Affinage

MIS12

Protein MIS12 homolog · UniProt Q9H081

Length
205 aa
Mass
24.1 kDa
Annotated
2026-06-10
28 papers in source corpus 21 papers cited in narrative 21 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2003 High

    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

    PMID:12515822 PMID:14602074

    Open questions at the time
    • Did not define the subunit composition of the human complex
    • Mechanism of kinetochore targeting unresolved
  2. 2006 High

    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

    PMID:16585270

    Open questions at the time
    • Structural organization of the complex not yet resolved
    • Direct vs indirect dependence of downstream proteins not separated
  3. 2010 High

    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)

    PMID:20819937 PMID:21075115

    Open questions at the time
    • Atomic-resolution contacts not yet defined
    • Regulation of interface engagement unknown
  4. 2010 Medium

    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

    PMID:20404110

    Open questions at the time
    • Which subunit is the direct chaperone client unresolved
    • Single lab
  5. 2011 High

    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

    PMID:21353556

    Open questions at the time
    • Structural basis of the interaction not yet solved
    • Regulation of the contact unknown
  6. 2014 High

    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

    PMID:24530301

    Open questions at the time
    • Residue-level interface not defined
    • Phosphoregulation of KNL1 recruitment not addressed
  7. 2015 High

    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

    PMID:26430240

    Open questions at the time
    • Conformational mechanism of NDC80 activation inferred not proven
    • Conservation in human complex not directly tested here
  8. 2016 High

    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

    PMID:27881301

    Open questions at the time
    • Dynamics of phosphoregulation in cells not fully resolved
    • Did not address CENP-T contribution
  9. 2016 Medium

    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

    PMID:26743940

    Open questions at the time
    • Direct vs indirect Cep57-MIS12 binding not biochemically isolated
    • Single lab
  10. 2020 Medium

    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

    PMID:33035345

    Open questions at the time
    • Whether senescence is solely MIS12-dependent not isolated
    • Single lab
  11. 2020 Medium

    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

    PMID:32341029

    Open questions at the time
    • Molecular mechanism connecting MIS12 to Cdc14B unknown
    • Conservation in other cell types untested
  12. 2024 Medium

    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

    PMID:39433344 PMID:39628583

    Open questions at the time
    • Quantitative contribution of CENP-C vs CENP-T pathways not fully partitioned
    • Feedback loop kinetics not measured
  13. 2025 Medium

    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

    PMID:40560426

    Open questions at the time
    • How Ser177 phosphorylation mechanically alters corona architecture unresolved
    • Single lab
  14. 2025 Medium

    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)

    PMID:bio_10.1101_2025.06.03.657598

    Open questions at the time
    • Not yet peer-reviewed
    • Direct conservation of the auto-inhibitory mechanism in human MIS12 not shown here

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • No unified spatiotemporal model linking the distinct phospho-switches
  • Relationship between mitotic scaffold function and the meiotic cyclin B1 role unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0005198 structural molecule activity 2 GO:0008092 cytoskeletal protein binding 1
Localization
GO:0005694 chromosome 3 GO:0005654 nucleoplasm 1 GO:0005815 microtubule organizing center 1
Pathway
R-HSA-1640170 Cell Cycle 4 R-HSA-8953897 Cellular responses to stimuli 1
Partners
CENP-CCENP-TCEP57DSN1KNL1NDC80NSL1PMF1
Complex memberships
KMN networkMIS12 complex (MIS12-DSN1-NSL1-PMF1/NNF1)kinetochore

Evidence

Reading pass · 21 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2003 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. RNAi knockdown in HeLa cells, immunofluorescence, live cell imaging The Journal of cell biology High 12515822
2004 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. Co-immunoprecipitation, mass spectrometry, RNAi in HeLa cells, immunofluorescence Nature cell biology High 15502821
2006 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. Bacterial coexpression/reconstitution, mitotic extract fractionation, RNAi in human and chicken cells, immunofluorescence The Journal of cell biology High 16585270
2010 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. Negative-stain electron microscopy, biochemical cross-linking, mass spectrometry, pulldown assays The Journal of cell biology High 20819937
2010 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. Co-immunoprecipitation, Hsp90/Sgt1 inhibition, immunofluorescence in human cells The Journal of cell biology Medium 20404110
2011 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. In vitro binding assay, dominant-negative expression in HeLa cells, immunofluorescence, chromosome segregation assay Current biology : CB High 21353556
2014 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. Biochemical pulldown, negative-stain electron microscopy 3D reconstruction, in vivo kinetochore targeting assay Molecular cell High 24530301
2015 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. Single-molecule biophysics (microtubule-binding assay), in vitro reconstitution, biochemical interaction mapping Proceedings of the National Academy of Sciences of the United States of America High 26430240
2016 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. X-ray crystallography, in vitro binding/mutagenesis, Aurora B kinase phosphorylation assay Cell High 27881301
2016 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. Co-immunoprecipitation, RNAi knockdown in human cells, immunofluorescence, SAC signaling assays Nature communications Medium 26743940
2020 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. m6A transcriptome profiling, METTL3 knockout/overexpression, IGF2BP2 identification by RIP/pulldown, mRNA stability assay in hMSCs Nucleic acids research Medium 33035345
2020 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. RNAi depletion in mouse oocytes, rescue by cyclin B1 overexpression or Cdc14B/Cdh1 co-depletion, immunofluorescence, GVBD assay Development (Cambridge, England) Medium 32341029
2019 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. Western blotting for phospho/ubiquitin-Mis12, overexpression and siRNA knockdown in human cancer cells, cell cycle analysis Scientific reports Low 31222108
2024 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. FTO overexpression/knockdown in VSMCs, proteasome inhibitor assays, Western blotting, flow cytometry, SA-β-gal staining Journal of inflammation research Low 38523689
2024 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. CENP-C Mis12-binding domain deletion/mutation in mouse and human RPE-1 cells, Aurora B localization assay, chromosome missegregation quantification Life science alliance Medium 39433344
2024 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. AlphaFold2 structure prediction, biochemical binding assays, cell biological validation in DT40 cells lacking CENP-C-Mis12C interaction, phosphorylation analysis iScience Medium 39628583
2025 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. In vitro kinase assay (NEK2A), phospho-specific antibodies, PP1 dephosphorylation assay, super-resolution imaging of kinetochore architecture, phospho-mutant cell lines Molecular biology of the cell Medium 40560426
2025 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. Cryo-EM structure determination, biochemical binding assays, genetic experiments in S. cerevisiae bioRxivpreprint Medium bio_10.1101_2025.06.03.657598
2003 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. Genetic epistasis (mtw1 ipl1 double mutants), dosage suppressor screen, co-immunoprecipitation in S. cerevisiae Developmental cell High 14602074
2010 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. Biochemical reconstitution, negative-stain electron microscopy, in vitro pulldown assays Journal of molecular biology High 21075115
2018 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. Fluorescence cross-correlation spectroscopy (FCCS) in living human cells PloS one Medium 29509805

Source papers

Stage 0 corpus · 28 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2011 Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Current biology : CB 222 21353556
2004 A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1. Nature cell biology 222 15502821
2003 Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway. The Journal of cell biology 201 12515822
2010 The MIS12 complex is a protein interaction hub for outer kinetochore assembly. The Journal of cell biology 191 20819937
1999 Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation. Genes & development 179 10398680
2006 The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation. The Journal of cell biology 178 16585270
2020 METTL3 counteracts premature aging via m6A-dependent stabilization of MIS12 mRNA. Nucleic acids research 157 33035345
2016 Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores. Cell 133 27881301
2014 Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization. Molecular cell 108 24530301
2003 An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase. Developmental cell 96 14602074
2010 Molecular architecture and connectivity of the budding yeast Mtw1 kinetochore complex. Journal of molecular biology 52 21075115
2010 Hsp90-Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore-microtubule binding sites. The Journal of cell biology 50 20404110
2015 Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex. Proceedings of the National Academy of Sciences of the United States of America 35 26430240
2011 CaMtw1, a member of the evolutionarily conserved Mis12 kinetochore protein family, is required for efficient inner kinetochore assembly in the pathogenic yeast Candida albicans. Molecular microbiology 28 21276093
2016 Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1-Mad2. Nature communications 27 26743940
2010 Drosophila Mis12 complex acts as a single functional unit essential for anaphase chromosome movement and a robust spindle assembly checkpoint. Genetics 24 20980244
2019 Antigen-driven selection of antibodies against SSA, SSB and the centromere 'complex', including a novel antigen, MIS12 complex, in human salivary glands. Annals of the rheumatic diseases 21 31611218
2020 Mis12 controls cyclin B1 stabilization via Cdc14B-mediated APC/CCdh1 regulation during meiotic G2/M transition in mouse oocytes. Development (Cambridge, England) 15 32341029
2018 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. PloS one 11 29509805
2020 C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast. Cell reports 7 32997987
2024 FTO Stabilizes MIS12 to Inhibit Vascular Smooth Muscle Cell Senescence in Atherosclerotic Plaque. Journal of inflammation research 6 38523689
2016 MIS12/MIND Control at the Kinetochore. Cell 5 27814517
2009 Characterization of the two centromeric proteins CENP-C and MIS12 in Nicotiana species. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 5 19697146
2024 CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation. Life science alliance 3 39433344
2024 Molecular details and phosphoregulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells. iScience 2 39628583
2019 Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12. Scientific reports 2 31222108
2026 Screening, validation, and transcriptional regulation analysis of oxidative stress-related biomarkers in gestational diabetes mellitus: SH3BP5, ITGAM, PRRG1, and MIS12. European journal of medical research 0 41787525
2025 Dynamic phosphorylation of MIS12 ensures accurate kinetochore-microtubule attachment by expanding the fibrous corona. Molecular biology of the cell 0 40560426

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