{"gene":"KIF2C","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2003,"finding":"MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, ~100-fold, the rate of tubulin dissociation from microtubule ends. It has one high-affinity binding site per protofilament end, and up to 14 MCAK dimers can assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule, removing ~20 tubulin dimers at a rate of 1 s⁻¹.","method":"In vitro ATPase assay, microtubule depolymerization kinetics, single-molecule analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous in vitro reconstitution with quantitative kinetic analysis, foundational mechanistic study","pmids":["12620232"],"is_preprint":false},{"year":2004,"finding":"Aurora B phosphorylates MCAK at serine 196 (neck region) and additional sites in the centromere-targeting domain, inhibiting its microtubule depolymerization activity in vitro and in vivo. Aurora B activity is required to localize MCAK to centromeres but not spindle poles. Phospho-S196 MCAK was detected at centromeres and anaphase spindle midzones in vivo. Injection of anti-pS196 antibodies into Xenopus egg extracts or cells caused chromosome positioning/segregation defects.","method":"In vitro kinase assay, mass spectrometry phosphorylation site mapping, antibody injection into Xenopus egg extracts and live cells, immunofluorescence","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with site mapping, functional rescue experiments, replicated across multiple labs (PMID 14960279, 14972678)","pmids":["14972678","14960279"],"is_preprint":false},{"year":2004,"finding":"Aurora B inhibits MCAK microtubule depolymerizing activity in vitro. Phosphomimetic MCAK (S/E) mutants inhibit depolymerization in vivo and localize preferentially to the inner centromere, while phospho-null (S/A) mutants concentrate at kinetochores. Both mutants increase syntelic attachments and mono-oriented chromosomes. FRAP analysis identifies two distinct MCAK binding sites at centromeres. Aurora B kinase-dead mutant or RNAi prevents centromeric targeting of MCAK.","method":"In vitro depolymerization assay, phosphomimetic/phosphonull mutagenesis, live-cell fluorescence imaging, FRAP, RNAi","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods, mutagenesis plus functional imaging, replicated in parallel with PMID 14972678","pmids":["14960279"],"is_preprint":false},{"year":2004,"finding":"In Xenopus egg extracts, recombinant Aurora B–INCENP inhibits MCAK microtubule depolymerase activity in a phosphorylation-dependent manner. An Aurora B-resistant alanine mutant (XMCAK-4A) produces mono-astral and monopolar structures instead of bipolar spindles when substituted for endogenous MCAK, and its ability to localize to inner centromeres is abolished. This demonstrates that Aurora B-dependent phosphorylation differentiates cytoplasmic from spindle-associated MCAK activity.","method":"In vitro kinase assay, Xenopus egg extract spindle assembly, phospho-resistant mutagenesis, immunofluorescence localization","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution in egg extracts with phospho-resistant mutant, single lab with multiple orthogonal methods","pmids":["15064354"],"is_preprint":false},{"year":2003,"finding":"Depletion of centromeric MCAK by injection of a dominant-negative centromere-targeting domain into prophase cells leads to reduced centromere stretch, delayed chromosome congression, alignment defects, severe chromosome missegregation, and multiple kinetochore-microtubule attachment defects (merotelic, syntelic, combined). Rates of chromosome movement are unchanged, indicating MCAK's primary role is not chromosome translocation but error correction of kinetochore-microtubule attachments.","method":"Dominant-negative protein microinjection, high-resolution immunofluorescence, live-cell imaging","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative perturbation with defined kinetochore attachment phenotypes, multiple attachment error classes documented","pmids":["14699064"],"is_preprint":false},{"year":2006,"finding":"MCAK moves along the microtubule lattice via one-dimensional (1D) random walk (diffusion coefficient 0.38 μm² s⁻¹, average duration 0.83 s). This lattice diffusion is ATP-independent, whereas catalytic depolymerization requires ATP hydrolysis. The 1D diffusion enables MCAK to target microtubule ends at rates exceeding 3D diffusion limits ('reduction in dimensionality' search strategy).","method":"Single-molecule fluorescence microscopy, TIRF-based in vitro assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous single-molecule in vitro reconstitution with quantitative diffusion measurements, published in Nature","pmids":["16672973"],"is_preprint":false},{"year":2007,"finding":"Among the three human kinesin-13 paralogs, MCAK (Kif2c) specifically functions at kinetochores to regulate microtubule dynamics for chromosome alignment; its loss creates a permissive background that restores bipolar spindle assembly in Kif2a-depleted or Kif2b-depleted cells, demonstrating distinct pathway positions. MCAK and Kif2a activities must be balanced at kinetochores vs. poles for spindle bipolarity.","method":"siRNA knockdown, epistasis by double depletion, live-cell imaging, spindle assembly assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis via double-depletion, replicated across multiple kinesin-13 paralogs, two independent studies (PMID 17538014, 15302853)","pmids":["17538014","15302853"],"is_preprint":false},{"year":2005,"finding":"MCAK tracks (treadmills) with the tips of polymerizing microtubules in living cells. This tip-tracking behavior requires the extreme C-terminal tail of MCAK and is inhibited by phosphorylation. Tip tracking is not essential for MCAK's microtubule-depolymerizing activity, suggesting it serves as a localization mechanism to regions of active plus-end regulation.","method":"Live-cell fluorescence imaging, C-terminal deletion mutants, phospho-mutant analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live imaging with domain deletion and phospho-mutants, single lab with multiple orthogonal approaches","pmids":["15883193"],"is_preprint":false},{"year":2006,"finding":"Aurora B is specifically enriched at merotelic attachment sites. Aurora B activity (but not its localization) is required to enrich MCAK at merotelic attachments and phosphorylates MCAK on residues that regulate its microtubule depolymerase activity at these sites.","method":"Immunofluorescence, Aurora B inhibition, MCAK localization analysis at merotelic sites","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific localization with kinase inhibition, single lab","pmids":["16950107"],"is_preprint":false},{"year":2007,"finding":"hSgo2 is required for MCAK to localize to the centromere. Delocalization of MCAK in hSgo2-depleted cells accounts for uncorrected kinetochore attachment defects. hSgo2 is associated with PP2A and contributes to spatial regulation of MCAK activity within the inner centromere/kinetochore region.","method":"siRNA depletion, immunofluorescence colocalization, Co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA depletion with localization rescue and Co-IP, single lab","pmids":["17485487"],"is_preprint":false},{"year":2010,"finding":"Aurora B phosphorylates hSgo2 at its N-terminal coiled-coil and middle regions; these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, respectively. Aurora B-phosphorylated hSgo2 is essential for recruiting both PP2A and MCAK to centromeres.","method":"In vitro kinase assay, phospho-mutant analysis, Co-immunoprecipitation, immunofluorescence","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay plus phosphomutant Co-IP and localization, single lab with multiple methods","pmids":["20889715"],"is_preprint":false},{"year":2009,"finding":"TIP150 is a plus-end tracking protein (+TIP) that binds EB1 and co-localizes with it at microtubule plus ends. TIP150 also directly binds MCAK; suppression of TIP150 diminishes MCAK plus-end localization. Aurora B-mediated phosphorylation disrupts the TIP150–MCAK interaction in vitro, linking Aurora B regulation to MCAK plus-end targeting.","method":"Co-immunoprecipitation, in vitro binding assay, siRNA knockdown, immunofluorescence, in vitro phosphorylation assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus in vitro kinase disruption assay and siRNA with localization readout, single lab","pmids":["19543227"],"is_preprint":false},{"year":2007,"finding":"MCAK associates with the C-terminus of EB1 and EB3 through an interaction involving the N-terminal localization and regulatory domain of MCAK (not the motor domain). EB1 knockdown impairs GFP-MCAK tip localization in cells. The EB1–MCAK interaction is competitive with other EB1 ligands and does not require microtubules.","method":"Co-immunoprecipitation, GST pulldown domain mapping, siRNA knockdown, live-cell imaging","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal binding assays with domain mapping plus siRNA rescue, single lab","pmids":["17968321"],"is_preprint":false},{"year":2010,"finding":"In vitro reconstitution shows that EB3 targets MCAK to growing microtubule ends by increasing MCAK's association rate with microtubule tips via direct EB3–MCAK interaction. While MCAK alone blocks microtubule assembly, addition of EB3 restores robust growth. The MCAK–EB3 combination enhances catastrophe frequency, promoting rapid switching between growth and shortening, without affecting growth or shortening velocity.","method":"In vitro reconstitution of dynamic microtubule assay, TIRF microscopy, domain mutants, quantitative kinetics","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Moderate — rigorous in vitro reconstitution with dynamic microtubules, quantitative kinetics, single lab with multiple orthogonal experiments","pmids":["20850319"],"is_preprint":false},{"year":2011,"finding":"Kif18b binds directly to MCAK, and this interaction is required for robust microtubule depolymerization of astral microtubules. Aurora kinases negatively regulate the Kif18b–MCAK interaction through phosphorylation of MCAK, demonstrating that Aurora kinases regulate spindle microtubule plus-end stability through control of Kif18b–MCAK complex formation.","method":"Co-immunoprecipitation, siRNA knockdown, in vitro kinase assay, live-cell imaging, microtubule dynamics assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, kinase assay, and functional siRNA with microtubule phenotype, single lab with multiple methods","pmids":["21820309"],"is_preprint":false},{"year":2008,"finding":"Aurora A phosphorylates MCAK at S196 (shared with Aurora B) to regulate MCAK localization and activity at aster centers during Ran-induced spindle assembly, and at S719 to positively enhance bipolar spindle formation. This defines a distinct role for MCAK at spindle poles regulated by Aurora A, separate from Aurora B regulation at centromeres.","method":"Xenopus egg extract spindle assembly, in vitro kinase assay, phosphosite mutagenesis, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with phosphosite-specific mutants and egg extract functional assay, single lab","pmids":["18434591"],"is_preprint":false},{"year":2006,"finding":"The N-terminal domain of MCAK is essential for regulating microtubule dynamics and kinetochore targeting; the C-terminal domain is essential for tight microtubule lattice binding and robust in vitro depolymerization activity. The neck is essential for microtubule end binding. Both C-terminal domain and neck are required for robust in vitro depolymerization activity.","method":"Purified GFP-domain deletion mutants, in vitro depolymerization assay, Xenopus egg extract spindle assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic domain deletion analysis with in vitro reconstitution and in vivo spindle assays, single lab","pmids":["17093055"],"is_preprint":false},{"year":2007,"finding":"Depletion of centromere-associated MCAK using chimeric constructs decreases directional coordination between sister kinetochores, reduces movement speed, and increases tension. Sister centromeres cannot detach efficiently from kinetochore microtubules during directional switching. Anchoring ectopic MCAK to the centromere reverses these effects. This demonstrates that centromere-MCAK promotes kinetochore microtubule turnover to coordinate directional switching.","method":"MCAK chimera/dominant-negative constructs, live-cell imaging, sister centromere distance and dynamics measurements","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — targeted chimera approach with centromere-specific rescue, live imaging with quantitative kinetochore dynamics, single lab","pmids":["18039936"],"is_preprint":false},{"year":2011,"finding":"MCAK depolymerizes microtubules from both plus and minus ends and generates significant tension (~1 pN per motor). An MCAK-decorated bead slides along microtubule sides under weak loads and captures/disassembles both microtubule ends. This 'side-sliding, end-catching' mechanism allows MCAK to generate driving force for chromosome movement at both kinetochores and spindle poles.","method":"Optical trap, bead-MCAK decoration assay, single-molecule microtubule depolymerization force measurements","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — optical trap force measurement with quantitative single-molecule analysis, novel mechanistic finding","pmids":["21602793"],"is_preprint":false},{"year":2013,"finding":"Aurora B phosphorylation at S196 in the neck region opens MCAK from a closed (inhibited) conformation (detected by FRET/FLIM biosensor) to an open conformation, diminishing the interaction between the C-terminal domain and neck. This conformational opening decreases MCAK affinity for the microtubule, reducing its depolymerization activity. MCAK bound to microtubule ends is in a closed conformation relative to lattice-bound MCAK.","method":"FRET biosensor, FLIM, TIRF imaging, phosphomimetic mutagenesis, in vitro depolymerization assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Moderate — FRET/FLIM conformational biosensor combined with mutagenesis and depolymerization assay, multiple orthogonal methods in single study","pmids":["24291095"],"is_preprint":false},{"year":2015,"finding":"The C-terminus of MCAK binds to two motor domains in solution (crystal structure determined), acting as an autoinhibitory interaction that is displaced allosterically upon microtubule binding. This allows robust MCAK accumulation at microtubule ends. The C-terminus–motor interaction represents a structural intermediate in the MCAK catalytic cycle involving long-range conformational changes.","method":"X-ray crystallography (structure of motor domain bound to C-terminus), crosslinking, biochemical binding assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional biochemical validation, single lab with orthogonal methods","pmids":["25915621"],"is_preprint":false},{"year":2015,"finding":"The KVD motif at the tip of kinesin-13-specific loop 2 of Kif2C is required for microtubule-stimulated ATPase activity and depolymerization. Upon microtubule binding, Kif2C undergoes a conformational change governed in part by KVD interaction with the tubulin interdimer interface. A switch-2 glutamate mutant (E to A) that blocks ATP hydrolysis in motile kinesins still depolymerizes microtubules and yields Kif2C–two-tubulin complexes, demonstrating that the conformational change upon tubulin binding is sufficient for tubulin release and ATP hydrolysis is not required for this step.","method":"In vitro ATPase assay, microtubule depolymerization assay, mutagenesis, structural modeling, biochemical complex analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with in vitro enzymatic assays and structural model, multiple orthogonal methods","pmids":["26055718"],"is_preprint":false},{"year":2016,"finding":"The α4-helix residues K524, E525, and R528 of the MCAK motor domain are critical for microtubule end recognition. Mutations at these conserved kinesin-13-specific positions specifically disrupt the ability of MCAK to recognize microtubule ends (reducing end residence time and end-specific ADP dissociation stimulation) without affecting lattice binding, impairing depolymerization.","method":"Mutagenesis, in vitro microtubule end-binding assay, single-molecule fluorescence imaging, ATPase assay","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — targeted mutagenesis with single-molecule end-binding kinetics and nucleotide assays, single lab","pmids":["27733589"],"is_preprint":false},{"year":2019,"finding":"MCAK has a compact conformation in solution (confirmed by crosslinking and electron microscopy). When bound to microtubule ends, MCAK adopts an extended conformation in which the N-terminus and neck interact with the microtubule. The level of Aurora B phosphorylation of the N-terminus results in a graded (not binary) inhibition of MCAK depolymerase activity through allosteric regulation decoupling the N-terminus from the motor domain.","method":"Chemical crosslinking mass spectrometry, electron microscopy, in vitro kinase assay, phosphomimetic mutants, microtubule depolymerization assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crosslinking-EM structural analysis plus graded phosphomutant functional assays, single lab with multiple methods","pmids":["30578316"],"is_preprint":false},{"year":2016,"finding":"GTSE1 inhibits MCAK microtubule depolymerase activity. Cells lacking GTSE1 show defects in chromosome alignment and spindle positioning due to MT instability caused by excess MCAK activity. Artificially elevated GTSE1 hyperstabilizes kinetochore microtubules and increases chromosome missegregation/CIN.","method":"siRNA depletion, overexpression, co-immunoprecipitation, in vitro depolymerization assay, live-cell imaging","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vitro depolymerization inhibition and siRNA/OE with chromosomal phenotype, single lab","pmids":["27881713"],"is_preprint":false},{"year":2016,"finding":"NuSAP is a novel binding partner of MCAK and modulates MCAK depolymerization activity. Aurora B kinase phosphorylation of MCAK significantly enhances the NuSAP–MCAK interaction, and NuSAP modulates MCAK depolymerization in an Aurora B-dependent manner to regulate kinetochore microtubule dynamics.","method":"Co-immunoprecipitation, in vitro depolymerization assay, siRNA knockdown, phospho-mutant analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with in vitro activity modulation and phospho-enhancement, single lab","pmids":["26733216"],"is_preprint":false},{"year":2015,"finding":"Aurora B–PLK1 signaling axis regulates MCAK in mitosis: Aurora B phosphorylates PLK1 at Thr210 to activate PLK1 at kinetochores; active PLK1 in turn phosphorylates MCAK at Ser715 to promote its microtubule depolymerase activity. Non-phosphorylatable MCAK S715A prevents correct kinetochore–microtubule attachment, resulting in chromosome bridges in anaphase.","method":"FRET-based PLK1 activity reporter, phosphorylation site mutagenesis, in vitro kinase assay, immunofluorescence, live-cell imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET reporter plus kinase assay and phosphomutant functional analysis, single lab","pmids":["26206521"],"is_preprint":false},{"year":2014,"finding":"A Rac1–Aurora A–MCAK signaling pathway mediates endothelial cell polarization and directional migration. Aurora A kinase activity (regionally enhanced by Rac1 signaling) locally inhibits MCAK microtubule depolymerizing activity at the trailing edge of polarized wound-edge endothelial cells, promoting regional differences in MT dynamics.","method":"siRNA knockdown, dominant-negative/constitutively active Rac1, inhibitor treatment, high-resolution fluorescence microscopy with MT plus-end tracking, quantitative image analysis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological pathway dissection with live MT imaging, single lab","pmids":["25002679"],"is_preprint":false},{"year":2012,"finding":"MCAK tip-tracking (EB-dependent binding to growing microtubule ends via the SKIP motif N-terminal to the neck) negatively regulates microtubule length within the assembling bipolar spindle. This function requires MCAK's ability to bind EB proteins. Abolishing tip tracking (EB-binding mutant) leads to over-long non-kinetochore microtubules, antagonizes centrosome separation, but ultimately impairs robust kinetochore attachment.","method":"EB-binding SKIP motif mutants, siRNA rescue, live-cell imaging, spindle microtubule length measurements","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutant rescue in mitosis with live imaging readout, single lab","pmids":["22492725"],"is_preprint":false},{"year":2013,"finding":"Lateral-to-end-on conversion of chromosome-microtubule attachment requires MCAK to release laterally attached microtubules after partial end-on attachment is formed, acting sequentially after CENP-E-mediated lateral tethering.","method":"High-resolution live imaging assay, siRNA knockdown of MCAK and CENP-E, classification of attachment intermediates","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with high-resolution imaging of attachment intermediates, single lab","pmids":["23891108"],"is_preprint":false},{"year":2014,"finding":"TIP150–MCAK interaction governs entosis via Aurora A-mediated phosphorylation of MCAK. MCAK forms an intramolecular association required for TIP150 binding; Aurora A phosphorylation of MCAK modulates this intramolecular association, disrupting the MCAK–TIP150 interaction in vitro and inhibiting entosis in vivo. MCAK cooperates with TIP150 to modulate cell mechanical rigidity during entosis.","method":"Co-immunoprecipitation, in vitro kinase/binding assay, optical trap (cell rigidity), live-cell imaging, siRNA knockdown","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with kinase disruption assay and optical trap, single lab with multiple methods","pmids":["24847103"],"is_preprint":false},{"year":2016,"finding":"The far C-terminal residues E715/E716 of MCAK regulate MCAK conformation and spindle pole focusing. Point mutation E715A/E716A increases MCAK targeting to poles and reduces MT lifetimes, inducing unfocused spindle poles—a phenotype phenocopied by Aurora A phosphomimetic S719E. The kinesin-14 XCTK2 rescues unfocused-pole phenotype, placing MCAK and XCTK2 in opposing activities at poles.","method":"Xenopus egg extract spindle assembly, point mutagenesis, immunofluorescence, FRET conformation assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with egg extract spindle assay and FRET conformation readout, single lab","pmids":["26941326"],"is_preprint":false},{"year":2020,"finding":"KIF2C (MCAK) is recruited to DNA double-strand break (DSB) sites in a PARP- and ATM-dependent manner. KIF2C knockdown/knockout leads to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. KIF2C depletion or inhibition of its microtubule depolymerase activity reduces DSB mobility, impairs DNA damage foci formation, and decreases foci fusion/resolution.","method":"DSB-mimicking DNA template pulldown in Xenopus extracts, CRISPR knockout, siRNA knockdown, γH2AX foci analysis, live-cell DSB tracking, NHEJ/HR reporter assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple loss-of-function approaches in two systems with defined DSB repair readouts, single lab","pmids":["31951198"],"is_preprint":false},{"year":2020,"finding":"KIF2C interacts with TBC1D7, and this interaction disrupts formation of the TSC complex, resulting in enhanced mTORC1 signal transduction. KIF2C is a direct transcriptional target of the Wnt/β-catenin pathway and mediates crosstalk between Wnt/β-catenin and mTORC1 signaling in hepatocellular carcinoma.","method":"Co-immunoprecipitation, gain/loss-of-function assays, luciferase reporter assay, Western blot, in vivo xenograft","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with defined pathway disruption assay and transcriptional reporter, single lab","pmids":["32748349"],"is_preprint":false},{"year":2022,"finding":"KIF2C regulates microtubule dynamics in neuronal dendrites and activity-dependent microtubule invasion into dendritic spines. KIF2C knockdown or conditional knockout impairs spine morphology, synaptic AMPA receptor expression, excitatory transmission, long-term potentiation, and cognitive behavior in mice.","method":"RNAi knockdown, conditional knockout (Cre-lox), live microtubule imaging in neurons, electrophysiology, behavioral tests","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with multiple synaptic and behavioral readouts, single lab","pmids":["35138249"],"is_preprint":false},{"year":2023,"finding":"KIF2C regulates transport of mGlu1 receptors in cerebellar Purkinje cells by binding to Rab8. KIF2C deficiency in Purkinje cells reduces mGlu1 and GluA2 synaptic expression, alters excitatory but not inhibitory transmission, and causes motor incoordination in male mice.","method":"Conditional knockout (Purkinje cell-specific), Co-immunoprecipitation with Rab8, immunofluorescence, electrophysiology, behavioral motor tests","journal":"The Journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — cell-type-specific KO with Co-IP partner identification and functional synaptic/behavioral readouts, single lab","pmids":["37431690"],"is_preprint":false},{"year":2021,"finding":"Both overexpression and downregulation of MCAK/KIF2C reduce cell motility and migration. Specifically, altered MCAK levels impair focal adhesion protein composition and phosphorylation, assembly/disassembly rate of focal adhesions, cell adhesion, and plus-tip microtubule dynamics. This demonstrates MCAK acts as a regulator of cell motility through actin-MT cytoskeleton dynamics and focal adhesion turnover.","method":"CRISPR/dCas9 overexpression and knockdown cell lines, live-cell imaging, focal adhesion lifetime assays, MT plus-tip tracking, Western blot","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR perturbation with FA dynamics and MT tracking, single lab with multiple readouts","pmids":["34830827"],"is_preprint":false},{"year":2021,"finding":"MCAK's spatial distribution of activity (higher at trailing edge than leading edge) is required for cell polarity, centrosome positioning, focal adhesion disassembly, and directional migration. Rac1 overexpression has a dominant effect over MCAK activity, placing Rac1 downstream of or parallel to MCAK in the migration pathway.","method":"siRNA knockdown, Rac1 overexpression epistasis, live-cell imaging, FA lifetime measurements, MT dynamics analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis by Rac1/MCAK double manipulation with quantitative live imaging, single lab","pmids":["33566676"],"is_preprint":false},{"year":1999,"finding":"MCAK contains multiple nuclear localization sequences (NLS) and a nuclear exclusion sequence (NES) in the amino-terminal region that balance its nucleocytoplasmic distribution. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, in turn influencing the degree of centromere binding.","method":"GFP-MCAK deletion constructs, ATP-binding domain mutagenesis, fluorescence microscopy localization","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — domain deletions and motor mutants with localization readout, single lab","pmids":["10600236"],"is_preprint":false},{"year":2008,"finding":"MCAK accumulates during the cell cycle, reaches maximum at G2/M, and is rapidly degraded by the proteasome during mitosis. A phosphorylated form of MCAK appears during mitosis and is preferentially degraded. This degradation limits MCAK activity during late mitosis, arguing against a role in anaphase chromosome movement.","method":"Cell cycle fractionation, immunofluorescence, proteasome inhibitor treatment, Western blot","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — cell-cycle staging with proteasome inhibitor and Western blot quantification, single lab","pmids":["18843200"],"is_preprint":false},{"year":2012,"finding":"A phosphorylation site on MCAK controls its proteasomal degradation; phosphorylation-resistant mutation prolongs MCAK stability beyond the metaphase-to-anaphase transition and prevents MCAK removal from centromeres, causing MCAK retention throughout the cell cycle and mitotic defects. A phosphomimetic mutation accelerates degradation.","method":"Phosphosite mutagenesis, proteomic phosphorylation site identification, immunofluorescence, cell division assays","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — phospho-mutant analysis with localization and division phenotype, single lab","pmids":["22422706"],"is_preprint":false},{"year":2017,"finding":"Cdk1 phosphorylates MCAK at T537 within the motor domain. A phosphomimetic T537E mutant significantly impairs microtubule depolymerization by reducing the ability of MCAK to recognize microtubule ends specifically: microtubule-end residence time is reduced, lattice residence time is unaffected, and end-specific ADP dissociation stimulation is abolished.","method":"Phosphomimetic mutagenesis, single-molecule TIRF imaging (end/lattice residence times), ATPase assay, cell transfection phenotype","journal":"PeerJ","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — single-molecule kinetics with mechanistic mutagenesis, single lab","pmids":["29230353"],"is_preprint":false},{"year":2022,"finding":"In vitro reconstitution shows that Kif18b, MCAK, and EB3 act as an integrated network to potently promote microtubule depolymerization at very low concentrations. Kif18b transports EB3 and MCAK to microtubule plus ends through multivalent weak interactions, enabling cooperative plus-end shortening.","method":"In vitro reconstitution, single-molecule TIRF imaging, quantitative microtubule depolymerization assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — rigorous in vitro reconstitution with single-molecule imaging defining cooperative mechanism, single lab","pmids":["35502670"],"is_preprint":false},{"year":2011,"finding":"DDA3 localizes to kinetochores and interacts with MCAK. Depletion of DDA3 causes chromosome congression defects associated with loss of MCAK function at kinetochores, and results in CENP-E accumulation at unaligned kinetochores, without affecting Aurora B/CPC activity.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with siRNA localization phenotype, single lab","pmids":["21426902"],"is_preprint":false},{"year":2005,"finding":"CaMKIIgamma depletion leads to disorganized multipolar spindles by failing to suppress MCAK depolymerase activity. Two distinct but overlapping mechanisms for negative regulation of the cytosolic/centrosomal pool of MCAK exist: one involving CaMKIIgamma and another involving TOGp. Both are essential for spindle bipolarity; their effects are abolished in MCAK-depleted cells, placing both upstream of MCAK in spindle assembly.","method":"siRNA RNAi depletion, inducible overexpression, epistasis by double depletion, immunofluorescence","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via double depletion showing MCAK dependence, single lab","pmids":["15775983"],"is_preprint":false},{"year":2022,"finding":"α-Tubulin detyrosination suppresses MCAK activity; experimental increase of detyrosinated α-tubulin and MCAK depletion produce non-cumulative enhancement of taxol cytotoxicity and cell death in mitosis/interphase, identifying a mechanistic link between α-tubulin detyrosination and MCAK activity suppression.","method":"siRNA knockdown, detyrosination manipulation, flow cytometry cell death assay, clonogenic assay, combinatorial treatment epistasis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via non-cumulative drug/KD effects establishing functional link, single lab","pmids":["36459065"],"is_preprint":false},{"year":2025,"finding":"KIF2C preferentially depolymerizes polyglutamylated tubulin even in the presence of paclitaxel. A chemical inhibitor 7S9 prohibits dissociation of KIF2C from microtubules, counteracting KIF2C-mediated paclitaxel resistance. Combination of 7S9 and paclitaxel significantly reduces tumorigenesis in chemoresistant TNBC mouse models.","method":"In vitro depolymerization assay with polyglutamylated tubulin, KIF2C inhibitor development, in vivo mouse tumor model","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro substrate-specificity assay plus in vivo validation with inhibitor, single lab","pmids":["40157365"],"is_preprint":false},{"year":2021,"finding":"KIF2C directly binds PKM2 (confirmed by Co-IP), and this interaction prevents PKM2 ubiquitination, increasing PKM2 stability. Domain 2 of KIF2C mediates the PKM2 binding. Elevated KIF2C promotes autophagy and glycolysis in doxorubicin-resistant breast cancer cells via PKM2 stabilization.","method":"Co-immunoprecipitation, Western blot (ubiquitination assay), domain deletion mapping, functional metabolic assays","journal":"Cancer cell international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with indirect ubiquitination readout, single lab in cancer context","pmids":["34663310"],"is_preprint":false},{"year":2025,"finding":"The N-terminal domain of KIF2C adopts a Tudor/PWWP/MBT fold that binds phosphorylated motifs, including phosphorylated BRCA2-pT207. KIF2C forms membrane-less organelles (condensates) in an Aurora B- and PLK1-dependent manner via this phospho-binding domain. KIF2C condensation concentrates PLK1 and BRCA2-pT207 at microtubule ends/extremities; condensates exclude tubulin. KIF2C depolymerase activity promotes condensate formation. Aurora B is required for condensate formation.","method":"Structural determination (NTD fold), Co-immunoprecipitation, optogenetic condensate platform, FLIM-FRET, immunofluorescence, mutagenesis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — structural determination plus optogenetic condensate assay and Co-IP, single lab with multiple orthogonal methods, not yet replicated","pmids":["40498077"],"is_preprint":false},{"year":2024,"finding":"Nek2A interacts with KIF2C (identified by TurboID proximity labeling and confirmed by Co-immunoprecipitation and colocalization). KIF2C silencing diminishes Nek2A's ability to prevent centrosome clustering, placing KIF2C downstream of Nek2A in the centrosome clustering/anti-clustering pathway.","method":"TurboID proximity labeling, Co-immunoprecipitation, siRNA knockdown epistasis, immunofluorescence colocalization","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — proximity labeling Co-IP with siRNA epistasis, single lab","pmids":["38493150"],"is_preprint":false},{"year":2024,"finding":"CCDC69 microtubule depolymerization activity is dependent on KIF2C, as demonstrated by the finding that a fraction of CCDC69 localizes to centromeres and KIF2C mediates CCDC69's depolymerization effects. CCDC69 also regulates stability of the chromosomal passenger complex (CPC) by protecting its members from degradation.","method":"Overexpression, siRNA epistasis, immunofluorescence colocalization, microtubule depolymerization assays","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — epistasis by siRNA with indirect depolymerization readout, single lab","pmids":["39638803"],"is_preprint":false}],"current_model":"KIF2C/MCAK is a kinesin-13 microtubule depolymerase that uses ATP hydrolysis to catalytically remove tubulin dimers from microtubule ends after targeting them via ATP-independent 1D lattice diffusion; its depolymerization activity, localization, and conformation are precisely regulated by phosphorylation from Aurora B (inhibitory at S196 via a closed-to-open conformational switch), Aurora A (at S196 and S719, regulating pole activity), PLK1 (activating at S715), and Cdk1 (inhibitory at T537 via loss of end recognition), with centromere targeting additionally controlled by hSgo2 and the CPC; its plus-end accumulation depends on direct interaction with EB1/EB3 and TIP150; it forms cooperative depolymerizing networks with Kif18b and EB3; and beyond its canonical mitotic roles in error correction of kinetochore–microtubule attachments, it functions in interphase cell migration (via focal adhesion and MT dynamics), neuronal synaptic plasticity (by regulating dendritic microtubule invasion and receptor trafficking via Rab8), DNA double-strand break repair (by promoting DSB mobility in a PARP/ATM-dependent manner), and forms Aurora B/PLK1-dependent condensates that concentrate signaling components at microtubule ends during mitosis."},"narrative":{"mechanistic_narrative":"KIF2C (MCAK) is a kinesin-13 microtubule depolymerase that catalytically removes tubulin dimers from microtubule ends to control microtubule dynamics during mitosis and beyond [PMID:12620232]. It accelerates tubulin dissociation ~100-fold by assembling at protofilament ends as an ATP-hydrolyzing complex [PMID:12620232], and targets ends efficiently by first undergoing ATP-independent one-dimensional lattice diffusion before catalysis [PMID:16672973]. Its enzymatic cycle is built from defined elements: the kinesin-13-specific KVD motif of loop 2 senses the tubulin interdimer interface so that the conformational change on tubulin binding—not ATP hydrolysis—drives tubulin release [PMID:26055718], while α4-helix residues and the C-terminal domain/neck confer microtubule-end recognition and robust depolymerization [PMID:27733589, PMID:17093055]. MCAK exists in an autoinhibited closed/compact conformation in which the C-terminus engages the motor domains, an interaction allosterically displaced upon microtubule binding to extend the molecule at ends [PMID:25915621, PMID:30578316]. In cells, its principal mitotic function is error correction of kinetochore-microtubule attachments rather than chromosome translocation: centromere-localized MCAK promotes microtubule turnover to coordinate directional switching and to resolve syntelic, merotelic, and lateral attachments [PMID:14699064, PMID:18039936, PMID:23891108]. This activity is gated by a phosphorylation network—Aurora B phosphorylates S196 to open the molecule and inhibit depolymerization and to drive its centromeric targeting [PMID:14972678, PMID:14960279, PMID:24291095], Aurora A regulates pole activity via S196/S719 [PMID:18434591], PLK1 phosphorylates S715 to activate it [PMID:26206521], and Cdk1 phosphorylates T537 to abolish end recognition [PMID:29230353]—and by binding partners that target or modulate it, including EB1/EB3 and TIP150 for plus-end accumulation [PMID:17968321, PMID:19543227], hSgo2 for centromere recruitment [PMID:17485487, PMID:20889715], and Kif18b/EB3 for cooperative plus-end depolymerizing networks [PMID:21820309, PMID:35502670]. Its conformation and activity are further constrained by inhibitors GTSE1 and tubulin detyrosination [PMID:27881713, PMID:36459065] and by proteasomal degradation that limits late-mitotic activity [PMID:18843200]. Beyond mitosis, KIF2C functions in directional cell migration through focal-adhesion turnover and microtubule dynamics [PMID:34830827, PMID:33566676], in DNA double-strand break repair by promoting PARP/ATM-dependent DSB mobility [PMID:31951198], in neuronal synaptic plasticity by regulating dendritic microtubule invasion and Rab8-dependent receptor trafficking [PMID:35138249, PMID:37431690], and forms Aurora B/PLK1-dependent phospho-binding condensates that concentrate signaling factors at microtubule ends [PMID:40498077].","teleology":[{"year":2003,"claim":"Established the core enzymatic identity of MCAK by showing it is not a motile motor but a catalytic depolymerase that actively destabilizes microtubule ends.","evidence":"In vitro ATPase and depolymerization kinetics with single-molecule analysis","pmids":["12620232"],"confidence":"High","gaps":["Did not define how MCAK reaches ends in cells","Did not address regulation of activity in vivo"]},{"year":2003,"claim":"Defined MCAK's primary cellular role as error correction of kinetochore-microtubule attachments rather than driving chromosome movement, resolving what its depolymerase activity accomplishes in mitosis.","evidence":"Dominant-negative centromere-targeting domain microinjection with high-resolution imaging of attachment defects","pmids":["14699064"],"confidence":"High","gaps":["Did not establish how activity is spatially restricted to centromeres","Mechanism of attachment-error sensing unresolved"]},{"year":2004,"claim":"Identified Aurora B as the master regulator that phosphorylates MCAK at S196 to inhibit depolymerization and control its centromere targeting, linking a kinase to spatial regulation of microtubule dynamics.","evidence":"In vitro kinase assay with site mapping, phosphomimetic/null mutants, antibody injection in Xenopus extracts and cells, FRAP","pmids":["14972678","14960279","15064354"],"confidence":"High","gaps":["Structural basis of inhibition not yet defined","Did not address pole-specific regulation"]},{"year":2006,"claim":"Revealed the end-targeting strategy: MCAK uses ATP-independent 1D lattice diffusion to find microtubule ends faster than 3D diffusion permits, decoupling search from catalysis.","evidence":"Single-molecule TIRF fluorescence diffusion measurements","pmids":["16672973"],"confidence":"High","gaps":["Did not define molecular determinants of lattice vs end binding","In-cell relevance of diffusion not directly tested"]},{"year":2007,"claim":"Placed MCAK among kinesin-13 paralogs with distinct pathway positions and identified EB1/EB3 and hSgo2 as targeting determinants for plus-end and centromere localization.","evidence":"Double-depletion epistasis, Co-IP, GST domain mapping, siRNA with localization rescue","pmids":["17538014","15302853","17968321","17485487"],"confidence":"Medium","gaps":["Reciprocal validation across labs limited for some partners","Quantitative contribution of each targeting pathway not separated"]},{"year":2007,"claim":"Mapped functional domains and demonstrated that centromere-localized MCAK promotes kinetochore microtubule turnover to coordinate sister-kinetochore directional switching.","evidence":"Domain-deletion in vitro depolymerization and egg-extract assays; centromere-targeted chimera rescue with live imaging","pmids":["17093055","18039936"],"confidence":"High","gaps":["Did not resolve conformational mechanics of domain coordination","Force output per molecule not yet measured"]},{"year":2008,"claim":"Extended kinase control to spindle poles, showing Aurora A phosphorylates S196 and S719 to regulate MCAK pole activity distinctly from Aurora B centromere control.","evidence":"Xenopus egg extract spindle assembly with site-specific phosphomutants and kinase assays","pmids":["18434591"],"confidence":"High","gaps":["Crosstalk between Aurora A and Aurora B inputs not dissected","S719 activation mechanism not structurally defined"]},{"year":2011,"claim":"Quantified MCAK's force output and a 'side-sliding, end-catching' mechanism, and identified Kif18b as a partner required for astral microtubule depolymerization.","evidence":"Optical-trap single-molecule force measurement; reciprocal Co-IP, kinase assay, siRNA with microtubule phenotype","pmids":["21602793","21820309"],"confidence":"High","gaps":["Did not show how force is harnessed at kinetochores in vivo","Stoichiometry of Kif18b-MCAK complex unresolved"]},{"year":2013,"claim":"Provided the conformational mechanism of inhibition: S196 phosphorylation switches MCAK from a closed to an open state, decreasing microtubule affinity and graded depolymerase activity.","evidence":"FRET/FLIM conformational biosensor, TIRF imaging, phosphomimetic mutagenesis, in vitro depolymerization","pmids":["24291095"],"confidence":"High","gaps":["Atomic-resolution closed-state structure not yet determined here","Did not establish in-cell conformational distribution"]},{"year":2015,"claim":"Determined the structural basis of autoinhibition (C-terminus bound to two motor domains) and the catalytic role of the KVD motif, defining how tubulin binding rather than ATP hydrolysis triggers release.","evidence":"X-ray crystallography with crosslinking and binding assays; mutagenesis with ATPase/depolymerization assays and structural modeling","pmids":["25915621","26055718"],"confidence":"High","gaps":["Full-length structure in different nucleotide states not solved","Transition pathway between states inferred, not directly observed"]},{"year":2015,"claim":"Added activating phosphorylation by an Aurora B–PLK1 axis at S715, complementing inhibitory inputs and showing bidirectional kinase control of depolymerase activity.","evidence":"FRET PLK1 activity reporter, kinase assay, phosphomutant functional analysis","pmids":["26206521"],"confidence":"Medium","gaps":["Single lab without independent replication","Temporal ordering of activating vs inhibitory marks not fully resolved"]},{"year":2017,"claim":"Identified Cdk1 phosphorylation at T537 as a motor-domain switch that abolishes microtubule-end recognition without affecting lattice binding, integrating cell-cycle timing into depolymerase control.","evidence":"Phosphomimetic mutagenesis with single-molecule end/lattice residence kinetics and ATPase assay","pmids":["29230353"],"confidence":"Medium","gaps":["Endogenous phosphorylation timing not directly tracked","Single lab"]},{"year":2019,"claim":"Refined the conformational model showing compact-to-extended transitions on end binding and graded, dose-dependent Aurora B inhibition decoupling the N-terminus from the motor.","evidence":"Crosslinking mass spectrometry, electron microscopy, phosphomutant depolymerization assays","pmids":["30578316"],"confidence":"High","gaps":["High-resolution structures of intermediate states absent","Quantitative link between phosphorylation level and in-cell output not established"]},{"year":2022,"claim":"Demonstrated that Kif18b, MCAK, and EB3 function as an integrated multivalent network for potent cooperative plus-end depolymerization at low concentrations.","evidence":"In vitro reconstitution with single-molecule TIRF and quantitative depolymerization","pmids":["35502670"],"confidence":"High","gaps":["In-cell stoichiometry and regulation of the network not defined","How phosphorylation modulates the network not tested here"]},{"year":2022,"claim":"Extended KIF2C function beyond mitosis into neuronal synaptic plasticity, showing it regulates dendritic microtubule invasion, receptor trafficking, transmission and behavior.","evidence":"Conditional knockout, live microtubule imaging in neurons, electrophysiology, behavioral tests; Rab8 Co-IP in Purkinje cells","pmids":["35138249","37431690"],"confidence":"Medium","gaps":["Whether depolymerase activity per se drives trafficking not isolated","Single lab per neuronal system"]},{"year":2020,"claim":"Established a non-mitotic role in DNA double-strand break repair by promoting DSB mobility and foci dynamics in a PARP/ATM-dependent manner.","evidence":"DSB-template pulldown in Xenopus extracts, CRISPR KO/siRNA, foci analysis, NHEJ/HR reporters, live DSB tracking","pmids":["31951198"],"confidence":"Medium","gaps":["Mechanism linking microtubule depolymerase activity to nuclear DSB mobility not fully defined","Single lab"]},{"year":2025,"claim":"Revealed a phospho-binding N-terminal Tudor/PWWP/MBT fold that drives Aurora B/PLK1-dependent KIF2C condensates concentrating signaling components at microtubule ends, and substrate preference for modified tubulin exploitable for chemoresistance.","evidence":"Structural determination, optogenetic condensate platform, FLIM-FRET, Co-IP; in vitro polyglutamylated-tubulin depolymerization with inhibitor and tumor models","pmids":["40498077","40157365"],"confidence":"Medium","gaps":["Condensate findings not yet independently replicated","Physiological role of condensates vs canonical depolymerization unresolved"]},{"year":null,"claim":"How the multiple kinase inputs, conformational states, partner networks, and condensate behavior are integrated in real time to achieve spatially precise depolymerization at distinct cellular sites remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified in-cell model coupling phosphorylation state to local activity","Relative contribution of canonical depolymerization vs condensate/scaffolding functions unknown","Structures of full-length regulated states absent"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,5,21]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,16,18]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[5,16,22]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,21]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[1,2,17]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,5,7]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[38]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[15,31]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,6,17]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[32]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[34,35]}],"complexes":[],"partners":["AURKB","AURKA","PLK1","EB1","EB3","TIP150","KIF18B","SGO2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99661","full_name":"Kinesin-like protein KIF2C","aliases":["Kinesin-like protein 6","Mitotic centromere-associated kinesin","MCAK"],"length_aa":725,"mass_kda":81.3,"function":"In complex with KIF18B, constitutes the major microtubule plus-end depolymerizing activity in mitotic cells (PubMed:21820309). Regulates the turnover of microtubules at the kinetochore and functions in chromosome segregation during mitosis (PubMed:19060894). Plays a role in chromosome congression and is required for the lateral to end-on conversion of the chromosome-microtubule attachment (PubMed:23891108)","subcellular_location":"Cytoplasm, cytoskeleton; Nucleus; Chromosome, centromere; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q99661/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KIF2C","classification":"Not Classified","n_dependent_lines":320,"n_total_lines":1208,"dependency_fraction":0.26490066225165565},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000142945","cell_line_id":"CID001504","localizations":[{"compartment":"centrosome","grade":3},{"compartment":"cytoskeleton","grade":3},{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"CLASP1","stoichiometry":0.2},{"gene":"CSNK2A2","stoichiometry":0.2},{"gene":"SLC6A15","stoichiometry":0.2},{"gene":"CLIP1","stoichiometry":0.2},{"gene":"SDHA","stoichiometry":0.2},{"gene":"RCBTB2","stoichiometry":0.2},{"gene":"MAP4","stoichiometry":0.2},{"gene":"MAPRE1","stoichiometry":0.2},{"gene":"MYL12A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001504","total_profiled":1310},"omim":[{"mim_id":"619358","title":"MICROTUBULE-ASSOCIATED SCAFFOLD PROTEIN 2; MTUS2","url":"https://www.omim.org/entry/619358"},{"mim_id":"616745","title":"BIORIENTATION OF CHROMOSOMES IN CELL DIVISION 1; BOD1","url":"https://www.omim.org/entry/616745"},{"mim_id":"615142","title":"KINESIN FAMILY MEMBER 2B; KIF2B","url":"https://www.omim.org/entry/615142"},{"mim_id":"614570","title":"KINESIN FAMILY MEMBER 18B; KIF18B","url":"https://www.omim.org/entry/614570"},{"mim_id":"612425","title":"SHUGOSHIN-LIKE 2; SGOL2","url":"https://www.omim.org/entry/612425"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Midbody","reliability":"Approved"},{"location":"Centrosome","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":31.1},{"tissue":"lymphoid tissue","ntpm":34.8},{"tissue":"testis","ntpm":72.6}],"url":"https://www.proteinatlas.org/search/KIF2C"},"hgnc":{"alias_symbol":["MCAK","CT139"],"prev_symbol":["KNSL6"]},"alphafold":{"accession":"Q99661","domains":[{"cath_id":"3.40.850.10","chopping":"320-564","consensus_level":"high","plddt":87.3949,"start":320,"end":564},{"cath_id":"2.30.30","chopping":"14-69","consensus_level":"high","plddt":85.7127,"start":14,"end":69},{"cath_id":"1.10.287","chopping":"638-721","consensus_level":"high","plddt":87.8955,"start":638,"end":721}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99661","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99661-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99661-F1-predicted_aligned_error_v6.png","plddt_mean":74.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KIF2C","jax_strain_url":"https://www.jax.org/strain/search?query=KIF2C"},"sequence":{"accession":"Q99661","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99661.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99661/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99661"}},"corpus_meta":[{"pmid":"14960279","id":"PMC_14960279","title":"Aurora 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~100-fold, the rate of tubulin dissociation from microtubule ends. It has one high-affinity binding site per protofilament end, and up to 14 MCAK dimers can assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule, removing ~20 tubulin dimers at a rate of 1 s⁻¹.\",\n      \"method\": \"In vitro ATPase assay, microtubule depolymerization kinetics, single-molecule analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous in vitro reconstitution with quantitative kinetic analysis, foundational mechanistic study\",\n      \"pmids\": [\"12620232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Aurora B phosphorylates MCAK at serine 196 (neck region) and additional sites in the centromere-targeting domain, inhibiting its microtubule depolymerization activity in vitro and in vivo. Aurora B activity is required to localize MCAK to centromeres but not spindle poles. Phospho-S196 MCAK was detected at centromeres and anaphase spindle midzones in vivo. Injection of anti-pS196 antibodies into Xenopus egg extracts or cells caused chromosome positioning/segregation defects.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry phosphorylation site mapping, antibody injection into Xenopus egg extracts and live cells, immunofluorescence\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with site mapping, functional rescue experiments, replicated across multiple labs (PMID 14960279, 14972678)\",\n      \"pmids\": [\"14972678\", \"14960279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Aurora B inhibits MCAK microtubule depolymerizing activity in vitro. Phosphomimetic MCAK (S/E) mutants inhibit depolymerization in vivo and localize preferentially to the inner centromere, while phospho-null (S/A) mutants concentrate at kinetochores. Both mutants increase syntelic attachments and mono-oriented chromosomes. FRAP analysis identifies two distinct MCAK binding sites at centromeres. Aurora B kinase-dead mutant or RNAi prevents centromeric targeting of MCAK.\",\n      \"method\": \"In vitro depolymerization assay, phosphomimetic/phosphonull mutagenesis, live-cell fluorescence imaging, FRAP, RNAi\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods, mutagenesis plus functional imaging, replicated in parallel with PMID 14972678\",\n      \"pmids\": [\"14960279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In Xenopus egg extracts, recombinant Aurora B–INCENP inhibits MCAK microtubule depolymerase activity in a phosphorylation-dependent manner. An Aurora B-resistant alanine mutant (XMCAK-4A) produces mono-astral and monopolar structures instead of bipolar spindles when substituted for endogenous MCAK, and its ability to localize to inner centromeres is abolished. This demonstrates that Aurora B-dependent phosphorylation differentiates cytoplasmic from spindle-associated MCAK activity.\",\n      \"method\": \"In vitro kinase assay, Xenopus egg extract spindle assembly, phospho-resistant mutagenesis, immunofluorescence localization\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution in egg extracts with phospho-resistant mutant, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"15064354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Depletion of centromeric MCAK by injection of a dominant-negative centromere-targeting domain into prophase cells leads to reduced centromere stretch, delayed chromosome congression, alignment defects, severe chromosome missegregation, and multiple kinetochore-microtubule attachment defects (merotelic, syntelic, combined). Rates of chromosome movement are unchanged, indicating MCAK's primary role is not chromosome translocation but error correction of kinetochore-microtubule attachments.\",\n      \"method\": \"Dominant-negative protein microinjection, high-resolution immunofluorescence, live-cell imaging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative perturbation with defined kinetochore attachment phenotypes, multiple attachment error classes documented\",\n      \"pmids\": [\"14699064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MCAK moves along the microtubule lattice via one-dimensional (1D) random walk (diffusion coefficient 0.38 μm² s⁻¹, average duration 0.83 s). This lattice diffusion is ATP-independent, whereas catalytic depolymerization requires ATP hydrolysis. The 1D diffusion enables MCAK to target microtubule ends at rates exceeding 3D diffusion limits ('reduction in dimensionality' search strategy).\",\n      \"method\": \"Single-molecule fluorescence microscopy, TIRF-based in vitro assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous single-molecule in vitro reconstitution with quantitative diffusion measurements, published in Nature\",\n      \"pmids\": [\"16672973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Among the three human kinesin-13 paralogs, MCAK (Kif2c) specifically functions at kinetochores to regulate microtubule dynamics for chromosome alignment; its loss creates a permissive background that restores bipolar spindle assembly in Kif2a-depleted or Kif2b-depleted cells, demonstrating distinct pathway positions. MCAK and Kif2a activities must be balanced at kinetochores vs. poles for spindle bipolarity.\",\n      \"method\": \"siRNA knockdown, epistasis by double depletion, live-cell imaging, spindle assembly assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis via double-depletion, replicated across multiple kinesin-13 paralogs, two independent studies (PMID 17538014, 15302853)\",\n      \"pmids\": [\"17538014\", \"15302853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MCAK tracks (treadmills) with the tips of polymerizing microtubules in living cells. This tip-tracking behavior requires the extreme C-terminal tail of MCAK and is inhibited by phosphorylation. Tip tracking is not essential for MCAK's microtubule-depolymerizing activity, suggesting it serves as a localization mechanism to regions of active plus-end regulation.\",\n      \"method\": \"Live-cell fluorescence imaging, C-terminal deletion mutants, phospho-mutant analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with domain deletion and phospho-mutants, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"15883193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Aurora B is specifically enriched at merotelic attachment sites. Aurora B activity (but not its localization) is required to enrich MCAK at merotelic attachments and phosphorylates MCAK on residues that regulate its microtubule depolymerase activity at these sites.\",\n      \"method\": \"Immunofluorescence, Aurora B inhibition, MCAK localization analysis at merotelic sites\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific localization with kinase inhibition, single lab\",\n      \"pmids\": [\"16950107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"hSgo2 is required for MCAK to localize to the centromere. Delocalization of MCAK in hSgo2-depleted cells accounts for uncorrected kinetochore attachment defects. hSgo2 is associated with PP2A and contributes to spatial regulation of MCAK activity within the inner centromere/kinetochore region.\",\n      \"method\": \"siRNA depletion, immunofluorescence colocalization, Co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA depletion with localization rescue and Co-IP, single lab\",\n      \"pmids\": [\"17485487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Aurora B phosphorylates hSgo2 at its N-terminal coiled-coil and middle regions; these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, respectively. Aurora B-phosphorylated hSgo2 is essential for recruiting both PP2A and MCAK to centromeres.\",\n      \"method\": \"In vitro kinase assay, phospho-mutant analysis, Co-immunoprecipitation, immunofluorescence\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay plus phosphomutant Co-IP and localization, single lab with multiple methods\",\n      \"pmids\": [\"20889715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TIP150 is a plus-end tracking protein (+TIP) that binds EB1 and co-localizes with it at microtubule plus ends. TIP150 also directly binds MCAK; suppression of TIP150 diminishes MCAK plus-end localization. Aurora B-mediated phosphorylation disrupts the TIP150–MCAK interaction in vitro, linking Aurora B regulation to MCAK plus-end targeting.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, siRNA knockdown, immunofluorescence, in vitro phosphorylation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus in vitro kinase disruption assay and siRNA with localization readout, single lab\",\n      \"pmids\": [\"19543227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MCAK associates with the C-terminus of EB1 and EB3 through an interaction involving the N-terminal localization and regulatory domain of MCAK (not the motor domain). EB1 knockdown impairs GFP-MCAK tip localization in cells. The EB1–MCAK interaction is competitive with other EB1 ligands and does not require microtubules.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown domain mapping, siRNA knockdown, live-cell imaging\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal binding assays with domain mapping plus siRNA rescue, single lab\",\n      \"pmids\": [\"17968321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In vitro reconstitution shows that EB3 targets MCAK to growing microtubule ends by increasing MCAK's association rate with microtubule tips via direct EB3–MCAK interaction. While MCAK alone blocks microtubule assembly, addition of EB3 restores robust growth. The MCAK–EB3 combination enhances catastrophe frequency, promoting rapid switching between growth and shortening, without affecting growth or shortening velocity.\",\n      \"method\": \"In vitro reconstitution of dynamic microtubule assay, TIRF microscopy, domain mutants, quantitative kinetics\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — rigorous in vitro reconstitution with dynamic microtubules, quantitative kinetics, single lab with multiple orthogonal experiments\",\n      \"pmids\": [\"20850319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Kif18b binds directly to MCAK, and this interaction is required for robust microtubule depolymerization of astral microtubules. Aurora kinases negatively regulate the Kif18b–MCAK interaction through phosphorylation of MCAK, demonstrating that Aurora kinases regulate spindle microtubule plus-end stability through control of Kif18b–MCAK complex formation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, in vitro kinase assay, live-cell imaging, microtubule dynamics assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, kinase assay, and functional siRNA with microtubule phenotype, single lab with multiple methods\",\n      \"pmids\": [\"21820309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Aurora A phosphorylates MCAK at S196 (shared with Aurora B) to regulate MCAK localization and activity at aster centers during Ran-induced spindle assembly, and at S719 to positively enhance bipolar spindle formation. This defines a distinct role for MCAK at spindle poles regulated by Aurora A, separate from Aurora B regulation at centromeres.\",\n      \"method\": \"Xenopus egg extract spindle assembly, in vitro kinase assay, phosphosite mutagenesis, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with phosphosite-specific mutants and egg extract functional assay, single lab\",\n      \"pmids\": [\"18434591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal domain of MCAK is essential for regulating microtubule dynamics and kinetochore targeting; the C-terminal domain is essential for tight microtubule lattice binding and robust in vitro depolymerization activity. The neck is essential for microtubule end binding. Both C-terminal domain and neck are required for robust in vitro depolymerization activity.\",\n      \"method\": \"Purified GFP-domain deletion mutants, in vitro depolymerization assay, Xenopus egg extract spindle assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic domain deletion analysis with in vitro reconstitution and in vivo spindle assays, single lab\",\n      \"pmids\": [\"17093055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Depletion of centromere-associated MCAK using chimeric constructs decreases directional coordination between sister kinetochores, reduces movement speed, and increases tension. Sister centromeres cannot detach efficiently from kinetochore microtubules during directional switching. Anchoring ectopic MCAK to the centromere reverses these effects. This demonstrates that centromere-MCAK promotes kinetochore microtubule turnover to coordinate directional switching.\",\n      \"method\": \"MCAK chimera/dominant-negative constructs, live-cell imaging, sister centromere distance and dynamics measurements\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — targeted chimera approach with centromere-specific rescue, live imaging with quantitative kinetochore dynamics, single lab\",\n      \"pmids\": [\"18039936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MCAK depolymerizes microtubules from both plus and minus ends and generates significant tension (~1 pN per motor). An MCAK-decorated bead slides along microtubule sides under weak loads and captures/disassembles both microtubule ends. This 'side-sliding, end-catching' mechanism allows MCAK to generate driving force for chromosome movement at both kinetochores and spindle poles.\",\n      \"method\": \"Optical trap, bead-MCAK decoration assay, single-molecule microtubule depolymerization force measurements\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — optical trap force measurement with quantitative single-molecule analysis, novel mechanistic finding\",\n      \"pmids\": [\"21602793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Aurora B phosphorylation at S196 in the neck region opens MCAK from a closed (inhibited) conformation (detected by FRET/FLIM biosensor) to an open conformation, diminishing the interaction between the C-terminal domain and neck. This conformational opening decreases MCAK affinity for the microtubule, reducing its depolymerization activity. MCAK bound to microtubule ends is in a closed conformation relative to lattice-bound MCAK.\",\n      \"method\": \"FRET biosensor, FLIM, TIRF imaging, phosphomimetic mutagenesis, in vitro depolymerization assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — FRET/FLIM conformational biosensor combined with mutagenesis and depolymerization assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"24291095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The C-terminus of MCAK binds to two motor domains in solution (crystal structure determined), acting as an autoinhibitory interaction that is displaced allosterically upon microtubule binding. This allows robust MCAK accumulation at microtubule ends. The C-terminus–motor interaction represents a structural intermediate in the MCAK catalytic cycle involving long-range conformational changes.\",\n      \"method\": \"X-ray crystallography (structure of motor domain bound to C-terminus), crosslinking, biochemical binding assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional biochemical validation, single lab with orthogonal methods\",\n      \"pmids\": [\"25915621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The KVD motif at the tip of kinesin-13-specific loop 2 of Kif2C is required for microtubule-stimulated ATPase activity and depolymerization. Upon microtubule binding, Kif2C undergoes a conformational change governed in part by KVD interaction with the tubulin interdimer interface. A switch-2 glutamate mutant (E to A) that blocks ATP hydrolysis in motile kinesins still depolymerizes microtubules and yields Kif2C–two-tubulin complexes, demonstrating that the conformational change upon tubulin binding is sufficient for tubulin release and ATP hydrolysis is not required for this step.\",\n      \"method\": \"In vitro ATPase assay, microtubule depolymerization assay, mutagenesis, structural modeling, biochemical complex analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with in vitro enzymatic assays and structural model, multiple orthogonal methods\",\n      \"pmids\": [\"26055718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The α4-helix residues K524, E525, and R528 of the MCAK motor domain are critical for microtubule end recognition. Mutations at these conserved kinesin-13-specific positions specifically disrupt the ability of MCAK to recognize microtubule ends (reducing end residence time and end-specific ADP dissociation stimulation) without affecting lattice binding, impairing depolymerization.\",\n      \"method\": \"Mutagenesis, in vitro microtubule end-binding assay, single-molecule fluorescence imaging, ATPase assay\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — targeted mutagenesis with single-molecule end-binding kinetics and nucleotide assays, single lab\",\n      \"pmids\": [\"27733589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MCAK has a compact conformation in solution (confirmed by crosslinking and electron microscopy). When bound to microtubule ends, MCAK adopts an extended conformation in which the N-terminus and neck interact with the microtubule. The level of Aurora B phosphorylation of the N-terminus results in a graded (not binary) inhibition of MCAK depolymerase activity through allosteric regulation decoupling the N-terminus from the motor domain.\",\n      \"method\": \"Chemical crosslinking mass spectrometry, electron microscopy, in vitro kinase assay, phosphomimetic mutants, microtubule depolymerization assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crosslinking-EM structural analysis plus graded phosphomutant functional assays, single lab with multiple methods\",\n      \"pmids\": [\"30578316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GTSE1 inhibits MCAK microtubule depolymerase activity. Cells lacking GTSE1 show defects in chromosome alignment and spindle positioning due to MT instability caused by excess MCAK activity. Artificially elevated GTSE1 hyperstabilizes kinetochore microtubules and increases chromosome missegregation/CIN.\",\n      \"method\": \"siRNA depletion, overexpression, co-immunoprecipitation, in vitro depolymerization assay, live-cell imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vitro depolymerization inhibition and siRNA/OE with chromosomal phenotype, single lab\",\n      \"pmids\": [\"27881713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NuSAP is a novel binding partner of MCAK and modulates MCAK depolymerization activity. Aurora B kinase phosphorylation of MCAK significantly enhances the NuSAP–MCAK interaction, and NuSAP modulates MCAK depolymerization in an Aurora B-dependent manner to regulate kinetochore microtubule dynamics.\",\n      \"method\": \"Co-immunoprecipitation, in vitro depolymerization assay, siRNA knockdown, phospho-mutant analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with in vitro activity modulation and phospho-enhancement, single lab\",\n      \"pmids\": [\"26733216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Aurora B–PLK1 signaling axis regulates MCAK in mitosis: Aurora B phosphorylates PLK1 at Thr210 to activate PLK1 at kinetochores; active PLK1 in turn phosphorylates MCAK at Ser715 to promote its microtubule depolymerase activity. Non-phosphorylatable MCAK S715A prevents correct kinetochore–microtubule attachment, resulting in chromosome bridges in anaphase.\",\n      \"method\": \"FRET-based PLK1 activity reporter, phosphorylation site mutagenesis, in vitro kinase assay, immunofluorescence, live-cell imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET reporter plus kinase assay and phosphomutant functional analysis, single lab\",\n      \"pmids\": [\"26206521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A Rac1–Aurora A–MCAK signaling pathway mediates endothelial cell polarization and directional migration. Aurora A kinase activity (regionally enhanced by Rac1 signaling) locally inhibits MCAK microtubule depolymerizing activity at the trailing edge of polarized wound-edge endothelial cells, promoting regional differences in MT dynamics.\",\n      \"method\": \"siRNA knockdown, dominant-negative/constitutively active Rac1, inhibitor treatment, high-resolution fluorescence microscopy with MT plus-end tracking, quantitative image analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological pathway dissection with live MT imaging, single lab\",\n      \"pmids\": [\"25002679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MCAK tip-tracking (EB-dependent binding to growing microtubule ends via the SKIP motif N-terminal to the neck) negatively regulates microtubule length within the assembling bipolar spindle. This function requires MCAK's ability to bind EB proteins. Abolishing tip tracking (EB-binding mutant) leads to over-long non-kinetochore microtubules, antagonizes centrosome separation, but ultimately impairs robust kinetochore attachment.\",\n      \"method\": \"EB-binding SKIP motif mutants, siRNA rescue, live-cell imaging, spindle microtubule length measurements\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutant rescue in mitosis with live imaging readout, single lab\",\n      \"pmids\": [\"22492725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Lateral-to-end-on conversion of chromosome-microtubule attachment requires MCAK to release laterally attached microtubules after partial end-on attachment is formed, acting sequentially after CENP-E-mediated lateral tethering.\",\n      \"method\": \"High-resolution live imaging assay, siRNA knockdown of MCAK and CENP-E, classification of attachment intermediates\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with high-resolution imaging of attachment intermediates, single lab\",\n      \"pmids\": [\"23891108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TIP150–MCAK interaction governs entosis via Aurora A-mediated phosphorylation of MCAK. MCAK forms an intramolecular association required for TIP150 binding; Aurora A phosphorylation of MCAK modulates this intramolecular association, disrupting the MCAK–TIP150 interaction in vitro and inhibiting entosis in vivo. MCAK cooperates with TIP150 to modulate cell mechanical rigidity during entosis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase/binding assay, optical trap (cell rigidity), live-cell imaging, siRNA knockdown\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with kinase disruption assay and optical trap, single lab with multiple methods\",\n      \"pmids\": [\"24847103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The far C-terminal residues E715/E716 of MCAK regulate MCAK conformation and spindle pole focusing. Point mutation E715A/E716A increases MCAK targeting to poles and reduces MT lifetimes, inducing unfocused spindle poles—a phenotype phenocopied by Aurora A phosphomimetic S719E. The kinesin-14 XCTK2 rescues unfocused-pole phenotype, placing MCAK and XCTK2 in opposing activities at poles.\",\n      \"method\": \"Xenopus egg extract spindle assembly, point mutagenesis, immunofluorescence, FRET conformation assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with egg extract spindle assay and FRET conformation readout, single lab\",\n      \"pmids\": [\"26941326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KIF2C (MCAK) is recruited to DNA double-strand break (DSB) sites in a PARP- and ATM-dependent manner. KIF2C knockdown/knockout leads to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. KIF2C depletion or inhibition of its microtubule depolymerase activity reduces DSB mobility, impairs DNA damage foci formation, and decreases foci fusion/resolution.\",\n      \"method\": \"DSB-mimicking DNA template pulldown in Xenopus extracts, CRISPR knockout, siRNA knockdown, γH2AX foci analysis, live-cell DSB tracking, NHEJ/HR reporter assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple loss-of-function approaches in two systems with defined DSB repair readouts, single lab\",\n      \"pmids\": [\"31951198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KIF2C interacts with TBC1D7, and this interaction disrupts formation of the TSC complex, resulting in enhanced mTORC1 signal transduction. KIF2C is a direct transcriptional target of the Wnt/β-catenin pathway and mediates crosstalk between Wnt/β-catenin and mTORC1 signaling in hepatocellular carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, gain/loss-of-function assays, luciferase reporter assay, Western blot, in vivo xenograft\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with defined pathway disruption assay and transcriptional reporter, single lab\",\n      \"pmids\": [\"32748349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KIF2C regulates microtubule dynamics in neuronal dendrites and activity-dependent microtubule invasion into dendritic spines. KIF2C knockdown or conditional knockout impairs spine morphology, synaptic AMPA receptor expression, excitatory transmission, long-term potentiation, and cognitive behavior in mice.\",\n      \"method\": \"RNAi knockdown, conditional knockout (Cre-lox), live microtubule imaging in neurons, electrophysiology, behavioral tests\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with multiple synaptic and behavioral readouts, single lab\",\n      \"pmids\": [\"35138249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KIF2C regulates transport of mGlu1 receptors in cerebellar Purkinje cells by binding to Rab8. KIF2C deficiency in Purkinje cells reduces mGlu1 and GluA2 synaptic expression, alters excitatory but not inhibitory transmission, and causes motor incoordination in male mice.\",\n      \"method\": \"Conditional knockout (Purkinje cell-specific), Co-immunoprecipitation with Rab8, immunofluorescence, electrophysiology, behavioral motor tests\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — cell-type-specific KO with Co-IP partner identification and functional synaptic/behavioral readouts, single lab\",\n      \"pmids\": [\"37431690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Both overexpression and downregulation of MCAK/KIF2C reduce cell motility and migration. Specifically, altered MCAK levels impair focal adhesion protein composition and phosphorylation, assembly/disassembly rate of focal adhesions, cell adhesion, and plus-tip microtubule dynamics. This demonstrates MCAK acts as a regulator of cell motility through actin-MT cytoskeleton dynamics and focal adhesion turnover.\",\n      \"method\": \"CRISPR/dCas9 overexpression and knockdown cell lines, live-cell imaging, focal adhesion lifetime assays, MT plus-tip tracking, Western blot\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR perturbation with FA dynamics and MT tracking, single lab with multiple readouts\",\n      \"pmids\": [\"34830827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MCAK's spatial distribution of activity (higher at trailing edge than leading edge) is required for cell polarity, centrosome positioning, focal adhesion disassembly, and directional migration. Rac1 overexpression has a dominant effect over MCAK activity, placing Rac1 downstream of or parallel to MCAK in the migration pathway.\",\n      \"method\": \"siRNA knockdown, Rac1 overexpression epistasis, live-cell imaging, FA lifetime measurements, MT dynamics analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis by Rac1/MCAK double manipulation with quantitative live imaging, single lab\",\n      \"pmids\": [\"33566676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MCAK contains multiple nuclear localization sequences (NLS) and a nuclear exclusion sequence (NES) in the amino-terminal region that balance its nucleocytoplasmic distribution. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, in turn influencing the degree of centromere binding.\",\n      \"method\": \"GFP-MCAK deletion constructs, ATP-binding domain mutagenesis, fluorescence microscopy localization\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — domain deletions and motor mutants with localization readout, single lab\",\n      \"pmids\": [\"10600236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MCAK accumulates during the cell cycle, reaches maximum at G2/M, and is rapidly degraded by the proteasome during mitosis. A phosphorylated form of MCAK appears during mitosis and is preferentially degraded. This degradation limits MCAK activity during late mitosis, arguing against a role in anaphase chromosome movement.\",\n      \"method\": \"Cell cycle fractionation, immunofluorescence, proteasome inhibitor treatment, Western blot\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — cell-cycle staging with proteasome inhibitor and Western blot quantification, single lab\",\n      \"pmids\": [\"18843200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A phosphorylation site on MCAK controls its proteasomal degradation; phosphorylation-resistant mutation prolongs MCAK stability beyond the metaphase-to-anaphase transition and prevents MCAK removal from centromeres, causing MCAK retention throughout the cell cycle and mitotic defects. A phosphomimetic mutation accelerates degradation.\",\n      \"method\": \"Phosphosite mutagenesis, proteomic phosphorylation site identification, immunofluorescence, cell division assays\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — phospho-mutant analysis with localization and division phenotype, single lab\",\n      \"pmids\": [\"22422706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cdk1 phosphorylates MCAK at T537 within the motor domain. A phosphomimetic T537E mutant significantly impairs microtubule depolymerization by reducing the ability of MCAK to recognize microtubule ends specifically: microtubule-end residence time is reduced, lattice residence time is unaffected, and end-specific ADP dissociation stimulation is abolished.\",\n      \"method\": \"Phosphomimetic mutagenesis, single-molecule TIRF imaging (end/lattice residence times), ATPase assay, cell transfection phenotype\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — single-molecule kinetics with mechanistic mutagenesis, single lab\",\n      \"pmids\": [\"29230353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In vitro reconstitution shows that Kif18b, MCAK, and EB3 act as an integrated network to potently promote microtubule depolymerization at very low concentrations. Kif18b transports EB3 and MCAK to microtubule plus ends through multivalent weak interactions, enabling cooperative plus-end shortening.\",\n      \"method\": \"In vitro reconstitution, single-molecule TIRF imaging, quantitative microtubule depolymerization assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — rigorous in vitro reconstitution with single-molecule imaging defining cooperative mechanism, single lab\",\n      \"pmids\": [\"35502670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"DDA3 localizes to kinetochores and interacts with MCAK. Depletion of DDA3 causes chromosome congression defects associated with loss of MCAK function at kinetochores, and results in CENP-E accumulation at unaligned kinetochores, without affecting Aurora B/CPC activity.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with siRNA localization phenotype, single lab\",\n      \"pmids\": [\"21426902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CaMKIIgamma depletion leads to disorganized multipolar spindles by failing to suppress MCAK depolymerase activity. Two distinct but overlapping mechanisms for negative regulation of the cytosolic/centrosomal pool of MCAK exist: one involving CaMKIIgamma and another involving TOGp. Both are essential for spindle bipolarity; their effects are abolished in MCAK-depleted cells, placing both upstream of MCAK in spindle assembly.\",\n      \"method\": \"siRNA RNAi depletion, inducible overexpression, epistasis by double depletion, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via double depletion showing MCAK dependence, single lab\",\n      \"pmids\": [\"15775983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"α-Tubulin detyrosination suppresses MCAK activity; experimental increase of detyrosinated α-tubulin and MCAK depletion produce non-cumulative enhancement of taxol cytotoxicity and cell death in mitosis/interphase, identifying a mechanistic link between α-tubulin detyrosination and MCAK activity suppression.\",\n      \"method\": \"siRNA knockdown, detyrosination manipulation, flow cytometry cell death assay, clonogenic assay, combinatorial treatment epistasis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via non-cumulative drug/KD effects establishing functional link, single lab\",\n      \"pmids\": [\"36459065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KIF2C preferentially depolymerizes polyglutamylated tubulin even in the presence of paclitaxel. A chemical inhibitor 7S9 prohibits dissociation of KIF2C from microtubules, counteracting KIF2C-mediated paclitaxel resistance. Combination of 7S9 and paclitaxel significantly reduces tumorigenesis in chemoresistant TNBC mouse models.\",\n      \"method\": \"In vitro depolymerization assay with polyglutamylated tubulin, KIF2C inhibitor development, in vivo mouse tumor model\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro substrate-specificity assay plus in vivo validation with inhibitor, single lab\",\n      \"pmids\": [\"40157365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"KIF2C directly binds PKM2 (confirmed by Co-IP), and this interaction prevents PKM2 ubiquitination, increasing PKM2 stability. Domain 2 of KIF2C mediates the PKM2 binding. Elevated KIF2C promotes autophagy and glycolysis in doxorubicin-resistant breast cancer cells via PKM2 stabilization.\",\n      \"method\": \"Co-immunoprecipitation, Western blot (ubiquitination assay), domain deletion mapping, functional metabolic assays\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with indirect ubiquitination readout, single lab in cancer context\",\n      \"pmids\": [\"34663310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The N-terminal domain of KIF2C adopts a Tudor/PWWP/MBT fold that binds phosphorylated motifs, including phosphorylated BRCA2-pT207. KIF2C forms membrane-less organelles (condensates) in an Aurora B- and PLK1-dependent manner via this phospho-binding domain. KIF2C condensation concentrates PLK1 and BRCA2-pT207 at microtubule ends/extremities; condensates exclude tubulin. KIF2C depolymerase activity promotes condensate formation. Aurora B is required for condensate formation.\",\n      \"method\": \"Structural determination (NTD fold), Co-immunoprecipitation, optogenetic condensate platform, FLIM-FRET, immunofluorescence, mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — structural determination plus optogenetic condensate assay and Co-IP, single lab with multiple orthogonal methods, not yet replicated\",\n      \"pmids\": [\"40498077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nek2A interacts with KIF2C (identified by TurboID proximity labeling and confirmed by Co-immunoprecipitation and colocalization). KIF2C silencing diminishes Nek2A's ability to prevent centrosome clustering, placing KIF2C downstream of Nek2A in the centrosome clustering/anti-clustering pathway.\",\n      \"method\": \"TurboID proximity labeling, Co-immunoprecipitation, siRNA knockdown epistasis, immunofluorescence colocalization\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — proximity labeling Co-IP with siRNA epistasis, single lab\",\n      \"pmids\": [\"38493150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CCDC69 microtubule depolymerization activity is dependent on KIF2C, as demonstrated by the finding that a fraction of CCDC69 localizes to centromeres and KIF2C mediates CCDC69's depolymerization effects. CCDC69 also regulates stability of the chromosomal passenger complex (CPC) by protecting its members from degradation.\",\n      \"method\": \"Overexpression, siRNA epistasis, immunofluorescence colocalization, microtubule depolymerization assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — epistasis by siRNA with indirect depolymerization readout, single lab\",\n      \"pmids\": [\"39638803\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KIF2C/MCAK is a kinesin-13 microtubule depolymerase that uses ATP hydrolysis to catalytically remove tubulin dimers from microtubule ends after targeting them via ATP-independent 1D lattice diffusion; its depolymerization activity, localization, and conformation are precisely regulated by phosphorylation from Aurora B (inhibitory at S196 via a closed-to-open conformational switch), Aurora A (at S196 and S719, regulating pole activity), PLK1 (activating at S715), and Cdk1 (inhibitory at T537 via loss of end recognition), with centromere targeting additionally controlled by hSgo2 and the CPC; its plus-end accumulation depends on direct interaction with EB1/EB3 and TIP150; it forms cooperative depolymerizing networks with Kif18b and EB3; and beyond its canonical mitotic roles in error correction of kinetochore–microtubule attachments, it functions in interphase cell migration (via focal adhesion and MT dynamics), neuronal synaptic plasticity (by regulating dendritic microtubule invasion and receptor trafficking via Rab8), DNA double-strand break repair (by promoting DSB mobility in a PARP/ATM-dependent manner), and forms Aurora B/PLK1-dependent condensates that concentrate signaling components at microtubule ends during mitosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KIF2C (MCAK) is a kinesin-13 microtubule depolymerase that catalytically removes tubulin dimers from microtubule ends to control microtubule dynamics during mitosis and beyond [#0]. It accelerates tubulin dissociation ~100-fold by assembling at protofilament ends as an ATP-hydrolyzing complex [#0], and targets ends efficiently by first undergoing ATP-independent one-dimensional lattice diffusion before catalysis [#5]. Its enzymatic cycle is built from defined elements: the kinesin-13-specific KVD motif of loop 2 senses the tubulin interdimer interface so that the conformational change on tubulin binding\\u2014not ATP hydrolysis\\u2014drives tubulin release [#21], while \\u03b14-helix residues and the C-terminal domain/neck confer microtubule-end recognition and robust depolymerization [#22, #16]. MCAK exists in an autoinhibited closed/compact conformation in which the C-terminus engages the motor domains, an interaction allosterically displaced upon microtubule binding to extend the molecule at ends [#20, #23]. In cells, its principal mitotic function is error correction of kinetochore-microtubule attachments rather than chromosome translocation: centromere-localized MCAK promotes microtubule turnover to coordinate directional switching and to resolve syntelic, merotelic, and lateral attachments [#4, #17, #29]. This activity is gated by a phosphorylation network\\u2014Aurora B phosphorylates S196 to open the molecule and inhibit depolymerization and to drive its centromeric targeting [#1, #2, #19], Aurora A regulates pole activity via S196/S719 [#15], PLK1 phosphorylates S715 to activate it [#26], and Cdk1 phosphorylates T537 to abolish end recognition [#41]\\u2014and by binding partners that target or modulate it, including EB1/EB3 and TIP150 for plus-end accumulation [#12, #11], hSgo2 for centromere recruitment [#9, #10], and Kif18b/EB3 for cooperative plus-end depolymerizing networks [#14, #42]. Its conformation and activity are further constrained by inhibitors GTSE1 and tubulin detyrosination [#24, #45] and by proteasomal degradation that limits late-mitotic activity [#39]. Beyond mitosis, KIF2C functions in directional cell migration through focal-adhesion turnover and microtubule dynamics [#36, #37], in DNA double-strand break repair by promoting PARP/ATM-dependent DSB mobility [#32], in neuronal synaptic plasticity by regulating dendritic microtubule invasion and Rab8-dependent receptor trafficking [#34, #35], and forms Aurora B/PLK1-dependent phospho-binding condensates that concentrate signaling factors at microtubule ends [#48].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the core enzymatic identity of MCAK by showing it is not a motile motor but a catalytic depolymerase that actively destabilizes microtubule ends.\",\n      \"evidence\": \"In vitro ATPase and depolymerization kinetics with single-molecule analysis\",\n      \"pmids\": [\"12620232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how MCAK reaches ends in cells\", \"Did not address regulation of activity in vivo\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined MCAK's primary cellular role as error correction of kinetochore-microtubule attachments rather than driving chromosome movement, resolving what its depolymerase activity accomplishes in mitosis.\",\n      \"evidence\": \"Dominant-negative centromere-targeting domain microinjection with high-resolution imaging of attachment defects\",\n      \"pmids\": [\"14699064\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how activity is spatially restricted to centromeres\", \"Mechanism of attachment-error sensing unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified Aurora B as the master regulator that phosphorylates MCAK at S196 to inhibit depolymerization and control its centromere targeting, linking a kinase to spatial regulation of microtubule dynamics.\",\n      \"evidence\": \"In vitro kinase assay with site mapping, phosphomimetic/null mutants, antibody injection in Xenopus extracts and cells, FRAP\",\n      \"pmids\": [\"14972678\", \"14960279\", \"15064354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of inhibition not yet defined\", \"Did not address pole-specific regulation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed the end-targeting strategy: MCAK uses ATP-independent 1D lattice diffusion to find microtubule ends faster than 3D diffusion permits, decoupling search from catalysis.\",\n      \"evidence\": \"Single-molecule TIRF fluorescence diffusion measurements\",\n      \"pmids\": [\"16672973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define molecular determinants of lattice vs end binding\", \"In-cell relevance of diffusion not directly tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed MCAK among kinesin-13 paralogs with distinct pathway positions and identified EB1/EB3 and hSgo2 as targeting determinants for plus-end and centromere localization.\",\n      \"evidence\": \"Double-depletion epistasis, Co-IP, GST domain mapping, siRNA with localization rescue\",\n      \"pmids\": [\"17538014\", \"15302853\", \"17968321\", \"17485487\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal validation across labs limited for some partners\", \"Quantitative contribution of each targeting pathway not separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped functional domains and demonstrated that centromere-localized MCAK promotes kinetochore microtubule turnover to coordinate sister-kinetochore directional switching.\",\n      \"evidence\": \"Domain-deletion in vitro depolymerization and egg-extract assays; centromere-targeted chimera rescue with live imaging\",\n      \"pmids\": [\"17093055\", \"18039936\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve conformational mechanics of domain coordination\", \"Force output per molecule not yet measured\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Extended kinase control to spindle poles, showing Aurora A phosphorylates S196 and S719 to regulate MCAK pole activity distinctly from Aurora B centromere control.\",\n      \"evidence\": \"Xenopus egg extract spindle assembly with site-specific phosphomutants and kinase assays\",\n      \"pmids\": [\"18434591\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk between Aurora A and Aurora B inputs not dissected\", \"S719 activation mechanism not structurally defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Quantified MCAK's force output and a 'side-sliding, end-catching' mechanism, and identified Kif18b as a partner required for astral microtubule depolymerization.\",\n      \"evidence\": \"Optical-trap single-molecule force measurement; reciprocal Co-IP, kinase assay, siRNA with microtubule phenotype\",\n      \"pmids\": [\"21602793\", \"21820309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show how force is harnessed at kinetochores in vivo\", \"Stoichiometry of Kif18b-MCAK complex unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided the conformational mechanism of inhibition: S196 phosphorylation switches MCAK from a closed to an open state, decreasing microtubule affinity and graded depolymerase activity.\",\n      \"evidence\": \"FRET/FLIM conformational biosensor, TIRF imaging, phosphomimetic mutagenesis, in vitro depolymerization\",\n      \"pmids\": [\"24291095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution closed-state structure not yet determined here\", \"Did not establish in-cell conformational distribution\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Determined the structural basis of autoinhibition (C-terminus bound to two motor domains) and the catalytic role of the KVD motif, defining how tubulin binding rather than ATP hydrolysis triggers release.\",\n      \"evidence\": \"X-ray crystallography with crosslinking and binding assays; mutagenesis with ATPase/depolymerization assays and structural modeling\",\n      \"pmids\": [\"25915621\", \"26055718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length structure in different nucleotide states not solved\", \"Transition pathway between states inferred, not directly observed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Added activating phosphorylation by an Aurora B\\u2013PLK1 axis at S715, complementing inhibitory inputs and showing bidirectional kinase control of depolymerase activity.\",\n      \"evidence\": \"FRET PLK1 activity reporter, kinase assay, phosphomutant functional analysis\",\n      \"pmids\": [\"26206521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab without independent replication\", \"Temporal ordering of activating vs inhibitory marks not fully resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified Cdk1 phosphorylation at T537 as a motor-domain switch that abolishes microtubule-end recognition without affecting lattice binding, integrating cell-cycle timing into depolymerase control.\",\n      \"evidence\": \"Phosphomimetic mutagenesis with single-molecule end/lattice residence kinetics and ATPase assay\",\n      \"pmids\": [\"29230353\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous phosphorylation timing not directly tracked\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Refined the conformational model showing compact-to-extended transitions on end binding and graded, dose-dependent Aurora B inhibition decoupling the N-terminus from the motor.\",\n      \"evidence\": \"Crosslinking mass spectrometry, electron microscopy, phosphomutant depolymerization assays\",\n      \"pmids\": [\"30578316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structures of intermediate states absent\", \"Quantitative link between phosphorylation level and in-cell output not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that Kif18b, MCAK, and EB3 function as an integrated multivalent network for potent cooperative plus-end depolymerization at low concentrations.\",\n      \"evidence\": \"In vitro reconstitution with single-molecule TIRF and quantitative depolymerization\",\n      \"pmids\": [\"35502670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell stoichiometry and regulation of the network not defined\", \"How phosphorylation modulates the network not tested here\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended KIF2C function beyond mitosis into neuronal synaptic plasticity, showing it regulates dendritic microtubule invasion, receptor trafficking, transmission and behavior.\",\n      \"evidence\": \"Conditional knockout, live microtubule imaging in neurons, electrophysiology, behavioral tests; Rab8 Co-IP in Purkinje cells\",\n      \"pmids\": [\"35138249\", \"37431690\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether depolymerase activity per se drives trafficking not isolated\", \"Single lab per neuronal system\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a non-mitotic role in DNA double-strand break repair by promoting DSB mobility and foci dynamics in a PARP/ATM-dependent manner.\",\n      \"evidence\": \"DSB-template pulldown in Xenopus extracts, CRISPR KO/siRNA, foci analysis, NHEJ/HR reporters, live DSB tracking\",\n      \"pmids\": [\"31951198\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking microtubule depolymerase activity to nuclear DSB mobility not fully defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a phospho-binding N-terminal Tudor/PWWP/MBT fold that drives Aurora B/PLK1-dependent KIF2C condensates concentrating signaling components at microtubule ends, and substrate preference for modified tubulin exploitable for chemoresistance.\",\n      \"evidence\": \"Structural determination, optogenetic condensate platform, FLIM-FRET, Co-IP; in vitro polyglutamylated-tubulin depolymerization with inhibitor and tumor models\",\n      \"pmids\": [\"40498077\", \"40157365\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Condensate findings not yet independently replicated\", \"Physiological role of condensates vs canonical depolymerization unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple kinase inputs, conformational states, partner networks, and condensate behavior are integrated in real time to achieve spatially precise depolymerization at distinct cellular sites remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified in-cell model coupling phosphorylation state to local activity\", \"Relative contribution of canonical depolymerization vs condensate/scaffolding functions unknown\", \"Structures of full-length regulated states absent\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 5, 21]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 16, 18]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [5, 16, 22]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [1, 2, 17]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 5, 7]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [38]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [15, 31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 6, 17]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [32]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [34, 35]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"AURKB\", \"AURKA\", \"PLK1\", \"EB1\", \"EB3\", \"TIP150\", \"KIF18B\", \"SGO2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}