HEC1, Zwint-1, PMF1, HP1α/γ) revealed that MIS12 operates as a hub connecting centromeric heterochromatin to outer kinetochore components.\",\n      \"evidence\": \"Co-immunoprecipitation and mass spectrometry in HeLa cells, HP1 double RNAi abolishing MIS12 kinetochore localization\",\n      \"pmids\": [\"15502821\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and minimal complex composition not resolved\", \"Direct versus bridged interactions not distinguished\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Reconstitution of a defined four-subunit MIS12 complex (MIS12, DSN1, PMF1, NSL1) established the minimal functional unit and showed it is required for recruitment of outer kinetochore components including NDC80/HEC1.\",\n      \"evidence\": \"Bacterial co-expression, mitotic extract fractionation, RNAi in human and chicken DT40 cells\",\n      \"pmids\": [\"16585270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural architecture of the complex unknown\", \"How MIS12 complex connects to inner kinetochore not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Structural and biochemical dissection revealed the elongated ~22 nm shape of the MIS12 complex and identified NSL1 as the scaffold bridging NDC80 and KNL1 complexes, defining MIS12 complex topology within the KMN network.\",\n      \"evidence\": \"Negative-stain EM, cross-linking mass spectrometry, biochemical reconstitution\",\n      \"pmids\": [\"20819937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure lacking\", \"CENP-C binding interface not mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Discovery that CENP-C directly binds the MIS12 complex through a conserved N-terminal motif answered how the inner kinetochore physically connects to the outer KMN network.\",\n      \"evidence\": \"In vitro binding assays and dominant-negative CENP-C fragment expression in HeLa cells\",\n      \"pmids\": [\"21353556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the CENP-C–MIS12 interface unresolved\", \"Regulatory mechanism controlling this interaction unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstration that RWD domains of KNL1 bind the MIS12 complex, together with 3D EM of the KMN network, established MIS12 as the central interaction hub organizing outer kinetochore topology.\",\n      \"evidence\": \"Biochemical interaction assays and 3D EM reconstruction of the KMN network\",\n      \"pmids\": [\"24530301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Allosteric consequences of hub assembly on microtubule binding not yet tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Single-molecule reconstitution showed the MIS12 (MIND) complex allosterically enhances Ndc80 microtubule-binding affinity fourfold without contacting microtubules itself, revealing a non-obvious activation mechanism.\",\n      \"evidence\": \"Single-molecule microtubule-binding assays with purified budding yeast complexes\",\n      \"pmids\": [\"26430240\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of allosteric activation not determined\", \"Whether human MIS12 complex has identical quantitative effect untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Crystal structures of the human MIS12 complex bound to CENP-C revealed atomic-level architecture and showed Aurora B phosphorylation of DSN1 regulates the CENP-C interaction, providing the structural and regulatory logic of inner–outer kinetochore coupling.\",\n      \"evidence\": \"X-ray crystallography, in vitro kinase assays, mutagenesis, and cell-based assays\",\n      \"pmids\": [\"27881301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length complex structure with all binding partners not obtained\", \"How phosphorylation dynamics are temporally controlled in vivo unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Epitranscriptomic regulation of MIS12 was established: METTL3-deposited m6A marks on MIS12 mRNA are read by IGF2BP2 to stabilize the transcript, linking RNA modification to kinetochore protein abundance and cellular senescence.\",\n      \"evidence\": \"m6A profiling, METTL3 KO/overexpression, IGF2BP2 reader identification, mRNA stability assays in human mesenchymal stem cells\",\n      \"pmids\": [\"33035345\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific m6A sites on MIS12 mRNA are functionally critical not mapped\", \"Whether this regulation operates in mitotic cells beyond stem cell senescence unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A positive feedback loop was uncovered in which CENP-C binding to MIS12 complex recruits Aurora B, and Aurora B reinforces the CENP-C–MIS12 interaction, ensuring error correction and biorientation; simultaneously, CENP-T was shown to engage MIS12 complex through three phospho-regulated binding surfaces, establishing dual inner-kinetochore receptor logic.\",\n      \"evidence\": \"CENP-C mutant cell lines with Aurora B localization assays; AlphaFold2 predictions validated by biochemical binding and DT40 genetic analysis\",\n      \"pmids\": [\"39433344\", \"39628583\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative contribution of CENP-T versus CENP-C pathways to MIS12 recruitment in different organisms not resolved\", \"How Aurora B feedback is terminated upon biorientation unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Phosphorylation of MIS12-Ser177 by NEK2A was shown to expand the fibrous corona for initial microtubule capture, with PP1 dephosphorylation compacting the kinetochore for stable end-on attachment—establishing MIS12 as a direct regulatory target controlling kinetochore structural dynamics.\",\n      \"evidence\": \"Phosphosite mapping, in vitro NEK2A kinase assay, phospho-specific antibody, PP1 functional assay, super-resolution imaging in human cells\",\n      \"pmids\": [\"40560426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism by which Ser177 phosphorylation causes corona expansion not determined\", \"Whether other MIS12 phosphosites cooperate with Ser177 untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A complete atomic model of the MIS12 complex simultaneously engaged with CENP-C, CENP-T, NDC80, and KNL1 in the context of the full kinetochore is still lacking, and the interplay between DSN1 auto-inhibition relief, MIS12-Ser177 phosphorylation, and Aurora B feedback in dictating kinetochore maturation timing remains to be dissected.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full reconstituted kinetochore structure with all MIS12 complex interfaces resolved\", \"Temporal coordination of NEK2A, Aurora B, and PP1 phospho-regulation on MIS12 complex not integrated\", \"In vivo stoichiometry of CENP-C vs CENP-T pathways for MIS12 recruitment unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3, 6, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 2, 5, 8]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 2, 16]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"complexes\": [\n      \"MIS12 complex (MIS12/DSN1/NSL1/PMF1)\",\n      \"KMN network\"\n    ],\n    \"partners\": [\n      \"DSN1\",\n      \"NSL1\",\n      \"PMF1\",\n      \"CENP-C\",\n      \"CENP-T\",\n      \"KNL1\",\n      \"NEK2A\",\n      \"IGF2BP2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}