{"gene":"MIS12","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2003,"finding":"hMis12 localizes to kinetochores in a manner independent of CENP-A loading pathway; RNAi depletion of hMis12 causes misaligned metaphase chromosomes, lagging anaphase chromosomes, and extended metaphase spindle length without mitotic delay, while CENP-A remains at kinetochores.","method":"RNAi knockdown in HeLa cells, immunofluorescence, live cell imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KD with defined cellular phenotype, replicated epistasis with CENP-A pathway, multiple orthogonal readouts","pmids":["12515822"],"is_preprint":false},{"year":2004,"finding":"Human hMis12 forms a conserved core complex with nine polypeptides including HEC1, Zwint-1, c20orf172, DC8, PMF1, and KIAA1570; additionally, hMis12 forms a stable complex with centromeric heterochromatin components HP1alpha and HP1gamma, and double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8.","method":"Co-immunoprecipitation, mass spectrometry, RNAi in HeLa cells, immunofluorescence","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry identification, RNAi epistasis with functional localization consequence, multiple orthogonal methods","pmids":["15502821"],"is_preprint":false},{"year":2006,"finding":"hMis12 forms a stable four-subunit complex with hDsn1, hNnf1 (PMF1), and hNsl1 (DC31) in human cells; depletion of Mis12 complex subunits causes mitotic delay, chromosome misalignment, reduced centromere stretch, diminished kinetochore microtubule bundles, and severely reduces kinetochore localization of Ndc80/HEC1, BubR1, CENPE, CENP-A, and CENP-H.","method":"Bacterial coexpression/reconstitution, mitotic extract fractionation, RNAi in human and chicken cells, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical reconstitution combined with RNAi epistasis and multiple downstream localization readouts, independently validated in two vertebrate cell systems","pmids":["16585270"],"is_preprint":false},{"year":2010,"finding":"The human MIS12 complex has an elongated structure (~22 nm long axis) and is organized as a scaffold in which the NSL1 subunit mediates interactions with both the NDC80 and KNL1 complexes within the KMN network.","method":"Negative-stain electron microscopy, biochemical cross-linking, mass spectrometry, pulldown assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — EM structure combined with cross-linking mass spectrometry and biochemical interaction mapping, multiple orthogonal methods in single study","pmids":["20819937"],"is_preprint":false},{"year":2010,"finding":"Human Hsp90-Sgt1 chaperone complex interacts with the Mis12 complex; inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule-binding sites.","method":"Co-immunoprecipitation, Hsp90/Sgt1 inhibition, immunofluorescence in human cells","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP identification of interaction plus functional inhibition studies with cellular phenotype, single lab","pmids":["20404110"],"is_preprint":false},{"year":2011,"finding":"Direct binding of the N-terminal region of CENP-C to the Mis12 complex connects the inner and outer kinetochore; expression of the isolated CENP-C N-terminal motif in HeLa cells prevents outer kinetochore assembly and causes chromosome missegregation and spindle assembly checkpoint impairment.","method":"In vitro binding assay, dominant-negative expression in HeLa cells, immunofluorescence, chromosome segregation assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding assay combined with dominant-negative functional validation and multiple cellular phenotype readouts","pmids":["21353556"],"is_preprint":false},{"year":2014,"finding":"RWD domains in Knl1 bind directly to the Mis12 complex and mediate kinetochore targeting of Knl1; the first 3D EM structure of the full KMN network shows that RWD-domain interactions with Mis12 complex shape KMN network topology.","method":"Biochemical pulldown, negative-stain electron microscopy 3D reconstruction, in vivo kinetochore targeting assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — structural EM combined with biochemical binding assays and in vivo functional validation","pmids":["24530301"],"is_preprint":false},{"year":2015,"finding":"The yeast Mis12/MIND complex (Mtw1, Nsl1, Nnf1, Dsn1) enhances microtubule-binding affinity of a single Ndc80 complex by fourfold in single-molecule assays; MIND itself does not bind microtubules but acts far from the microtubule-binding domain of Ndc80, and its activation is redundant with a Ndc80 mutation that may alter its folded conformation.","method":"Single-molecule biophysics (microtubule-binding assay), in vitro reconstitution, biochemical interaction mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — single-molecule reconstitution with quantitative readout, in vitro assay plus mutagenesis, single lab","pmids":["26430240"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of human MIS12 complex bound to a CENP-C fragment reveals the structural basis of the MIS12C–CENP-C interaction; Aurora B kinase phosphorylation regulates this interaction; the structure allows building a near-complete structural model of the KMN assembly.","method":"X-ray crystallography, in vitro binding/mutagenesis, Aurora B kinase phosphorylation assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with mutagenesis and kinase regulation validated biochemically","pmids":["27881301"],"is_preprint":false},{"year":2016,"finding":"Cep57 binds to Mis12 (a KMN component) and also interacts with Mad1; depletion of Cep57 reduces kinetochore localization of Mad1-Mad2, weakens spindle assembly checkpoint signaling, and increases chromosome segregation errors; microtubule-binding activity of Cep57 is involved in timely removal of Mad1 from kinetochores.","method":"Co-immunoprecipitation, RNAi knockdown in human cells, immunofluorescence, SAC signaling assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP of interaction with Mis12, RNAi functional studies with downstream SAC readouts, single lab","pmids":["26743940"],"is_preprint":false},{"year":2020,"finding":"METTL3-mediated m6A modification stabilizes MIS12 mRNA; loss of m6A modifications accelerates MIS12 mRNA turnover and decreases MIS12 expression, accelerating cellular senescence; the m6A reader IGF2BP2 recognizes and stabilizes m6A-modified MIS12 mRNA.","method":"m6A transcriptome profiling, METTL3 knockout/overexpression, IGF2BP2 identification by RIP/pulldown, mRNA stability assay in hMSCs","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple orthogonal methods (m6A-seq, KO, rescue, reader identification) in single lab","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 meiotic G2/M transition by regulating cyclin B1 accumulation through Cdc14B-mediated APC/CCdh1 regulation; impaired GVBD after Mis12 depletion is rescued by overexpressing cyclin B1 or by depleting Cdc14B or Cdh1.","method":"RNAi depletion in mouse oocytes, rescue by cyclin B1 overexpression or Cdc14B/Cdh1 co-depletion, immunofluorescence, GVBD assay","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (double depletion rescue), multiple rescue conditions, localization by immunofluorescence, single lab","pmids":["32341029"],"is_preprint":false},{"year":2019,"finding":"BITC (benzyl isothiocyanate) increases phosphorylated and ubiquitinated Mis12 levels and reduces total Mis12 protein, suggesting Mis12 degradation through the ubiquitin-proteasome system; overexpression of Mis12 suppresses BITC antiproliferative effects in HCT-116 cells, and knockdown enhances them.","method":"Western blotting for phospho/ubiquitin-Mis12, overexpression and siRNA knockdown in human cancer cells, cell cycle analysis","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — western blot for modification with pharmacological agent, no direct E3 ligase identification, single lab and single method per claim","pmids":["31222108"],"is_preprint":false},{"year":2024,"finding":"FTO stabilizes MIS12 protein in vascular smooth muscle cells through a proteasome-mediated pathway; FTO upregulation inhibits VSMC senescence induced by ox-LDL, and this effect is dependent on MIS12 stabilization.","method":"FTO overexpression/knockdown in VSMCs, proteasome inhibitor assays, Western blotting, flow cytometry, SA-β-gal staining","journal":"Journal of inflammation research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — proteasome pathway inferred from inhibitor assays, no direct biochemical reconstitution of FTO-MIS12 interaction, single lab","pmids":["38523689"],"is_preprint":false},{"year":2024,"finding":"CENP-C binding to the outer kinetochore Mis12 complex facilitates centromeric recruitment of Aurora B; Aurora B in turn reinforces the CENP-C-Mis12C interaction, establishing a positive regulatory loop that ensures chromosome biorientation and error correction of kinetochore-microtubule attachments.","method":"CENP-C Mis12-binding domain deletion/mutation in mouse and human RPE-1 cells, Aurora B localization assay, chromosome missegregation quantification","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional deletion mutant combined with Aurora B localization and mitotic error quantification, positive feedback loop established by multiple epistatic experiments","pmids":["39433344"],"is_preprint":false},{"year":2024,"finding":"CENP-T binds the Mis12 complex through three interaction surfaces (identified by AlphaFold predictions validated biochemically and cell biologically); this interaction is cooperatively regulated by dual phosphorylation of Dsn1 (a Mis12C component) and CENP-T, ensuring robust Mis12C recruitment and proper mitotic progression.","method":"AlphaFold2 structure prediction, biochemical binding assays, cell biological validation in DT40 cells lacking CENP-C-Mis12C interaction, phosphorylation analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — computational prediction validated by biochemical and cell biological orthogonal methods in a clean genetic background, single lab","pmids":["39628583"],"is_preprint":false},{"year":2025,"finding":"MIS12 is phosphorylated at Ser177 by NEK2A from prophase to prometaphase; this phosphorylation expands the fibrous corona of the outer kinetochore, facilitating microtubule attachment; Ser177 is subsequently dephosphorylated by PP1 upon chromosome alignment, enabling kinetochore compaction and end-on attachment conversion.","method":"In vitro kinase assay (NEK2A), phospho-specific antibodies, PP1 dephosphorylation assay, super-resolution imaging of kinetochore architecture, phospho-mutant cell lines","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — kinase assay and phosphatase assay identifying writer/eraser, phospho-mutant functional rescue, kinetochore structural readout, single lab","pmids":["40560426"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structures of budding yeast KMN complex reveal that α-helical C-terminal motifs of Mis12c (Mtw1c) 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 on the Mis12c head domain; Aurora B/Ipl1 phosphorylation of Dsn1-AI releases this 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 / Moderate — cryo-EM structure with biochemical and genetic validation, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.06.03.657598"],"is_preprint":true},{"year":2003,"finding":"The budding yeast Mtw1 complex (comprising Mtw1, Dsn1, Nnf1, and Nsl1) is required for kinetochore biorientation; the spindle checkpoint activation in mtw1-1 mutants requires Ipl1/Aurora kinase, suggesting Mtw1 promotes tension at kinetochores; Dsn1 co-immunoprecipitates with Mif2/CENP-C, Cse4/CENP-A, Mtw1, Nnf1, and Nsl1.","method":"Genetic epistasis (mtw1 ipl1 double mutants), dosage suppressor screen, co-immunoprecipitation in S. cerevisiae","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis combined with Co-IP, dosage suppressor screen, multiple orthogonal approaches","pmids":["14602074"],"is_preprint":false},{"year":2010,"finding":"The budding yeast Mtw1 complex can be biochemically reconstituted as two stable heterodimers (Mtw1-Nnf1 and Dsn1-Nsl1) forming an elongated bilobed structure (~25 nm); the complex interacts directly with the Ndc80 complex via Spc24/Spc25 head domain and directly associates with a partial Ctf19 complex in vitro; Ndc80 and Ctf19 complexes do not compete for Mtw1 complex binding.","method":"Biochemical reconstitution, negative-stain electron microscopy, in vitro pulldown assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full in vitro reconstitution with EM structure and direct binding assays, single lab","pmids":["21075115"],"is_preprint":false},{"year":2018,"finding":"In living human interphase cells outside centromeres, hMis12 co-migrates with CENP-C/H/I/K/M/T/W/N/L proteins by fluorescence cross-correlation spectroscopy, indicating that hMis12, Nsl1, Dsn1, and Nnf1 form a complex in the nucleoplasm outside centromeres.","method":"Fluorescence cross-correlation spectroscopy (FCCS) in living human cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell protein co-migration measurement by FCCS, single lab, single method","pmids":["29509805"],"is_preprint":false}],"current_model":"MIS12 is the central scaffold subunit of the conserved tetrameric MIS12 complex (MIS12, DSN1, NSL1, PMF1/NNF1) that occupies the core of the outer kinetochore KMN network, directly bridging the inner kinetochore (via high-affinity binding to CENP-C and CENP-T) to the microtubule-binding NDC80 complex and the checkpoint-scaffolding KNL1 complex through NSL1-mediated contacts; the CENP-C–MIS12 interaction is structurally defined by crystal structures and is regulated by Aurora B kinase phosphorylation of DSN1 (which relieves auto-inhibition) and by PP1-dependent dephosphorylation of MIS12-Ser177 (phosphorylated by NEK2A) to control fibrous corona expansion and compaction, while MIS12 complex stability is also regulated post-translationally by Hsp90-Sgt1 chaperones and by m6A/IGF2BP2-dependent mRNA stabilization via METTL3; in oocytes, MIS12 additionally plays a non-kinetochore role in meiotic G2/M transition by regulating cyclin B1 levels through the Cdc14B–APC/CCdh1 axis."},"narrative":{"mechanistic_narrative":"MIS12 is the central scaffold subunit of a conserved tetrameric kinetochore complex (with DSN1/Dsn1, NSL1/Nsl1, and PMF1/Nnf1) that constitutes the core of the outer-kinetochore KMN network and is required for accurate chromosome segregation [PMID:12515822, PMID:16585270, PMID:14602074]. The complex assembles as an elongated bilobed scaffold in which the NSL1 subunit mediates contacts with both the microtubule-binding NDC80 complex and the checkpoint-scaffolding KNL1 complex, the latter through direct binding of KNL1 RWD domains [PMID:20819937, PMID:24530301, PMID:21075115]; rather than binding microtubules itself, the MIS12 complex enhances the microtubule-binding affinity of NDC80 [PMID:26430240]. MIS12 bridges the inner kinetochore to this outer assembly by binding the N-terminal region of CENP-C, an interaction defined at atomic resolution by crystallography and required for outer-kinetochore assembly and checkpoint function [PMID:21353556, PMID:27881301]; CENP-T provides a second, cooperatively phosphoregulated docking surface for the complex [PMID:39628583]. Assembly of the inner–outer connection is gated by Aurora B phosphorylation of DSN1, which relieves an auto-inhibitory segment that otherwise occludes CENP-C binding, and the CENP-C–MIS12 interaction in turn recruits Aurora B to reinforce biorientation in a positive feedback loop [PMID:27881301, PMID:39433344, PMID:bio_10.1101_2025.06.03.657598]. Kinetochore architecture is further tuned by NEK2A phosphorylation of MIS12-Ser177, which expands the fibrous corona, and by PP1-dependent dephosphorylation that drives compaction and end-on attachment [PMID:40560426]. MIS12 complex stability is supported by the Hsp90-Sgt1 chaperone system and by METTL3/IGF2BP2-dependent m6A stabilization of MIS12 mRNA, whose loss accelerates senescence [PMID:20404110, PMID:33035345]. Beyond mitosis, in mouse oocytes MIS12 acts at the cytoplasm and spindle poles rather than kinetochores to drive the meiotic G2/M transition by regulating cyclin B1 through a Cdc14B–APC/C^Cdh1 axis [PMID:32341029].","teleology":[{"year":2003,"claim":"Established that MIS12 is a kinetochore component functioning independently of the CENP-A loading pathway and required for chromosome alignment and segregation, distinguishing it from inner-centromere assembly.","evidence":"RNAi depletion with immunofluorescence and live imaging in HeLa cells; genetic epistasis and Co-IP in budding yeast","pmids":["12515822","14602074"],"confidence":"High","gaps":["Did not define the subunit composition of the human complex","Mechanism of kinetochore targeting unresolved"]},{"year":2006,"claim":"Defined MIS12 as part of a stable four-subunit complex whose depletion abolishes outer-kinetochore recruitment of NDC80/HEC1 and checkpoint proteins, placing it upstream in kinetochore hierarchy.","evidence":"Bacterial reconstitution plus RNAi epistasis with multiple localization readouts in human and chicken cells","pmids":["16585270"],"confidence":"High","gaps":["Structural organization of the complex not yet resolved","Direct vs indirect dependence of downstream proteins not separated"]},{"year":2010,"claim":"Resolved the elongated scaffold architecture of the complex and assigned NSL1 as the subunit bridging NDC80 and KNL1 within the KMN network, and showed in yeast the bilobed two-heterodimer organization linking NDC80 and the Ctf19/CENP complex.","evidence":"Negative-stain EM, cross-linking mass spectrometry, and in vitro pulldowns (human and S. cerevisiae)","pmids":["20819937","21075115"],"confidence":"High","gaps":["Atomic-resolution contacts not yet defined","Regulation of interface engagement unknown"]},{"year":2010,"claim":"Identified post-translational stabilization of the MIS12 complex by the Hsp90-Sgt1 chaperone system, explaining how functional microtubule-binding sites are built.","evidence":"Co-IP and chaperone inhibition with cellular phenotype in human cells","pmids":["20404110"],"confidence":"Medium","gaps":["Which subunit is the direct chaperone client unresolved","Single lab"]},{"year":2011,"claim":"Demonstrated that direct CENP-C N-terminal binding to the MIS12 complex physically connects inner and outer kinetochore and is required for outer-kinetochore assembly and checkpoint integrity.","evidence":"In vitro binding plus dominant-negative expression and segregation assays in HeLa cells","pmids":["21353556"],"confidence":"High","gaps":["Structural basis of the interaction not yet solved","Regulation of the contact unknown"]},{"year":2014,"claim":"Mapped KNL1 RWD-domain binding to the MIS12 complex and showed it shapes overall KMN network topology, defining how the checkpoint scaffold is targeted.","evidence":"Biochemical pulldowns, 3D negative-stain EM of full KMN, and in vivo targeting assays","pmids":["24530301"],"confidence":"High","gaps":["Residue-level interface not defined","Phosphoregulation of KNL1 recruitment not addressed"]},{"year":2015,"claim":"Showed mechanistically that the complex enhances NDC80 microtubule affinity without itself binding microtubules, defining a non-microtubule activator role.","evidence":"Single-molecule microtubule-binding assays with reconstituted yeast MIND and NDC80 plus mutagenesis","pmids":["26430240"],"confidence":"High","gaps":["Conformational mechanism of NDC80 activation inferred not proven","Conservation in human complex not directly tested here"]},{"year":2016,"claim":"Provided the crystal structure of the human complex bound to CENP-C and showed Aurora B phosphorylation regulates the interaction, enabling a near-complete KMN structural model.","evidence":"X-ray crystallography, in vitro mutagenesis, and Aurora B kinase phosphorylation assay","pmids":["27881301"],"confidence":"High","gaps":["Dynamics of phosphoregulation in cells not fully resolved","Did not address CENP-T contribution"]},{"year":2016,"claim":"Linked the complex to checkpoint signaling via Cep57, which binds MIS12 and supports Mad1-Mad2 kinetochore localization.","evidence":"Co-IP, RNAi, and SAC signaling assays in human cells","pmids":["26743940"],"confidence":"Medium","gaps":["Direct vs indirect Cep57-MIS12 binding not biochemically isolated","Single lab"]},{"year":2020,"claim":"Established mRNA-level control of MIS12 by METTL3/IGF2BP2-dependent m6A stabilization, connecting MIS12 abundance to cellular senescence.","evidence":"m6A profiling, METTL3 KO/overexpression, reader identification, and mRNA stability assays in hMSCs","pmids":["33035345"],"confidence":"Medium","gaps":["Whether senescence is solely MIS12-dependent not isolated","Single lab"]},{"year":2020,"claim":"Revealed a non-kinetochore meiotic role in which oocyte MIS12 drives the G2/M transition through Cdc14B–APC/C^Cdh1 control of cyclin B1.","evidence":"RNAi depletion with cyclin B1 / Cdc14B / Cdh1 rescue epistasis and GVBD assays in mouse oocytes","pmids":["32341029"],"confidence":"Medium","gaps":["Molecular mechanism connecting MIS12 to Cdc14B unknown","Conservation in other cell types untested"]},{"year":2024,"claim":"Defined CENP-T as a second, phosphoregulated docking platform for the complex and established a CENP-C–MIS12–Aurora B positive feedback loop driving biorientation.","evidence":"AlphaFold prediction with biochemical/cellular validation in DT40 cells; CENP-C binding-domain mutants with Aurora B localization in mouse and RPE-1 cells","pmids":["39628583","39433344"],"confidence":"Medium","gaps":["Quantitative contribution of CENP-C vs CENP-T pathways not fully partitioned","Feedback loop kinetics not measured"]},{"year":2025,"claim":"Identified a NEK2A/PP1 phospho-switch on MIS12-Ser177 controlling fibrous corona expansion versus compaction during attachment maturation.","evidence":"NEK2A kinase and PP1 phosphatase assays, phospho-specific antibodies, phospho-mutant cell lines, and super-resolution kinetochore imaging","pmids":["40560426"],"confidence":"Medium","gaps":["How Ser177 phosphorylation mechanically alters corona architecture unresolved","Single lab"]},{"year":2025,"claim":"Provided cryo-EM evidence that a Dsn1 auto-inhibitory segment occludes inner-kinetochore binding sites on the MIS12c head and that Aurora B phosphorylation relieves this auto-inhibition to strengthen inner-outer connections.","evidence":"Cryo-EM of budding yeast KMN with biochemical and genetic validation (preprint)","pmids":["bio_10.1101_2025.06.03.657598"],"confidence":"Medium","gaps":["Not yet peer-reviewed","Direct conservation of the auto-inhibitory mechanism in human MIS12 not shown here"]},{"year":null,"claim":"How the multiple phosphoregulatory inputs (Aurora B on DSN1, NEK2A/PP1 on Ser177) and the CENP-C versus CENP-T receptor pathways are integrated temporally to control kinetochore assembly and maturation remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified spatiotemporal model linking the distinct phospho-switches","Relationship between mitotic scaffold function and the meiotic cyclin B1 role unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,5,6,7]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3,19]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,2,5]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[20]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,2,5,14]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[10]}],"complexes":["MIS12 complex (MIS12-DSN1-NSL1-PMF1/NNF1)","KMN network","kinetochore"],"partners":["CENP-C","CENP-T","NSL1","DSN1","PMF1","KNL1","NDC80","CEP57"],"other_free_text":[]}},"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":222,"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 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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":21,"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 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kinetochores in a manner independent of CENP-A loading pathway; RNAi depletion of hMis12 causes misaligned metaphase chromosomes, lagging anaphase chromosomes, and extended metaphase spindle length without mitotic delay, while CENP-A remains at kinetochores.\",\n      \"method\": \"RNAi knockdown in HeLa cells, immunofluorescence, live cell imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KD with defined cellular phenotype, replicated epistasis with CENP-A pathway, multiple orthogonal readouts\",\n      \"pmids\": [\"12515822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human hMis12 forms a conserved core complex with nine polypeptides including HEC1, Zwint-1, c20orf172, DC8, PMF1, and KIAA1570; additionally, hMis12 forms a stable complex with centromeric heterochromatin components HP1alpha and HP1gamma, and double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, RNAi in HeLa cells, immunofluorescence\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry identification, RNAi epistasis with functional localization consequence, multiple orthogonal methods\",\n      \"pmids\": [\"15502821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"hMis12 forms a stable four-subunit complex with hDsn1, hNnf1 (PMF1), and hNsl1 (DC31) in human cells; depletion of Mis12 complex subunits causes mitotic delay, chromosome misalignment, reduced centromere stretch, diminished kinetochore microtubule bundles, and severely reduces kinetochore localization of Ndc80/HEC1, BubR1, CENPE, CENP-A, and CENP-H.\",\n      \"method\": \"Bacterial coexpression/reconstitution, mitotic extract fractionation, RNAi in human and chicken cells, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical reconstitution combined with RNAi epistasis and multiple downstream localization readouts, independently validated in two vertebrate cell systems\",\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 is organized as a scaffold in which the NSL1 subunit mediates interactions with both the NDC80 and KNL1 complexes within the KMN network.\",\n      \"method\": \"Negative-stain electron microscopy, biochemical cross-linking, mass spectrometry, pulldown assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — EM structure combined with cross-linking mass spectrometry and biochemical interaction mapping, multiple orthogonal methods in single study\",\n      \"pmids\": [\"20819937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human Hsp90-Sgt1 chaperone complex interacts with the Mis12 complex; inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule-binding sites.\",\n      \"method\": \"Co-immunoprecipitation, Hsp90/Sgt1 inhibition, immunofluorescence in human cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP identification of interaction plus functional inhibition studies with cellular phenotype, single lab\",\n      \"pmids\": [\"20404110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Direct binding of the N-terminal region of CENP-C to the Mis12 complex connects the inner and outer kinetochore; expression of the isolated CENP-C N-terminal motif in HeLa cells prevents outer kinetochore assembly and causes chromosome missegregation and spindle assembly checkpoint impairment.\",\n      \"method\": \"In vitro binding assay, dominant-negative expression in HeLa cells, immunofluorescence, chromosome segregation assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding assay combined with dominant-negative functional validation and multiple cellular phenotype readouts\",\n      \"pmids\": [\"21353556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RWD domains in Knl1 bind directly to the Mis12 complex and mediate kinetochore targeting of Knl1; the first 3D EM structure of the full KMN network shows that RWD-domain interactions with Mis12 complex shape KMN network topology.\",\n      \"method\": \"Biochemical pulldown, negative-stain electron microscopy 3D reconstruction, in vivo kinetochore targeting assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — structural EM combined with biochemical binding assays and in vivo functional validation\",\n      \"pmids\": [\"24530301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The yeast Mis12/MIND complex (Mtw1, Nsl1, Nnf1, Dsn1) enhances microtubule-binding affinity of a single Ndc80 complex by fourfold in single-molecule assays; MIND itself does not bind microtubules but acts far from the microtubule-binding domain of Ndc80, and its activation is redundant with a Ndc80 mutation that may alter its folded conformation.\",\n      \"method\": \"Single-molecule biophysics (microtubule-binding assay), in vitro reconstitution, biochemical interaction mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule reconstitution with quantitative readout, in vitro assay plus mutagenesis, single lab\",\n      \"pmids\": [\"26430240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of human MIS12 complex bound to a CENP-C fragment reveals the structural basis of the MIS12C–CENP-C interaction; Aurora B kinase phosphorylation regulates this interaction; the structure allows building a near-complete structural model of the KMN assembly.\",\n      \"method\": \"X-ray crystallography, in vitro binding/mutagenesis, Aurora B kinase phosphorylation assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with mutagenesis and kinase regulation validated biochemically\",\n      \"pmids\": [\"27881301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cep57 binds to Mis12 (a KMN component) and also interacts with Mad1; depletion of Cep57 reduces kinetochore localization of Mad1-Mad2, weakens spindle assembly checkpoint signaling, and increases chromosome segregation errors; microtubule-binding activity of Cep57 is involved in timely removal of Mad1 from kinetochores.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown in human cells, immunofluorescence, SAC signaling assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP of interaction with Mis12, RNAi functional studies with downstream SAC readouts, single lab\",\n      \"pmids\": [\"26743940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"METTL3-mediated m6A modification stabilizes MIS12 mRNA; loss of m6A modifications accelerates MIS12 mRNA turnover and decreases MIS12 expression, accelerating cellular senescence; the m6A reader IGF2BP2 recognizes and stabilizes m6A-modified MIS12 mRNA.\",\n      \"method\": \"m6A transcriptome profiling, METTL3 knockout/overexpression, IGF2BP2 identification by RIP/pulldown, mRNA stability assay in hMSCs\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple orthogonal methods (m6A-seq, KO, rescue, reader identification) in single lab\",\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 meiotic G2/M transition by regulating cyclin B1 accumulation through Cdc14B-mediated APC/CCdh1 regulation; impaired GVBD after Mis12 depletion is rescued by overexpressing cyclin B1 or by depleting Cdc14B or Cdh1.\",\n      \"method\": \"RNAi depletion in mouse oocytes, rescue by cyclin B1 overexpression or Cdc14B/Cdh1 co-depletion, immunofluorescence, GVBD assay\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (double depletion rescue), multiple rescue conditions, localization by immunofluorescence, single lab\",\n      \"pmids\": [\"32341029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"BITC (benzyl isothiocyanate) increases phosphorylated and ubiquitinated Mis12 levels and reduces total Mis12 protein, suggesting Mis12 degradation through the ubiquitin-proteasome system; overexpression of Mis12 suppresses BITC antiproliferative effects in HCT-116 cells, and knockdown enhances them.\",\n      \"method\": \"Western blotting for phospho/ubiquitin-Mis12, overexpression and siRNA knockdown in human cancer cells, cell cycle analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — western blot for modification with pharmacological agent, no direct E3 ligase identification, single lab and single method per claim\",\n      \"pmids\": [\"31222108\"],\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 inhibits VSMC senescence induced by ox-LDL, and this effect is dependent on MIS12 stabilization.\",\n      \"method\": \"FTO overexpression/knockdown in VSMCs, proteasome inhibitor assays, Western blotting, flow cytometry, SA-β-gal staining\",\n      \"journal\": \"Journal of inflammation research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — proteasome pathway inferred from inhibitor assays, no direct biochemical reconstitution of FTO-MIS12 interaction, single lab\",\n      \"pmids\": [\"38523689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CENP-C binding to the outer kinetochore Mis12 complex facilitates centromeric recruitment of Aurora B; Aurora B in turn reinforces the CENP-C-Mis12C interaction, establishing a positive regulatory loop that ensures chromosome biorientation and error correction of kinetochore-microtubule attachments.\",\n      \"method\": \"CENP-C Mis12-binding domain deletion/mutation in mouse and human RPE-1 cells, Aurora B localization assay, chromosome missegregation quantification\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional deletion mutant combined with Aurora B localization and mitotic error quantification, positive feedback loop established by multiple epistatic experiments\",\n      \"pmids\": [\"39433344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CENP-T binds the Mis12 complex through three interaction surfaces (identified by AlphaFold predictions validated biochemically and cell biologically); this interaction is cooperatively regulated by dual phosphorylation of Dsn1 (a Mis12C component) and CENP-T, ensuring robust Mis12C recruitment and proper mitotic progression.\",\n      \"method\": \"AlphaFold2 structure prediction, biochemical binding assays, cell biological validation in DT40 cells lacking CENP-C-Mis12C interaction, phosphorylation analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — computational prediction validated by biochemical and cell biological orthogonal methods in a clean genetic background, single lab\",\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; this phosphorylation expands the fibrous corona of the outer kinetochore, facilitating microtubule attachment; Ser177 is subsequently dephosphorylated by PP1 upon chromosome alignment, enabling kinetochore compaction and end-on attachment conversion.\",\n      \"method\": \"In vitro kinase assay (NEK2A), phospho-specific antibodies, PP1 dephosphorylation assay, super-resolution imaging of kinetochore architecture, phospho-mutant cell lines\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — kinase assay and phosphatase assay identifying writer/eraser, phospho-mutant functional rescue, kinetochore structural readout, single lab\",\n      \"pmids\": [\"40560426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of budding yeast KMN complex reveal that α-helical C-terminal motifs of Mis12c (Mtw1c) 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 on the Mis12c head domain; Aurora B/Ipl1 phosphorylation of Dsn1-AI releases this 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 / Moderate — cryo-EM structure with biochemical and genetic validation, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The budding yeast Mtw1 complex (comprising Mtw1, Dsn1, Nnf1, and Nsl1) is required for kinetochore biorientation; the spindle checkpoint activation in mtw1-1 mutants requires Ipl1/Aurora kinase, suggesting Mtw1 promotes tension at kinetochores; Dsn1 co-immunoprecipitates with Mif2/CENP-C, Cse4/CENP-A, Mtw1, Nnf1, and Nsl1.\",\n      \"method\": \"Genetic epistasis (mtw1 ipl1 double mutants), dosage suppressor screen, co-immunoprecipitation in S. cerevisiae\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis combined with Co-IP, dosage suppressor screen, multiple orthogonal approaches\",\n      \"pmids\": [\"14602074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The budding yeast Mtw1 complex can be biochemically reconstituted as two stable heterodimers (Mtw1-Nnf1 and Dsn1-Nsl1) forming an elongated bilobed structure (~25 nm); the complex interacts directly with the Ndc80 complex via Spc24/Spc25 head domain and directly associates with a partial Ctf19 complex in vitro; Ndc80 and Ctf19 complexes do not compete for Mtw1 complex binding.\",\n      \"method\": \"Biochemical reconstitution, negative-stain electron microscopy, in vitro pulldown assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full in vitro reconstitution with EM structure and direct binding assays, single lab\",\n      \"pmids\": [\"21075115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In living human interphase cells outside centromeres, hMis12 co-migrates with CENP-C/H/I/K/M/T/W/N/L proteins by fluorescence cross-correlation spectroscopy, indicating that hMis12, Nsl1, Dsn1, and Nnf1 form a complex in the nucleoplasm outside centromeres.\",\n      \"method\": \"Fluorescence cross-correlation spectroscopy (FCCS) in living human cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell protein co-migration measurement by FCCS, single lab, single method\",\n      \"pmids\": [\"29509805\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MIS12 is the central scaffold subunit of the conserved tetrameric MIS12 complex (MIS12, DSN1, NSL1, PMF1/NNF1) that occupies the core of the outer kinetochore KMN network, directly bridging the inner kinetochore (via high-affinity binding to CENP-C and CENP-T) to the microtubule-binding NDC80 complex and the checkpoint-scaffolding KNL1 complex through NSL1-mediated contacts; the CENP-C–MIS12 interaction is structurally defined by crystal structures and is regulated by Aurora B kinase phosphorylation of DSN1 (which relieves auto-inhibition) and by PP1-dependent dephosphorylation of MIS12-Ser177 (phosphorylated by NEK2A) to control fibrous corona expansion and compaction, while MIS12 complex stability is also regulated post-translationally by Hsp90-Sgt1 chaperones and by m6A/IGF2BP2-dependent mRNA stabilization via METTL3; in oocytes, MIS12 additionally plays a non-kinetochore role in meiotic G2/M transition by regulating cyclin B1 levels through the Cdc14B–APC/CCdh1 axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MIS12 is the central scaffold subunit of a conserved tetrameric kinetochore complex (with DSN1/Dsn1, NSL1/Nsl1, and PMF1/Nnf1) that constitutes the core of the outer-kinetochore KMN network and is required for accurate chromosome segregation [#0, #2, #18]. The complex assembles as an elongated bilobed scaffold in which the NSL1 subunit mediates contacts with both the microtubule-binding NDC80 complex and the checkpoint-scaffolding KNL1 complex, the latter through direct binding of KNL1 RWD domains [#3, #6, #19]; rather than binding microtubules itself, the MIS12 complex enhances the microtubule-binding affinity of NDC80 [#7]. MIS12 bridges the inner kinetochore to this outer assembly by binding the N-terminal region of CENP-C, an interaction defined at atomic resolution by crystallography and required for outer-kinetochore assembly and checkpoint function [#5, #8]; CENP-T provides a second, cooperatively phosphoregulated docking surface for the complex [#15]. Assembly of the inner–outer connection is gated by Aurora B phosphorylation of DSN1, which relieves an auto-inhibitory segment that otherwise occludes CENP-C binding, and the CENP-C–MIS12 interaction in turn recruits Aurora B to reinforce biorientation in a positive feedback loop [#8, #14, #17]. Kinetochore architecture is further tuned by NEK2A phosphorylation of MIS12-Ser177, which expands the fibrous corona, and by PP1-dependent dephosphorylation that drives compaction and end-on attachment [#16]. MIS12 complex stability is supported by the Hsp90-Sgt1 chaperone system and by METTL3/IGF2BP2-dependent m6A stabilization of MIS12 mRNA, whose loss accelerates senescence [#4, #10]. Beyond mitosis, in mouse oocytes MIS12 acts at the cytoplasm and spindle poles rather than kinetochores to drive the meiotic G2/M transition by regulating cyclin B1 through a Cdc14B–APC/C^Cdh1 axis [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that MIS12 is a kinetochore component functioning independently of the CENP-A loading pathway and required for chromosome alignment and segregation, distinguishing it from inner-centromere assembly.\",\n      \"evidence\": \"RNAi depletion with immunofluorescence and live imaging in HeLa cells; genetic epistasis and Co-IP in budding yeast\",\n      \"pmids\": [\"12515822\", \"14602074\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the subunit composition of the human complex\", \"Mechanism of kinetochore targeting unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined MIS12 as part of a stable four-subunit complex whose depletion abolishes outer-kinetochore recruitment of NDC80/HEC1 and checkpoint proteins, placing it upstream in kinetochore hierarchy.\",\n      \"evidence\": \"Bacterial reconstitution plus RNAi epistasis with multiple localization readouts in human and chicken cells\",\n      \"pmids\": [\"16585270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural organization of the complex not yet resolved\", \"Direct vs indirect dependence of downstream proteins not separated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved the elongated scaffold architecture of the complex and assigned NSL1 as the subunit bridging NDC80 and KNL1 within the KMN network, and showed in yeast the bilobed two-heterodimer organization linking NDC80 and the Ctf19/CENP complex.\",\n      \"evidence\": \"Negative-stain EM, cross-linking mass spectrometry, and in vitro pulldowns (human and S. cerevisiae)\",\n      \"pmids\": [\"20819937\", \"21075115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution contacts not yet defined\", \"Regulation of interface engagement unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified post-translational stabilization of the MIS12 complex by the Hsp90-Sgt1 chaperone system, explaining how functional microtubule-binding sites are built.\",\n      \"evidence\": \"Co-IP and chaperone inhibition with cellular phenotype in human cells\",\n      \"pmids\": [\"20404110\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which subunit is the direct chaperone client unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that direct CENP-C N-terminal binding to the MIS12 complex physically connects inner and outer kinetochore and is required for outer-kinetochore assembly and checkpoint integrity.\",\n      \"evidence\": \"In vitro binding plus dominant-negative expression and segregation assays in HeLa cells\",\n      \"pmids\": [\"21353556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the interaction not yet solved\", \"Regulation of the contact unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapped KNL1 RWD-domain binding to the MIS12 complex and showed it shapes overall KMN network topology, defining how the checkpoint scaffold is targeted.\",\n      \"evidence\": \"Biochemical pulldowns, 3D negative-stain EM of full KMN, and in vivo targeting assays\",\n      \"pmids\": [\"24530301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Residue-level interface not defined\", \"Phosphoregulation of KNL1 recruitment not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed mechanistically that the complex enhances NDC80 microtubule affinity without itself binding microtubules, defining a non-microtubule activator role.\",\n      \"evidence\": \"Single-molecule microtubule-binding assays with reconstituted yeast MIND and NDC80 plus mutagenesis\",\n      \"pmids\": [\"26430240\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational mechanism of NDC80 activation inferred not proven\", \"Conservation in human complex not directly tested here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided the crystal structure of the human complex bound to CENP-C and showed Aurora B phosphorylation regulates the interaction, enabling a near-complete KMN structural model.\",\n      \"evidence\": \"X-ray crystallography, in vitro mutagenesis, and Aurora B kinase phosphorylation assay\",\n      \"pmids\": [\"27881301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of phosphoregulation in cells not fully resolved\", \"Did not address CENP-T contribution\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked the complex to checkpoint signaling via Cep57, which binds MIS12 and supports Mad1-Mad2 kinetochore localization.\",\n      \"evidence\": \"Co-IP, RNAi, and SAC signaling assays in human cells\",\n      \"pmids\": [\"26743940\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect Cep57-MIS12 binding not biochemically isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established mRNA-level control of MIS12 by METTL3/IGF2BP2-dependent m6A stabilization, connecting MIS12 abundance to cellular senescence.\",\n      \"evidence\": \"m6A profiling, METTL3 KO/overexpression, reader identification, and mRNA stability assays in hMSCs\",\n      \"pmids\": [\"33035345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether senescence is solely MIS12-dependent not isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a non-kinetochore meiotic role in which oocyte MIS12 drives the G2/M transition through Cdc14B–APC/C^Cdh1 control of cyclin B1.\",\n      \"evidence\": \"RNAi depletion with cyclin B1 / Cdc14B / Cdh1 rescue epistasis and GVBD assays in mouse oocytes\",\n      \"pmids\": [\"32341029\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism connecting MIS12 to Cdc14B unknown\", \"Conservation in other cell types untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined CENP-T as a second, phosphoregulated docking platform for the complex and established a CENP-C–MIS12–Aurora B positive feedback loop driving biorientation.\",\n      \"evidence\": \"AlphaFold prediction with biochemical/cellular validation in DT40 cells; CENP-C binding-domain mutants with Aurora B localization in mouse and RPE-1 cells\",\n      \"pmids\": [\"39628583\", \"39433344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative contribution of CENP-C vs CENP-T pathways not fully partitioned\", \"Feedback loop kinetics not measured\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a NEK2A/PP1 phospho-switch on MIS12-Ser177 controlling fibrous corona expansion versus compaction during attachment maturation.\",\n      \"evidence\": \"NEK2A kinase and PP1 phosphatase assays, phospho-specific antibodies, phospho-mutant cell lines, and super-resolution kinetochore imaging\",\n      \"pmids\": [\"40560426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Ser177 phosphorylation mechanically alters corona architecture unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided cryo-EM evidence that a Dsn1 auto-inhibitory segment occludes inner-kinetochore binding sites on the MIS12c head and that Aurora B phosphorylation relieves this auto-inhibition to strengthen inner-outer connections.\",\n      \"evidence\": \"Cryo-EM of budding yeast KMN with biochemical and genetic validation (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not yet peer-reviewed\", \"Direct conservation of the auto-inhibitory mechanism in human MIS12 not shown here\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple phosphoregulatory inputs (Aurora B on DSN1, NEK2A/PP1 on Ser177) and the CENP-C versus CENP-T receptor pathways are integrated temporally to control kinetochore assembly and maturation remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified spatiotemporal model linking the distinct phospho-switches\", \"Relationship between mitotic scaffold function and the meiotic cyclin B1 role unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 5, 6, 7]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 2, 5, 14]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [\n      \"MIS12 complex (MIS12-DSN1-NSL1-PMF1/NNF1)\",\n      \"KMN network\",\n      \"kinetochore\"\n    ],\n    \"partners\": [\n      \"CENP-C\",\n      \"CENP-T\",\n      \"NSL1\",\n      \"DSN1\",\n      \"PMF1\",\n      \"KNL1\",\n      \"NDC80\",\n      \"Cep57\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}