{"gene":"CKAP5","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1998,"finding":"TOGp (CKAP5) is a microtubule-associated protein that co-sediments with taxol-stabilized microtubules in vitro, promotes microtubule assembly both in solution and from nucleation centers, and localizes to centrosomes and spindles in mitotic cells (ER during interphase), establishing it as the human homolog of XMAP215.","method":"Microtubule co-sedimentation assay, immunofluorescence microscopy, subcellular fractionation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct biochemical binding assay plus localization, replicated across multiple cell lines, foundational characterization paper","pmids":["9570755"],"is_preprint":false},{"year":2000,"finding":"The microtubule-binding domain of TOGp resides in a basic ~600 amino acid region near the N-terminus; full-length TOGp shows enhanced binding to microtubule ends and binds along individual protofilaments; the C-terminal region binds free tubulin dimer with low affinity.","method":"Microtubule co-pelleting assay with truncation constructs expressed in reticulocyte lysate","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic domain mapping by in vitro co-pelleting with multiple truncation constructs, clear domain attribution","pmids":["10770946"],"is_preprint":false},{"year":2000,"finding":"ch-TOGp is required for microtubule aster formation and robust microtubule polymerization in a mammalian mitotic extract; immunodepletion of ch-TOGp severely inhibits microtubule polymerization in an ATP-dependent manner.","method":"Immunodepletion from mammalian mitotic extract, microtubule aster assembly assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution-type depletion/add-back in cell-free extract with rigorous controls showing ATP dependence","pmids":["10766876"],"is_preprint":false},{"year":2000,"finding":"TOG/XMAP215 associates with cyclin B1 and p34cdc2 kinase; the C-terminal proline-rich region of TOG is sufficient to co-sediment cyclin B1 with microtubules; this interaction may target MPF to the microtubule cytoskeleton during mitosis.","method":"Co-immunoprecipitation, microtubule co-sedimentation with recombinant fragments, immunofluorescence co-localization","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus domain-mapping cosedimentation in a single lab; domain localization of interaction confirmed","pmids":["10640423"],"is_preprint":false},{"year":2002,"finding":"ch-TOG interacts with TACC1 through the C-terminus of ch-TOG, as identified by yeast two-hybrid screening and confirmed biochemically, suggesting ch-TOG participates in multiple TACC protein complexes.","method":"Yeast two-hybrid screen, biochemical interaction assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus some biochemical follow-up; single lab","pmids":["11903063"],"is_preprint":false},{"year":2003,"finding":"RNAi depletion of ch-TOG in HeLa cells causes highly disorganized spindles with disorganized spindle poles; ch-TOG plays a major role in organizing spindle poles and a minor role in stabilizing spindle microtubules. TACC3 depletion mislocalizes ch-TOG from spindle microtubules, indicating TACC3 is required to recruit ch-TOG to centrosomes/spindles.","method":"RNA interference (RNAi) in HeLa cells, immunofluorescence microscopy","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean RNAi knockdown with specific cellular phenotype (spindle pole disorganization), reciprocal dependency between ch-TOG and TACC3 demonstrated, widely replicated","pmids":["12569123"],"is_preprint":false},{"year":2004,"finding":"ch-TOG (TOGp) protects spindle microtubules from MCAK-mediated depolymerization at the centrosome to prevent multipolar spindles; TOGp also has an MCAK-independent role in protecting spindle MTs from MCAK at plus ends during mitosis.","method":"Inducible overexpression and shRNA knockdown in human leukemia cells, phenotypic spindle analysis","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic gain/loss-of-function with defined phenotypic readouts, single lab","pmids":["14749730"],"is_preprint":false},{"year":2006,"finding":"Purified TOGp shortens the nucleation phase of microtubule assembly, increases the apparent first-order rate constant of elongation, decreases tubulin critical concentration, and inhibits depolymerization during steady state — mechanistically acting as a promoter of both nucleation and elongation.","method":"In vitro kinetic analysis of tubulin polymerization with purified TOGp, turbidimetry, GTPase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified components and quantitative kinetic parameters; single lab","pmids":["17178729"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of a TOG domain (from C. elegans Zyg9) reveals a flat, paddle-like structure of six HEAT-repeat elements; the intra-HEAT repeat turns form a conserved, surface-exposed tubulin-binding face; mutagenesis of conserved residues in the TOG1 domain of Stu2p (yeast CKAP5 homolog) confirmed these turns participate in tubulin contact.","method":"X-ray crystallography (1.9 Å), site-directed mutagenesis of tubulin-binding residues","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutational validation of tubulin-binding surface; foundational structural paper for the family","pmids":["17355870"],"is_preprint":false},{"year":2008,"finding":"Aurora-A depletion causes ch-TOG to abnormally accumulate at spindle poles and MCAK to delocalize from poles; co-depletion of Aurora-A and ch-TOG mitigates the fragmented pole phenotype caused by Aurora-A loss, placing ch-TOG downstream of Aurora-A in spindle pole integrity control.","method":"RNAi co-depletion (Aurora-A and ch-TOG), immunofluorescence, genetic epistasis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via double knockdown with defined phenotypic rescue; single lab","pmids":["18663358"],"is_preprint":false},{"year":2008,"finding":"ch-TOG is required to produce/maintain long centrosomal microtubules after nuclear envelope breakdown and for proper microtubule dynamics at centrosomes; additionally, ch-TOG protects kinetochore microtubules from MCAK depolymerization. The centrosomal microtubule assembly function is independent of MCAK activity (co-depletion of MCAK does not rescue centrosomal MT defects).","method":"RNAi knockdown, co-depletion of ch-TOG and MCAK, real-time spindle assembly imaging","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — live-cell imaging plus co-depletion epistasis; two distinct mechanistic roles dissected","pmids":["18809577"],"is_preprint":false},{"year":2009,"finding":"TOGp depletion by siRNA slows spindle microtubule turnover ~2-fold (measured by FRAP of GFP-tubulin) and reduces microtubule regrowth after nocodazole washout at centrosomes and near chromatin, without affecting EB1-tracked plus-end growth rates; kinetochore oscillations during prometaphase/metaphase are also reduced.","method":"siRNA knockdown, FRAP of GFP-tubulin, EB1-GFP plus-end tracking, nocodazole washout assay","journal":"Cell motility and the cytoskeleton","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal live-cell assays post-knockdown; single lab","pmids":["19373773"],"is_preprint":false},{"year":2010,"finding":"Integrin-linked kinase (ILK) regulates centrosome clustering in cancer cells through ch-TOG and TACC3; ILK performs centrosome clustering activity in a focal adhesion-independent, centrosome-dependent manner via these microtubule-regulating proteins. A specific TACC3 phosphorylation is required and ILK regulates it via Aurora-A.","method":"RNAi knockdown, live-cell imaging, ILK inhibition, phosphorylation analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway epistasis by co-depletion, live-cell analysis; single lab","pmids":["20838383"],"is_preprint":false},{"year":2011,"finding":"ch-TOG, TACC3, and clathrin form a trimeric complex that constitutes inter-microtubule bridges in kinetochore fibers; TACC3 and ch-TOG anchor the complex to spindle microtubules; clathrin depletion or TACC3 depletion causes loss of short inter-MT bridges and general loss of kinetochore fiber microtubules; immunogold labeling confirmed clathrin in inter-MT bridges.","method":"Co-immunoprecipitation, electron microscopy ultrastructural analysis, immunogold labeling, RNAi knockdown","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying trimeric complex plus ultrastructural confirmation by immunogold EM; multiple orthogonal methods","pmids":["21297582"],"is_preprint":false},{"year":2011,"finding":"XMAP215 polymerase activity requires multiple tubulin-binding TOG domains plus a basic lattice-binding region; individual TOG domains differentially contribute to overall tubulin affinity, and overall affinity correlates with polymerase activity; an engineered 'bonsai' protein with two TOG domains and the basic region retains near-full polymerase activity.","method":"Site-directed mutagenesis of TOG domains, in vitro microtubule polymerization assays, tubulin binding assays","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis of each TOG domain with quantitative in vitro polymerization and binding assays; structure-function relationships established","pmids":["21282620"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of Stu2p TOG1 bound to yeast αβ-tubulin shows TOG1 binds curved (unpolymerized) αβ-tubulin conformation; TOG1 contacts α- and β-tubulin surfaces that do not participate in microtubule assembly; this conformation-selective binding explains how TOG polymerases discriminate unpolymerized from polymerized tubulin and recognize the growing microtubule end.","method":"X-ray crystallography of TOG1:αβ-tubulin complex","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution co-crystal structure of TOG domain bound to tubulin; defines catalytic mechanism","pmids":["22904013"],"is_preprint":false},{"year":2012,"finding":"ch-TOG accumulation sites at microtubule plus ends protrude ~100 nm beyond EB1 comets, indicating ch-TOG and EB1 recognize distinct regions at growing microtubule ends; ch-TOG and EB1 additively increase microtubule dynamicity but only EB1 exhibits microtubule-cell cortex attachment activity.","method":"High-resolution (TIRF/SIM) microscopy, overexpression in interphase HeLa cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — high-resolution nanoscale localization and functional overexpression analysis; single lab","pmids":["23251535"],"is_preprint":false},{"year":2012,"finding":"Clathrin (CHC17 isoform) stabilizes ch-TOG protein levels and promotes its localization to metaphase centrosomes; clathrin inactivation during S phase reduces both clathrin and ch-TOG at metaphase centrosomes, causing centrosome fragmentation, phenocopied by Aurora A inhibition, placing clathrin upstream of ch-TOG centrosomal stabilization.","method":"RNAi depletion, chemical SNAP-tag inactivation, Aurora A inhibitor (MLN8237), live-cell imaging","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — acute inactivation approach with staged cell cycle analysis; single lab, Aurora A link established","pmids":["22891263"],"is_preprint":false},{"year":2013,"finding":"Within the TACC3/ch-TOG/clathrin complex, TACC3 and clathrin are interdependent for spindle recruitment; the N-terminal domain of clathrin and the TACC domain of TACC3 form a cooperative microtubule interaction surface; a dileucine motif and Aurora A-phosphorylated S558 on TACC3 bind clathrin; a proposed sixth TOG domain (stutter in TACC3 coiled-coil interacts with ch-TOG TOG6) is required for microtubule localization of ch-TOG but not TACC3-clathrin.","method":"Interaction mapping by truncation/mutagenesis, Co-IP, microtubule binding assays, live-cell localization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic domain mapping with multiple orthogonal methods, interaction surfaces defined, localization consequences tested","pmids":["23918938"],"is_preprint":false},{"year":2013,"finding":"Rapid removal of TACC3/ch-TOG/clathrin complex from kinetochore fibers at metaphase (using 'knocksideways') reduces kinetochore fiber tension and prevents spindle checkpoint satisfaction without significant loss of kinetochore fiber microtubule number, indicating the complex is required for maintenance of K-fiber tension rather than microtubule stability per se.","method":"Knocksideways rapid inactivation, live-cell imaging, inter-kinetochore distance measurement, spindle checkpoint assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — acute, stage-specific inactivation with clear functional readouts; dissects assembly vs maintenance roles","pmids":["23532825"],"is_preprint":false},{"year":2014,"finding":"Crystal structures of ch-TOG TOG4 reveal a conserved domain bend architecturally distinct from TOG1 and TOG2, predicting novel engagement with α-tubulin; in vitro assays show differential tubulin-binding affinities across the TOG array; mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue of microtubule polymerization.","method":"X-ray crystallography of ch-TOG TOG4, in vitro tubulin binding assays, S2 cell rescue assay with point mutants","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of human ch-TOG TOG4 plus biochemical binding assays and cell-based rescue experiments","pmids":["24966168"],"is_preprint":false},{"year":2015,"finding":"TACC3 tracks plus-ends of microtubules via its interaction with ch-TOG, independently of EB1, EB3, Aurora-A phosphorylation, and clathrin binding; this defines a clathrin-independent pool of TACC3-ch-TOG at microtubule plus ends.","method":"Mutational analysis, live-cell fluorescence microscopy, co-immunoprecipitation","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutational dissection with live-cell tracking; single lab","pmids":["25596274"],"is_preprint":false},{"year":2015,"finding":"ch-TOG is required for directed cancer cell migration; eribulin depletes ch-TOG from microtubule plus ends, decreasing microtubule growth speed and cortical stabilization; ch-TOG knockdown phenocopies eribulin effects on microtubule dynamics and chemotaxis.","method":"siRNA knockdown of ch-TOG, live-cell microtubule dynamics imaging, chemotaxis assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi knockdown with defined microtubule dynamics readout and functional migration assay; single lab","pmids":["26497677"],"is_preprint":false},{"year":2016,"finding":"Stu2 (yeast CKAP5 ortholog) interacts with the Ndc80 kinetochore complex and its human ortholog ch-TOG shares this conserved interaction; Stu2 can stabilize or destabilize kinetochore-microtubule attachments depending on tension level and whether the microtubule tip is assembling or disassembling, independent of its regulation of microtubule dynamics.","method":"Reconstituted kinetochore-microtubule attachment assay, force measurements, biochemical pulldowns, genetic analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted kinetochore assay with quantitative force measurements, multiple orthogonal methods, conserved interaction shown for ch-TOG","pmids":["27156448"],"is_preprint":false},{"year":2017,"finding":"Conditional knockout of TOG (CKAP5) in myelinating glia causes severe CNS hypomyelination and reduced MBP protein without affecting MBP mRNA levels; in TOG KO oligodendrocytes, MBP mRNA is confined to cell bodies rather than being transported into processes/myelin compartment, indicating TOG is required for MBP mRNA transport to sites of translation.","method":"Conditional knockout in mice, in situ hybridization for MBP mRNA, immunoblotting, electron microscopy of myelin","journal":"Glia","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple biochemical and histological readouts establishing post-transcriptional mRNA transport role","pmids":["28063167"],"is_preprint":false},{"year":2018,"finding":"Structural and biochemical studies of Alp14 (fission yeast CKAP5 ortholog) TOG array show it binds four tubulins via dimeric TOG1-TOG2 subunits arranged in square assemblies; an X-ray structure reveals square-shaped pseudo-dimeric assemblies positioning four unpolymerized tubulins in a polarized wheel-like configuration; an 'unfurled' assembly binds two polymerized tubulins, supporting a model in which square assemblies unfurl to facilitate concerted tubulin polymerization into protofilaments.","method":"X-ray crystallography, crosslinking, electron microscopy, biochemical mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures plus EM and crosslinking in solution; mechanistic model for polymerase assembly","pmids":["30422110"],"is_preprint":false},{"year":2023,"finding":"TACC3-ch-TOG interaction controls TACC3 phosphorylation and TACC3-mediated stabilization of γ-TuRC at centrosomes; deletion of the ch-TOG-binding region in TACC3 enhances γ-TuRC recruitment to centrosomes; ch-TOG depletion increases γ-tubulin localization on spindle microtubules without affecting centrosomal γ-tubulin, indicating ch-TOG controls spatial regulation of γ-TuRC-dependent microtubule nucleation.","method":"TACC3 deletion mutants, co-immunoprecipitation, immunofluorescence quantification of γ-TuRC localization in human cells","journal":"Bioscience reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mutants with biochemical and localization readouts; single lab","pmids":["36790370"],"is_preprint":false},{"year":2024,"finding":"CKAP5 stabilizes CENP-E at kinetochores in human cells; CKAP5 depletion impairs CENP-E localization at metaphase kinetochores, increases kinetochore-microtubule stability, and causes attachment errors; the TOG4-TOG5 domain of CKAP5 mediates interaction with CENP-E-BubR1 and rescues CENP-E kinetochore localization; CKAP5 depletion facilitates PP1 recruitment to kinetochores, and a PP1 target site-specific CENP-E phosphomimetic mutant is stabilized at kinetochores in CKAP5-depleted cells.","method":"siRNA knockdown, domain rescue (TOG4-TOG5 fragment), co-immunoprecipitation (CENP-E-BubR1), PP1 localization assay, phosphomimetic mutant analysis, computational modeling","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (domain rescue, Co-IP, PP1 pathway analysis, phosphomimetic mutant), mechanistic pathway established","pmids":["38424231"],"is_preprint":false},{"year":2024,"finding":"The TOG5 domain of CKAP5 is necessary for binding and bundling actin filaments and for cross-linking microtubules with F-actin in vitro; this MT/F-actin cross-linking function does not require microtubule polymerase activity; in neuronal growth cones, CKAP5 requires both MT and F-actin binding (but not MT polymerization) to promote MT-F-actin alignment and axon outgrowth.","method":"In vitro TIRF reconstitution with purified proteins, TOG5 deletion/mutation, MT/F-actin co-sedimentation and bundling assays, Xenopus neuronal growth cone live imaging","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution assays with domain mutants plus in vivo neuronal validation; novel TOG5-specific actin-binding activity directly demonstrated","pmids":["39504455"],"is_preprint":false},{"year":2025,"finding":"The α-helical bundle domain of ch-TOG that mediates interaction with TACC3 was structurally characterized; inhibition of the TACC3-ch-TOG interaction (using Affimer tools that displace ch-TOG without affecting spindle localization of other complex components) causes fragmentation of pericentriolar material in metaphase cells and delays mitotic progression, revealing a role for TACC3-ch-TOG in maintaining pericentriolar material integrity.","method":"X-ray crystallography/structural modeling, biophysical assays, Affimer-mediated inhibition in live cells, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — structure of interaction domain plus specific molecular inhibition in live cells with defined phenotypic consequence; single lab with multiple orthogonal methods","pmids":["40105698"],"is_preprint":false},{"year":2025,"finding":"Stu2 (yeast CKAP5 ortholog) displays enzyme-like hyperbolic dependence of microtubule growth rate on tubulin concentration; the amount of Stu2 on the microtubule end does not vary with tubulin concentration; TOG:tubulin binding is high affinity (~10 nM) with slow dissociation (~0.03 s⁻¹); Stu2 operates as a tubulin-shuttling antenna on the microtubule end, primarily limited by the rate of tubulin:TOG association.","method":"In vitro microtubule dynamics assay with quantitative kinetic modeling, TOG:tubulin binding measurements","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro reconstitution with kinetic modeling; preprint, not yet peer reviewed","pmids":["bio_10.1101_2025.06.09.658552"],"is_preprint":true}],"current_model":"CKAP5/ch-TOG is a conserved microtubule polymerase that uses an array of structurally distinct TOG domains to bind free, curved (unpolymerized) αβ-tubulin and catalyze rapid microtubule plus-end growth by shuttling tubulin to the growing tip; it organizes mitotic spindle poles and kinetochore fibers through a trimeric complex with TACC3 and clathrin (anchored by Aurora A-phosphorylated TACC3 and controlled by the TOG6/TACC3-interacting stutter), stabilizes CENP-E at kinetochores via its TOG4-TOG5 domains to suppress attachment errors, cross-links microtubules with F-actin through its TOG5 domain to promote axon outgrowth, and in oligodendrocytes transports MBP mRNA into processes for local translation."},"narrative":{"mechanistic_narrative":"CKAP5 (ch-TOG/TOGp), the human homolog of XMAP215, is a microtubule-associated protein and microtubule polymerase that drives microtubule assembly at centrosomes, spindles, and growing plus ends [PMID:9570755, PMID:10766876, PMID:17178729]. Mechanistically, it uses an array of TOG domains—flat, HEAT-repeat paddles whose conserved intra-repeat turns form a tubulin-binding face [PMID:17355870]—to selectively bind curved, unpolymerized αβ-tubulin via surfaces not used in lattice contacts, allowing it to discriminate free tubulin and recognize the growing tip [PMID:22904013]. Polymerase activity requires multiple tubulin-binding TOG domains plus a basic lattice-binding region, with overall tubulin affinity correlating with activity; mutating tubulin contacts in any single TOG domain sharply reduces polymerization [PMID:21282620, PMID:24966168]. In dividing cells, ch-TOG organizes spindle poles and protects centrosomal and kinetochore microtubules from MCAK-mediated depolymerization, acting downstream of Aurora-A in spindle pole integrity [PMID:12569123, PMID:14749730, PMID:18809577, PMID:18663358]. It forms a trimeric complex with TACC3 and clathrin that builds inter-microtubule bridges within kinetochore fibers and maintains K-fiber tension; TACC3—stabilized by Aurora-A phosphorylation—recruits ch-TOG to spindle microtubules, and the TACC3 coiled-coil 'stutter' engages a sixth TOG-like region of ch-TOG [PMID:21297582, PMID:23918938, PMID:12569123, PMID:23532825]. Beyond polymerase activity, ch-TOG stabilizes CENP-E at kinetochores through its TOG4–TOG5 domains to suppress attachment errors [PMID:38424231], cross-links microtubules with F-actin via its TOG5 domain to promote axon outgrowth independently of polymerase activity [PMID:39504455], and is required in oligodendrocytes to transport MBP mRNA into processes for local translation [PMID:28063167].","teleology":[{"year":1998,"claim":"Established that the XMAP215 human homolog is a bona fide microtubule-associated protein that promotes assembly and localizes to centrosomes and spindles, defining its cellular arena.","evidence":"Microtubule co-sedimentation, immunofluorescence, and fractionation in human cells","pmids":["9570755"],"confidence":"High","gaps":["Did not resolve which protein regions bind microtubules versus free tubulin","No kinetic mechanism of assembly promotion"]},{"year":2000,"claim":"Mapped functional domains, separating an N-terminal basic microtubule-binding/end-binding region from a C-terminal free-tubulin-binding region, and demonstrated the protein is essential for microtubule polymerization in mitotic extract.","evidence":"Truncation co-pelleting assays and immunodepletion/add-back in mammalian mitotic extract","pmids":["10770946","10766876"],"confidence":"High","gaps":["Domain architecture of tubulin recognition not yet structurally defined","ATP-dependence of extract polymerization not mechanistically explained"]},{"year":2000,"claim":"Linked ch-TOG to mitotic kinase machinery by showing its C-terminal proline-rich region co-sediments cyclin B1/cdc2 with microtubules, raising the possibility it targets MPF to the cytoskeleton.","evidence":"Co-IP and fragment co-sedimentation in a single lab","pmids":["10640423"],"confidence":"Medium","gaps":["Functional consequence of MPF targeting not established","Single-lab interaction without reciprocal in vivo validation"]},{"year":2002,"claim":"Identified TACC-family proteins as ch-TOG partners via its C-terminus, opening the TACC axis that would prove central to its spindle recruitment.","evidence":"Yeast two-hybrid screen with biochemical confirmation","pmids":["11903063"],"confidence":"Medium","gaps":["Cellular function of the ch-TOG–TACC interaction not yet tested","Y2H interaction needs in vivo context"]},{"year":2003,"claim":"Defined ch-TOG's primary mitotic role as spindle pole organization and established the reciprocal dependency in which TACC3 is required to recruit ch-TOG to spindles.","evidence":"RNAi knockdown and immunofluorescence in HeLa cells","pmids":["12569123"],"confidence":"High","gaps":["Molecular basis of TACC3-dependent recruitment not yet mapped","Did not distinguish polymerase from scaffolding contributions"]},{"year":2004,"claim":"Showed ch-TOG protects spindle microtubules from MCAK depolymerization at both poles and plus ends, defining an antagonistic balance governing spindle stability.","evidence":"Inducible overexpression and shRNA in human leukemia cells with phenotypic spindle analysis","pmids":["14749730"],"confidence":"Medium","gaps":["Mechanism of MCAK antagonism unresolved","Single-lab phenotypic study"]},{"year":2006,"claim":"Provided the first quantitative in vitro mechanism, showing purified protein shortens nucleation, accelerates elongation, lowers critical concentration, and inhibits depolymerization.","evidence":"In vitro kinetic turbidimetry and GTPase assays with purified TOGp","pmids":["17178729"],"confidence":"High","gaps":["Structural basis of these kinetic effects not addressed","Single-lab kinetic study"]},{"year":2007,"claim":"Solved the TOG domain fold, revealing a flat HEAT-repeat paddle with a conserved surface-exposed tubulin-binding face validated by mutagenesis.","evidence":"X-ray crystallography of a TOG domain plus site-directed mutagenesis of orthologs","pmids":["17355870"],"confidence":"High","gaps":["Conformation of tubulin recognized by TOG not yet shown","How multiple TOGs cooperate not addressed"]},{"year":2008,"claim":"Placed ch-TOG downstream of Aurora-A in spindle pole integrity and dissected its dual roles in centrosomal microtubule production and MCAK-independent kinetochore microtubule protection.","evidence":"RNAi co-depletion epistasis and live-cell spindle imaging","pmids":["18663358","18809577"],"confidence":"High","gaps":["Direct Aurora-A regulation of ch-TOG not demonstrated","Mechanistic link between centrosomal and kinetochore functions unclear"]},{"year":2009,"claim":"Quantified ch-TOG's contribution to spindle microtubule turnover and kinetochore oscillation, showing it acts on turnover and regrowth rather than EB1-tracked plus-end growth rate.","evidence":"siRNA with FRAP, EB1 tracking, and nocodazole washout in human cells","pmids":["19373773"],"confidence":"Medium","gaps":["Discrepancy with in vitro elongation effects not reconciled","Single-lab live-cell study"]},{"year":2011,"claim":"Established the ch-TOG/TACC3/clathrin trimeric complex as the structural inter-microtubule bridges of kinetochore fibers, and defined the multi-TOG-plus-basic-region requirement for polymerase activity.","evidence":"Reciprocal Co-IP with immunogold EM, plus systematic TOG-domain mutagenesis with in vitro polymerization assays","pmids":["21297582","21282620"],"confidence":"High","gaps":["Stoichiometry and architecture of the bridge complex not fully resolved","How TOG affinity quantitatively translates to processivity unaddressed"]},{"year":2012,"claim":"Captured the catalytic basis of conformation selectivity by crystallizing a TOG domain bound to curved αβ-tubulin, and showed ch-TOG occupies a distinct plus-end zone beyond EB1.","evidence":"X-ray co-crystal structure of TOG1:αβ-tubulin and high-resolution plus-end localization microscopy","pmids":["22904013","23251535"],"confidence":"High","gaps":["How conformation-selective binding drives net polymerization in cells not shown","Functional division of labor between ch-TOG and EB1 zones partly inferred"]},{"year":2012,"claim":"Defined clathrin as an upstream stabilizer of ch-TOG protein levels and centrosomal localization, linking S-phase clathrin function and Aurora-A to centrosome integrity.","evidence":"RNAi, SNAP-tag chemical inactivation, and Aurora-A inhibition with live-cell imaging","pmids":["22891263"],"confidence":"Medium","gaps":["Mechanism by which clathrin stabilizes ch-TOG protein unknown","Single-lab study"]},{"year":2013,"claim":"Mapped the molecular interaction surfaces of the TACC3/ch-TOG/clathrin complex and dissected its requirement for kinetochore-fiber tension and checkpoint satisfaction rather than microtubule number.","evidence":"Truncation/mutagenesis interaction mapping plus knocksideways acute inactivation with checkpoint assays","pmids":["23918938","23532825"],"confidence":"High","gaps":["Physical basis of tension generation by the bridges not established","Whether the proposed TOG6/stutter interaction is a true sixth TOG domain unresolved"]},{"year":2014,"claim":"Showed the TOG array is functionally non-redundant, with structurally distinct domains (e.g., bent TOG4) and differential tubulin affinities, each individually required for full polymerase rescue.","evidence":"X-ray crystallography of ch-TOG TOG4, in vitro binding, and S2 cell rescue with point mutants","pmids":["24966168"],"confidence":"High","gaps":["How distinct TOG geometries are spatially arranged at the tip not shown","Specific tubulin-conformation engaged by TOG4 inferred from structure"]},{"year":2015,"claim":"Revealed a clathrin- and Aurora-A-independent TACC3–ch-TOG pool that tracks microtubule plus ends, and tied ch-TOG plus-end activity to cancer cell migration sensitive to eribulin.","evidence":"Mutational analysis with live-cell plus-end tracking, and siRNA with chemotaxis and microtubule dynamics assays","pmids":["25596274","26497677"],"confidence":"Medium","gaps":["Relative contributions of complexed versus free ch-TOG pools to dynamics unresolved","Single-lab studies"]},{"year":2016,"claim":"Identified a conserved Ndc80-kinetochore interaction and showed the ortholog can both stabilize and destabilize kinetochore-microtubule attachments in a tension- and tip-state-dependent manner, separate from polymerase activity.","evidence":"Reconstituted kinetochore-microtubule attachment assay with force measurements and genetics in yeast, with conserved interaction shown for ch-TOG","pmids":["27156448"],"confidence":"High","gaps":["Whether human ch-TOG recapitulates the bidirectional attachment behavior in cells not directly tested","Molecular switch governing stabilize-vs-destabilize unresolved"]},{"year":2017,"claim":"Uncovered a non-mitotic role in which ch-TOG transports MBP mRNA into oligodendrocyte processes for local translation, revealing a post-transcriptional function in myelination.","evidence":"Conditional knockout in mice with in situ hybridization, immunoblotting, and EM of myelin","pmids":["28063167"],"confidence":"High","gaps":["Mechanism linking ch-TOG to mRNA cargo transport not defined","Whether this depends on polymerase or scaffolding activity unknown"]},{"year":2018,"claim":"Proposed a polymerase assembly model in which TOG arrays form square pseudo-dimeric assemblies positioning four unpolymerized tubulins that unfurl to drive concerted protofilament polymerization.","evidence":"X-ray crystallography, crosslinking, EM, and mutagenesis of the fission yeast ortholog","pmids":["30422110"],"confidence":"High","gaps":["Whether human ch-TOG forms equivalent square assemblies not directly shown","Unfurling step not captured in real time"]},{"year":2023,"claim":"Showed the TACC3–ch-TOG interaction spatially regulates γ-TuRC-dependent nucleation, restraining γ-tubulin on spindle microtubules versus centrosomes.","evidence":"TACC3 deletion mutants, Co-IP, and γ-TuRC localization quantification in human cells","pmids":["36790370"],"confidence":"Medium","gaps":["Direct effect of ch-TOG on γ-TuRC activity not measured","Single-lab study"]},{"year":2024,"claim":"Defined a polymerase-independent kinetochore function in which TOG4–TOG5 stabilizes CENP-E via CENP-E-BubR1 binding and antagonizes PP1, suppressing attachment errors.","evidence":"siRNA, TOG4-TOG5 domain rescue, Co-IP, PP1 localization, and phosphomimetic analysis in human cells","pmids":["38424231"],"confidence":"High","gaps":["Direct structural basis of TOG4-TOG5/CENP-E-BubR1 binding not solved","How this role coordinates with polymerase activity unclear"]},{"year":2024,"claim":"Established a TOG5-mediated actin-binding/bundling activity that cross-links microtubules with F-actin, polymerase-independent, to drive growth-cone alignment and axon outgrowth.","evidence":"In vitro TIRF reconstitution with domain mutants and Xenopus neuronal growth-cone imaging","pmids":["39504455"],"confidence":"High","gaps":["Structural basis of TOG5 actin binding not solved","How MT/F-actin cross-linking is regulated in vivo unknown"]},{"year":2025,"claim":"Structurally characterized the ch-TOG α-helical bundle that binds TACC3 and showed selective disruption of this interface fragments pericentriolar material and delays mitosis, defining a PCM-integrity role.","evidence":"Crystallography/modeling with Affimer-mediated inhibition in live human cells","pmids":["40105698"],"confidence":"High","gaps":["How TACC3–ch-TOG maintains PCM integrity mechanistically unresolved","Single-lab study"]},{"year":2025,"claim":"Quantitatively modeled the polymerase as a tubulin-shuttling antenna with enzyme-like, saturable kinetics whose end occupancy is concentration-independent, limited by tubulin:TOG association.","evidence":"In vitro microtubule dynamics with kinetic modeling and TOG:tubulin binding measurements (preprint)","pmids":["bio_10.1101_2025.06.09.658552"],"confidence":"Medium","gaps":["Not yet peer reviewed","Whether human ch-TOG shares identical kinetics not directly shown"]},{"year":null,"claim":"How CKAP5's distinct activities—polymerase, kinetochore-attachment regulation, MT/F-actin cross-linking, and mRNA transport—are coordinated and switched within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model integrating polymerase versus scaffolding/transport functions","Regulatory inputs (phosphorylation, partner availability) that toggle between modes not mapped","Mechanism of MBP mRNA cargo recognition unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[7,14,20,25]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,28]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[13,18,27]},{"term_id":"GO:0003723","term_label":"RNA 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that co-sediments with taxol-stabilized microtubules in vitro, promotes microtubule assembly both in solution and from nucleation centers, and localizes to centrosomes and spindles in mitotic cells (ER during interphase), establishing it as the human homolog of XMAP215.\",\n      \"method\": \"Microtubule co-sedimentation assay, immunofluorescence microscopy, subcellular fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct biochemical binding assay plus localization, replicated across multiple cell lines, foundational characterization paper\",\n      \"pmids\": [\"9570755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The microtubule-binding domain of TOGp resides in a basic ~600 amino acid region near the N-terminus; full-length TOGp shows enhanced binding to microtubule ends and binds along individual protofilaments; the C-terminal region binds free tubulin dimer with low affinity.\",\n      \"method\": \"Microtubule co-pelleting assay with truncation constructs expressed in reticulocyte lysate\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic domain mapping by in vitro co-pelleting with multiple truncation constructs, clear domain attribution\",\n      \"pmids\": [\"10770946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"ch-TOGp is required for microtubule aster formation and robust microtubule polymerization in a mammalian mitotic extract; immunodepletion of ch-TOGp severely inhibits microtubule polymerization in an ATP-dependent manner.\",\n      \"method\": \"Immunodepletion from mammalian mitotic extract, microtubule aster assembly assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution-type depletion/add-back in cell-free extract with rigorous controls showing ATP dependence\",\n      \"pmids\": [\"10766876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"TOG/XMAP215 associates with cyclin B1 and p34cdc2 kinase; the C-terminal proline-rich region of TOG is sufficient to co-sediment cyclin B1 with microtubules; this interaction may target MPF to the microtubule cytoskeleton during mitosis.\",\n      \"method\": \"Co-immunoprecipitation, microtubule co-sedimentation with recombinant fragments, immunofluorescence co-localization\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus domain-mapping cosedimentation in a single lab; domain localization of interaction confirmed\",\n      \"pmids\": [\"10640423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ch-TOG interacts with TACC1 through the C-terminus of ch-TOG, as identified by yeast two-hybrid screening and confirmed biochemically, suggesting ch-TOG participates in multiple TACC protein complexes.\",\n      \"method\": \"Yeast two-hybrid screen, biochemical interaction assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus some biochemical follow-up; single lab\",\n      \"pmids\": [\"11903063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RNAi depletion of ch-TOG in HeLa cells causes highly disorganized spindles with disorganized spindle poles; ch-TOG plays a major role in organizing spindle poles and a minor role in stabilizing spindle microtubules. TACC3 depletion mislocalizes ch-TOG from spindle microtubules, indicating TACC3 is required to recruit ch-TOG to centrosomes/spindles.\",\n      \"method\": \"RNA interference (RNAi) in HeLa cells, immunofluorescence microscopy\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean RNAi knockdown with specific cellular phenotype (spindle pole disorganization), reciprocal dependency between ch-TOG and TACC3 demonstrated, widely replicated\",\n      \"pmids\": [\"12569123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ch-TOG (TOGp) protects spindle microtubules from MCAK-mediated depolymerization at the centrosome to prevent multipolar spindles; TOGp also has an MCAK-independent role in protecting spindle MTs from MCAK at plus ends during mitosis.\",\n      \"method\": \"Inducible overexpression and shRNA knockdown in human leukemia cells, phenotypic spindle analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic gain/loss-of-function with defined phenotypic readouts, single lab\",\n      \"pmids\": [\"14749730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Purified TOGp shortens the nucleation phase of microtubule assembly, increases the apparent first-order rate constant of elongation, decreases tubulin critical concentration, and inhibits depolymerization during steady state — mechanistically acting as a promoter of both nucleation and elongation.\",\n      \"method\": \"In vitro kinetic analysis of tubulin polymerization with purified TOGp, turbidimetry, GTPase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified components and quantitative kinetic parameters; single lab\",\n      \"pmids\": [\"17178729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of a TOG domain (from C. elegans Zyg9) reveals a flat, paddle-like structure of six HEAT-repeat elements; the intra-HEAT repeat turns form a conserved, surface-exposed tubulin-binding face; mutagenesis of conserved residues in the TOG1 domain of Stu2p (yeast CKAP5 homolog) confirmed these turns participate in tubulin contact.\",\n      \"method\": \"X-ray crystallography (1.9 Å), site-directed mutagenesis of tubulin-binding residues\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutational validation of tubulin-binding surface; foundational structural paper for the family\",\n      \"pmids\": [\"17355870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Aurora-A depletion causes ch-TOG to abnormally accumulate at spindle poles and MCAK to delocalize from poles; co-depletion of Aurora-A and ch-TOG mitigates the fragmented pole phenotype caused by Aurora-A loss, placing ch-TOG downstream of Aurora-A in spindle pole integrity control.\",\n      \"method\": \"RNAi co-depletion (Aurora-A and ch-TOG), immunofluorescence, genetic epistasis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via double knockdown with defined phenotypic rescue; single lab\",\n      \"pmids\": [\"18663358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ch-TOG is required to produce/maintain long centrosomal microtubules after nuclear envelope breakdown and for proper microtubule dynamics at centrosomes; additionally, ch-TOG protects kinetochore microtubules from MCAK depolymerization. The centrosomal microtubule assembly function is independent of MCAK activity (co-depletion of MCAK does not rescue centrosomal MT defects).\",\n      \"method\": \"RNAi knockdown, co-depletion of ch-TOG and MCAK, real-time spindle assembly imaging\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live-cell imaging plus co-depletion epistasis; two distinct mechanistic roles dissected\",\n      \"pmids\": [\"18809577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TOGp depletion by siRNA slows spindle microtubule turnover ~2-fold (measured by FRAP of GFP-tubulin) and reduces microtubule regrowth after nocodazole washout at centrosomes and near chromatin, without affecting EB1-tracked plus-end growth rates; kinetochore oscillations during prometaphase/metaphase are also reduced.\",\n      \"method\": \"siRNA knockdown, FRAP of GFP-tubulin, EB1-GFP plus-end tracking, nocodazole washout assay\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal live-cell assays post-knockdown; single lab\",\n      \"pmids\": [\"19373773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Integrin-linked kinase (ILK) regulates centrosome clustering in cancer cells through ch-TOG and TACC3; ILK performs centrosome clustering activity in a focal adhesion-independent, centrosome-dependent manner via these microtubule-regulating proteins. A specific TACC3 phosphorylation is required and ILK regulates it via Aurora-A.\",\n      \"method\": \"RNAi knockdown, live-cell imaging, ILK inhibition, phosphorylation analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway epistasis by co-depletion, live-cell analysis; single lab\",\n      \"pmids\": [\"20838383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ch-TOG, TACC3, and clathrin form a trimeric complex that constitutes inter-microtubule bridges in kinetochore fibers; TACC3 and ch-TOG anchor the complex to spindle microtubules; clathrin depletion or TACC3 depletion causes loss of short inter-MT bridges and general loss of kinetochore fiber microtubules; immunogold labeling confirmed clathrin in inter-MT bridges.\",\n      \"method\": \"Co-immunoprecipitation, electron microscopy ultrastructural analysis, immunogold labeling, RNAi knockdown\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying trimeric complex plus ultrastructural confirmation by immunogold EM; multiple orthogonal methods\",\n      \"pmids\": [\"21297582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"XMAP215 polymerase activity requires multiple tubulin-binding TOG domains plus a basic lattice-binding region; individual TOG domains differentially contribute to overall tubulin affinity, and overall affinity correlates with polymerase activity; an engineered 'bonsai' protein with two TOG domains and the basic region retains near-full polymerase activity.\",\n      \"method\": \"Site-directed mutagenesis of TOG domains, in vitro microtubule polymerization assays, tubulin binding assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis of each TOG domain with quantitative in vitro polymerization and binding assays; structure-function relationships established\",\n      \"pmids\": [\"21282620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of Stu2p TOG1 bound to yeast αβ-tubulin shows TOG1 binds curved (unpolymerized) αβ-tubulin conformation; TOG1 contacts α- and β-tubulin surfaces that do not participate in microtubule assembly; this conformation-selective binding explains how TOG polymerases discriminate unpolymerized from polymerized tubulin and recognize the growing microtubule end.\",\n      \"method\": \"X-ray crystallography of TOG1:αβ-tubulin complex\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution co-crystal structure of TOG domain bound to tubulin; defines catalytic mechanism\",\n      \"pmids\": [\"22904013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ch-TOG accumulation sites at microtubule plus ends protrude ~100 nm beyond EB1 comets, indicating ch-TOG and EB1 recognize distinct regions at growing microtubule ends; ch-TOG and EB1 additively increase microtubule dynamicity but only EB1 exhibits microtubule-cell cortex attachment activity.\",\n      \"method\": \"High-resolution (TIRF/SIM) microscopy, overexpression in interphase HeLa cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — high-resolution nanoscale localization and functional overexpression analysis; single lab\",\n      \"pmids\": [\"23251535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Clathrin (CHC17 isoform) stabilizes ch-TOG protein levels and promotes its localization to metaphase centrosomes; clathrin inactivation during S phase reduces both clathrin and ch-TOG at metaphase centrosomes, causing centrosome fragmentation, phenocopied by Aurora A inhibition, placing clathrin upstream of ch-TOG centrosomal stabilization.\",\n      \"method\": \"RNAi depletion, chemical SNAP-tag inactivation, Aurora A inhibitor (MLN8237), live-cell imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — acute inactivation approach with staged cell cycle analysis; single lab, Aurora A link established\",\n      \"pmids\": [\"22891263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Within the TACC3/ch-TOG/clathrin complex, TACC3 and clathrin are interdependent for spindle recruitment; the N-terminal domain of clathrin and the TACC domain of TACC3 form a cooperative microtubule interaction surface; a dileucine motif and Aurora A-phosphorylated S558 on TACC3 bind clathrin; a proposed sixth TOG domain (stutter in TACC3 coiled-coil interacts with ch-TOG TOG6) is required for microtubule localization of ch-TOG but not TACC3-clathrin.\",\n      \"method\": \"Interaction mapping by truncation/mutagenesis, Co-IP, microtubule binding assays, live-cell localization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic domain mapping with multiple orthogonal methods, interaction surfaces defined, localization consequences tested\",\n      \"pmids\": [\"23918938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Rapid removal of TACC3/ch-TOG/clathrin complex from kinetochore fibers at metaphase (using 'knocksideways') reduces kinetochore fiber tension and prevents spindle checkpoint satisfaction without significant loss of kinetochore fiber microtubule number, indicating the complex is required for maintenance of K-fiber tension rather than microtubule stability per se.\",\n      \"method\": \"Knocksideways rapid inactivation, live-cell imaging, inter-kinetochore distance measurement, spindle checkpoint assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — acute, stage-specific inactivation with clear functional readouts; dissects assembly vs maintenance roles\",\n      \"pmids\": [\"23532825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of ch-TOG TOG4 reveal a conserved domain bend architecturally distinct from TOG1 and TOG2, predicting novel engagement with α-tubulin; in vitro assays show differential tubulin-binding affinities across the TOG array; mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue of microtubule polymerization.\",\n      \"method\": \"X-ray crystallography of ch-TOG TOG4, in vitro tubulin binding assays, S2 cell rescue assay with point mutants\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of human ch-TOG TOG4 plus biochemical binding assays and cell-based rescue experiments\",\n      \"pmids\": [\"24966168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TACC3 tracks plus-ends of microtubules via its interaction with ch-TOG, independently of EB1, EB3, Aurora-A phosphorylation, and clathrin binding; this defines a clathrin-independent pool of TACC3-ch-TOG at microtubule plus ends.\",\n      \"method\": \"Mutational analysis, live-cell fluorescence microscopy, co-immunoprecipitation\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutational dissection with live-cell tracking; single lab\",\n      \"pmids\": [\"25596274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ch-TOG is required for directed cancer cell migration; eribulin depletes ch-TOG from microtubule plus ends, decreasing microtubule growth speed and cortical stabilization; ch-TOG knockdown phenocopies eribulin effects on microtubule dynamics and chemotaxis.\",\n      \"method\": \"siRNA knockdown of ch-TOG, live-cell microtubule dynamics imaging, chemotaxis assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi knockdown with defined microtubule dynamics readout and functional migration assay; single lab\",\n      \"pmids\": [\"26497677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Stu2 (yeast CKAP5 ortholog) interacts with the Ndc80 kinetochore complex and its human ortholog ch-TOG shares this conserved interaction; Stu2 can stabilize or destabilize kinetochore-microtubule attachments depending on tension level and whether the microtubule tip is assembling or disassembling, independent of its regulation of microtubule dynamics.\",\n      \"method\": \"Reconstituted kinetochore-microtubule attachment assay, force measurements, biochemical pulldowns, genetic analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted kinetochore assay with quantitative force measurements, multiple orthogonal methods, conserved interaction shown for ch-TOG\",\n      \"pmids\": [\"27156448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Conditional knockout of TOG (CKAP5) in myelinating glia causes severe CNS hypomyelination and reduced MBP protein without affecting MBP mRNA levels; in TOG KO oligodendrocytes, MBP mRNA is confined to cell bodies rather than being transported into processes/myelin compartment, indicating TOG is required for MBP mRNA transport to sites of translation.\",\n      \"method\": \"Conditional knockout in mice, in situ hybridization for MBP mRNA, immunoblotting, electron microscopy of myelin\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple biochemical and histological readouts establishing post-transcriptional mRNA transport role\",\n      \"pmids\": [\"28063167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Structural and biochemical studies of Alp14 (fission yeast CKAP5 ortholog) TOG array show it binds four tubulins via dimeric TOG1-TOG2 subunits arranged in square assemblies; an X-ray structure reveals square-shaped pseudo-dimeric assemblies positioning four unpolymerized tubulins in a polarized wheel-like configuration; an 'unfurled' assembly binds two polymerized tubulins, supporting a model in which square assemblies unfurl to facilitate concerted tubulin polymerization into protofilaments.\",\n      \"method\": \"X-ray crystallography, crosslinking, electron microscopy, biochemical mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures plus EM and crosslinking in solution; mechanistic model for polymerase assembly\",\n      \"pmids\": [\"30422110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TACC3-ch-TOG interaction controls TACC3 phosphorylation and TACC3-mediated stabilization of γ-TuRC at centrosomes; deletion of the ch-TOG-binding region in TACC3 enhances γ-TuRC recruitment to centrosomes; ch-TOG depletion increases γ-tubulin localization on spindle microtubules without affecting centrosomal γ-tubulin, indicating ch-TOG controls spatial regulation of γ-TuRC-dependent microtubule nucleation.\",\n      \"method\": \"TACC3 deletion mutants, co-immunoprecipitation, immunofluorescence quantification of γ-TuRC localization in human cells\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutants with biochemical and localization readouts; single lab\",\n      \"pmids\": [\"36790370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CKAP5 stabilizes CENP-E at kinetochores in human cells; CKAP5 depletion impairs CENP-E localization at metaphase kinetochores, increases kinetochore-microtubule stability, and causes attachment errors; the TOG4-TOG5 domain of CKAP5 mediates interaction with CENP-E-BubR1 and rescues CENP-E kinetochore localization; CKAP5 depletion facilitates PP1 recruitment to kinetochores, and a PP1 target site-specific CENP-E phosphomimetic mutant is stabilized at kinetochores in CKAP5-depleted cells.\",\n      \"method\": \"siRNA knockdown, domain rescue (TOG4-TOG5 fragment), co-immunoprecipitation (CENP-E-BubR1), PP1 localization assay, phosphomimetic mutant analysis, computational modeling\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (domain rescue, Co-IP, PP1 pathway analysis, phosphomimetic mutant), mechanistic pathway established\",\n      \"pmids\": [\"38424231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The TOG5 domain of CKAP5 is necessary for binding and bundling actin filaments and for cross-linking microtubules with F-actin in vitro; this MT/F-actin cross-linking function does not require microtubule polymerase activity; in neuronal growth cones, CKAP5 requires both MT and F-actin binding (but not MT polymerization) to promote MT-F-actin alignment and axon outgrowth.\",\n      \"method\": \"In vitro TIRF reconstitution with purified proteins, TOG5 deletion/mutation, MT/F-actin co-sedimentation and bundling assays, Xenopus neuronal growth cone live imaging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution assays with domain mutants plus in vivo neuronal validation; novel TOG5-specific actin-binding activity directly demonstrated\",\n      \"pmids\": [\"39504455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The α-helical bundle domain of ch-TOG that mediates interaction with TACC3 was structurally characterized; inhibition of the TACC3-ch-TOG interaction (using Affimer tools that displace ch-TOG without affecting spindle localization of other complex components) causes fragmentation of pericentriolar material in metaphase cells and delays mitotic progression, revealing a role for TACC3-ch-TOG in maintaining pericentriolar material integrity.\",\n      \"method\": \"X-ray crystallography/structural modeling, biophysical assays, Affimer-mediated inhibition in live cells, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — structure of interaction domain plus specific molecular inhibition in live cells with defined phenotypic consequence; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40105698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Stu2 (yeast CKAP5 ortholog) displays enzyme-like hyperbolic dependence of microtubule growth rate on tubulin concentration; the amount of Stu2 on the microtubule end does not vary with tubulin concentration; TOG:tubulin binding is high affinity (~10 nM) with slow dissociation (~0.03 s⁻¹); Stu2 operates as a tubulin-shuttling antenna on the microtubule end, primarily limited by the rate of tubulin:TOG association.\",\n      \"method\": \"In vitro microtubule dynamics assay with quantitative kinetic modeling, TOG:tubulin binding measurements\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro reconstitution with kinetic modeling; preprint, not yet peer reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.09.658552\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CKAP5/ch-TOG is a conserved microtubule polymerase that uses an array of structurally distinct TOG domains to bind free, curved (unpolymerized) αβ-tubulin and catalyze rapid microtubule plus-end growth by shuttling tubulin to the growing tip; it organizes mitotic spindle poles and kinetochore fibers through a trimeric complex with TACC3 and clathrin (anchored by Aurora A-phosphorylated TACC3 and controlled by the TOG6/TACC3-interacting stutter), stabilizes CENP-E at kinetochores via its TOG4-TOG5 domains to suppress attachment errors, cross-links microtubules with F-actin through its TOG5 domain to promote axon outgrowth, and in oligodendrocytes transports MBP mRNA into processes for local translation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CKAP5 (ch-TOG/TOGp), the human homolog of XMAP215, is a microtubule-associated protein and microtubule polymerase that drives microtubule assembly at centrosomes, spindles, and growing plus ends [#0, #2, #7]. Mechanistically, it uses an array of TOG domains—flat, HEAT-repeat paddles whose conserved intra-repeat turns form a tubulin-binding face [#8]—to selectively bind curved, unpolymerized αβ-tubulin via surfaces not used in lattice contacts, allowing it to discriminate free tubulin and recognize the growing tip [#15]. Polymerase activity requires multiple tubulin-binding TOG domains plus a basic lattice-binding region, with overall tubulin affinity correlating with activity; mutating tubulin contacts in any single TOG domain sharply reduces polymerization [#14, #20]. In dividing cells, ch-TOG organizes spindle poles and protects centrosomal and kinetochore microtubules from MCAK-mediated depolymerization, acting downstream of Aurora-A in spindle pole integrity [#5, #6, #10, #9]. It forms a trimeric complex with TACC3 and clathrin that builds inter-microtubule bridges within kinetochore fibers and maintains K-fiber tension; TACC3—stabilized by Aurora-A phosphorylation—recruits ch-TOG to spindle microtubules, and the TACC3 coiled-coil 'stutter' engages a sixth TOG-like region of ch-TOG [#13, #18, #5, #19]. Beyond polymerase activity, ch-TOG stabilizes CENP-E at kinetochores through its TOG4–TOG5 domains to suppress attachment errors [#27], cross-links microtubules with F-actin via its TOG5 domain to promote axon outgrowth independently of polymerase activity [#28], and is required in oligodendrocytes to transport MBP mRNA into processes for local translation [#24].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that the XMAP215 human homolog is a bona fide microtubule-associated protein that promotes assembly and localizes to centrosomes and spindles, defining its cellular arena.\",\n      \"evidence\": \"Microtubule co-sedimentation, immunofluorescence, and fractionation in human cells\",\n      \"pmids\": [\"9570755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which protein regions bind microtubules versus free tubulin\", \"No kinetic mechanism of assembly promotion\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Mapped functional domains, separating an N-terminal basic microtubule-binding/end-binding region from a C-terminal free-tubulin-binding region, and demonstrated the protein is essential for microtubule polymerization in mitotic extract.\",\n      \"evidence\": \"Truncation co-pelleting assays and immunodepletion/add-back in mammalian mitotic extract\",\n      \"pmids\": [\"10770946\", \"10766876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain architecture of tubulin recognition not yet structurally defined\", \"ATP-dependence of extract polymerization not mechanistically explained\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Linked ch-TOG to mitotic kinase machinery by showing its C-terminal proline-rich region co-sediments cyclin B1/cdc2 with microtubules, raising the possibility it targets MPF to the cytoskeleton.\",\n      \"evidence\": \"Co-IP and fragment co-sedimentation in a single lab\",\n      \"pmids\": [\"10640423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of MPF targeting not established\", \"Single-lab interaction without reciprocal in vivo validation\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified TACC-family proteins as ch-TOG partners via its C-terminus, opening the TACC axis that would prove central to its spindle recruitment.\",\n      \"evidence\": \"Yeast two-hybrid screen with biochemical confirmation\",\n      \"pmids\": [\"11903063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular function of the ch-TOG–TACC interaction not yet tested\", \"Y2H interaction needs in vivo context\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined ch-TOG's primary mitotic role as spindle pole organization and established the reciprocal dependency in which TACC3 is required to recruit ch-TOG to spindles.\",\n      \"evidence\": \"RNAi knockdown and immunofluorescence in HeLa cells\",\n      \"pmids\": [\"12569123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of TACC3-dependent recruitment not yet mapped\", \"Did not distinguish polymerase from scaffolding contributions\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed ch-TOG protects spindle microtubules from MCAK depolymerization at both poles and plus ends, defining an antagonistic balance governing spindle stability.\",\n      \"evidence\": \"Inducible overexpression and shRNA in human leukemia cells with phenotypic spindle analysis\",\n      \"pmids\": [\"14749730\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of MCAK antagonism unresolved\", \"Single-lab phenotypic study\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Provided the first quantitative in vitro mechanism, showing purified protein shortens nucleation, accelerates elongation, lowers critical concentration, and inhibits depolymerization.\",\n      \"evidence\": \"In vitro kinetic turbidimetry and GTPase assays with purified TOGp\",\n      \"pmids\": [\"17178729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of these kinetic effects not addressed\", \"Single-lab kinetic study\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Solved the TOG domain fold, revealing a flat HEAT-repeat paddle with a conserved surface-exposed tubulin-binding face validated by mutagenesis.\",\n      \"evidence\": \"X-ray crystallography of a TOG domain plus site-directed mutagenesis of orthologs\",\n      \"pmids\": [\"17355870\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformation of tubulin recognized by TOG not yet shown\", \"How multiple TOGs cooperate not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Placed ch-TOG downstream of Aurora-A in spindle pole integrity and dissected its dual roles in centrosomal microtubule production and MCAK-independent kinetochore microtubule protection.\",\n      \"evidence\": \"RNAi co-depletion epistasis and live-cell spindle imaging\",\n      \"pmids\": [\"18663358\", \"18809577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Aurora-A regulation of ch-TOG not demonstrated\", \"Mechanistic link between centrosomal and kinetochore functions unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Quantified ch-TOG's contribution to spindle microtubule turnover and kinetochore oscillation, showing it acts on turnover and regrowth rather than EB1-tracked plus-end growth rate.\",\n      \"evidence\": \"siRNA with FRAP, EB1 tracking, and nocodazole washout in human cells\",\n      \"pmids\": [\"19373773\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Discrepancy with in vitro elongation effects not reconciled\", \"Single-lab live-cell study\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established the ch-TOG/TACC3/clathrin trimeric complex as the structural inter-microtubule bridges of kinetochore fibers, and defined the multi-TOG-plus-basic-region requirement for polymerase activity.\",\n      \"evidence\": \"Reciprocal Co-IP with immunogold EM, plus systematic TOG-domain mutagenesis with in vitro polymerization assays\",\n      \"pmids\": [\"21297582\", \"21282620\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the bridge complex not fully resolved\", \"How TOG affinity quantitatively translates to processivity unaddressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Captured the catalytic basis of conformation selectivity by crystallizing a TOG domain bound to curved αβ-tubulin, and showed ch-TOG occupies a distinct plus-end zone beyond EB1.\",\n      \"evidence\": \"X-ray co-crystal structure of TOG1:αβ-tubulin and high-resolution plus-end localization microscopy\",\n      \"pmids\": [\"22904013\", \"23251535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How conformation-selective binding drives net polymerization in cells not shown\", \"Functional division of labor between ch-TOG and EB1 zones partly inferred\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined clathrin as an upstream stabilizer of ch-TOG protein levels and centrosomal localization, linking S-phase clathrin function and Aurora-A to centrosome integrity.\",\n      \"evidence\": \"RNAi, SNAP-tag chemical inactivation, and Aurora-A inhibition with live-cell imaging\",\n      \"pmids\": [\"22891263\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which clathrin stabilizes ch-TOG protein unknown\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped the molecular interaction surfaces of the TACC3/ch-TOG/clathrin complex and dissected its requirement for kinetochore-fiber tension and checkpoint satisfaction rather than microtubule number.\",\n      \"evidence\": \"Truncation/mutagenesis interaction mapping plus knocksideways acute inactivation with checkpoint assays\",\n      \"pmids\": [\"23918938\", \"23532825\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physical basis of tension generation by the bridges not established\", \"Whether the proposed TOG6/stutter interaction is a true sixth TOG domain unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed the TOG array is functionally non-redundant, with structurally distinct domains (e.g., bent TOG4) and differential tubulin affinities, each individually required for full polymerase rescue.\",\n      \"evidence\": \"X-ray crystallography of ch-TOG TOG4, in vitro binding, and S2 cell rescue with point mutants\",\n      \"pmids\": [\"24966168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How distinct TOG geometries are spatially arranged at the tip not shown\", \"Specific tubulin-conformation engaged by TOG4 inferred from structure\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed a clathrin- and Aurora-A-independent TACC3–ch-TOG pool that tracks microtubule plus ends, and tied ch-TOG plus-end activity to cancer cell migration sensitive to eribulin.\",\n      \"evidence\": \"Mutational analysis with live-cell plus-end tracking, and siRNA with chemotaxis and microtubule dynamics assays\",\n      \"pmids\": [\"25596274\", \"26497677\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contributions of complexed versus free ch-TOG pools to dynamics unresolved\", \"Single-lab studies\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a conserved Ndc80-kinetochore interaction and showed the ortholog can both stabilize and destabilize kinetochore-microtubule attachments in a tension- and tip-state-dependent manner, separate from polymerase activity.\",\n      \"evidence\": \"Reconstituted kinetochore-microtubule attachment assay with force measurements and genetics in yeast, with conserved interaction shown for ch-TOG\",\n      \"pmids\": [\"27156448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human ch-TOG recapitulates the bidirectional attachment behavior in cells not directly tested\", \"Molecular switch governing stabilize-vs-destabilize unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Uncovered a non-mitotic role in which ch-TOG transports MBP mRNA into oligodendrocyte processes for local translation, revealing a post-transcriptional function in myelination.\",\n      \"evidence\": \"Conditional knockout in mice with in situ hybridization, immunoblotting, and EM of myelin\",\n      \"pmids\": [\"28063167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking ch-TOG to mRNA cargo transport not defined\", \"Whether this depends on polymerase or scaffolding activity unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Proposed a polymerase assembly model in which TOG arrays form square pseudo-dimeric assemblies positioning four unpolymerized tubulins that unfurl to drive concerted protofilament polymerization.\",\n      \"evidence\": \"X-ray crystallography, crosslinking, EM, and mutagenesis of the fission yeast ortholog\",\n      \"pmids\": [\"30422110\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human ch-TOG forms equivalent square assemblies not directly shown\", \"Unfurling step not captured in real time\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed the TACC3–ch-TOG interaction spatially regulates γ-TuRC-dependent nucleation, restraining γ-tubulin on spindle microtubules versus centrosomes.\",\n      \"evidence\": \"TACC3 deletion mutants, Co-IP, and γ-TuRC localization quantification in human cells\",\n      \"pmids\": [\"36790370\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effect of ch-TOG on γ-TuRC activity not measured\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a polymerase-independent kinetochore function in which TOG4–TOG5 stabilizes CENP-E via CENP-E-BubR1 binding and antagonizes PP1, suppressing attachment errors.\",\n      \"evidence\": \"siRNA, TOG4-TOG5 domain rescue, Co-IP, PP1 localization, and phosphomimetic analysis in human cells\",\n      \"pmids\": [\"38424231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of TOG4-TOG5/CENP-E-BubR1 binding not solved\", \"How this role coordinates with polymerase activity unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a TOG5-mediated actin-binding/bundling activity that cross-links microtubules with F-actin, polymerase-independent, to drive growth-cone alignment and axon outgrowth.\",\n      \"evidence\": \"In vitro TIRF reconstitution with domain mutants and Xenopus neuronal growth-cone imaging\",\n      \"pmids\": [\"39504455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TOG5 actin binding not solved\", \"How MT/F-actin cross-linking is regulated in vivo unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Structurally characterized the ch-TOG α-helical bundle that binds TACC3 and showed selective disruption of this interface fragments pericentriolar material and delays mitosis, defining a PCM-integrity role.\",\n      \"evidence\": \"Crystallography/modeling with Affimer-mediated inhibition in live human cells\",\n      \"pmids\": [\"40105698\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TACC3–ch-TOG maintains PCM integrity mechanistically unresolved\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Quantitatively modeled the polymerase as a tubulin-shuttling antenna with enzyme-like, saturable kinetics whose end occupancy is concentration-independent, limited by tubulin:TOG association.\",\n      \"evidence\": \"In vitro microtubule dynamics with kinetic modeling and TOG:tubulin binding measurements (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.09.658552\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not yet peer reviewed\", \"Whether human ch-TOG shares identical kinetics not directly shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CKAP5's distinct activities—polymerase, kinetochore-attachment regulation, MT/F-actin cross-linking, and mRNA transport—are coordinated and switched within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model integrating polymerase versus scaffolding/transport functions\", \"Regulatory inputs (phosphorylation, partner availability) that toggle between modes not mapped\", \"Mechanism of MBP mRNA cargo recognition unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [7, 14, 20, 25]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 28]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [13, 18, 27]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 5, 10, 17, 29]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 16, 28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5, 13, 19, 27]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [26, 29]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [24, 28]}\n    ],\n    \"complexes\": [\n      \"TACC3/ch-TOG/clathrin complex\",\n      \"kinetochore fiber inter-microtubule bridges\"\n    ],\n    \"partners\": [\n      \"TACC3\",\n      \"CLTC\",\n      \"TACC1\",\n      \"CENP-E\",\n      \"BubR1\",\n      \"NDC80\",\n      \"MCAK\",\n      \"AURKA\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}