{"gene":"CEP135","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2002,"finding":"Cep135 is a 135-kDa coiled-coil centrosomal protein that localizes to the centrosome throughout the cell cycle, independent of the microtubule network. It distributes in association with electron-dense pericentriolar material. Three independent centrosome-targeting domains were identified by deletion construct analysis. Overexpression or RNAi suppression both caused disorganization of interphase and mitotic spindle microtubules, establishing a role in centrosomal microtubule organization.","method":"Monoclonal antibody identification, deletion construct overexpression, RNA interference, immunofluorescence microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (RNAi knockdown, overexpression of deletion constructs, immunofluorescence localization), replicated across conditions in a single rigorous study","pmids":["11781336"],"is_preprint":false},{"year":2000,"finding":"Overexpressed Cep135 assembles into filamentous polymers and whorl-like particles composed of ~7 nm parallel dense lines both at the centrosome and in the cytoplasm, with the polymer architecture dependent on specific domains of the protein, indicating Cep135 is a structural self-assembling component of the centrosome.","method":"Transient transfection of HA/GFP-tagged full-length and truncated constructs in CHO cells and baculovirus expression in Sf9 cells, electron microscopy","journal":"Microscopy research and technique","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct structural visualization by EM with domain-specific constructs, single lab","pmids":["10842375"],"is_preprint":false},{"year":2004,"finding":"Cep135 directly interacts with the p50 dynactin subunit (dynamitin) via the C-terminal sequence of Cep135 binding the central domain of p50. This interaction is required for centrosomal targeting of p50; exogenous p50 lacking the Cep135-binding domain failed to localize to the centrosome. Altered levels of either protein displaced the other as well as γ-tubulin and pericentrin, leading to microtubule disorganization.","method":"Yeast two-hybrid screen, co-immunoprecipitation, immunostaining of co-expressed binding domains in CHO cells, RNAi, overexpression","journal":"Cell motility and the cytoskeleton","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid discovery confirmed by co-IP and domain-mapping with functional consequences (centrosome targeting, microtubule organization), multiple orthogonal methods","pmids":["14983524"],"is_preprint":false},{"year":2008,"finding":"CEP135 acts as a platform protein for C-NAP1 at the centriole. Depletion of CEP135 caused premature centrosome splitting accompanied by specific reduction of centrosomal C-NAP1 levels. Ectopic expression of CEP135 mutant proteins also caused centrosome splitting with reduced centrosomal C-NAP1, establishing CEP135 as required for C-NAP1 centriolar localization.","method":"RNAi knockdown, overexpression of CEP135 mutants, immunofluorescence microscopy","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi and dominant-negative overexpression with defined phenotypic readout, single lab","pmids":["18851962"],"is_preprint":false},{"year":2012,"finding":"CEP135/Bld10 can bind and stabilize microtubules and is required for the early steps of central microtubule pair formation in Drosophila flagella. Assembly of the central MT pair begins prior to meiotic divisions with nucleation of a singlet MT within the basal body, and BLD10/CEP135 is essential for this early nucleation step.","method":"Drosophila genetic analysis, electron microscopy of spermatogenesis, MT binding/stabilization assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with defined ultrastructural phenotype plus biochemical MT binding assay, multiple orthogonal methods","pmids":["22898782"],"is_preprint":false},{"year":2012,"finding":"In Tetrahymena, Bld10/Cep135 is an outer cartwheel domain protein that stabilizes basal bodies to resist the forces generated by ciliary beating. Bld10 promotes stability of the A- and C-tubules of the triplet microtubules and proper positioning of the triplet MT blades. In bld10Δ cells, ciliary beating forces promote basal body disassembly, revealing a role in basal body maintenance distinct from its assembly role.","method":"Tetrahymena genetic deletion, electron microscopy, live-cell analysis of basal body dynamics","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined ultrastructural and functional phenotypes, multiple orthogonal methods (EM, live imaging)","pmids":["23115304"],"is_preprint":false},{"year":2012,"finding":"In Drosophila, Cep135/Bld10 is not essential for cartwheel formation or establishing the ninefold symmetry of centrioles. However, absence of Cep135/Bld10 leads to increased centriole width and progressive cartwheel disassembly over time. Cep135/Bld10 localizes between inner (SAS-6, Ana2) and outer (Asl, DSpd-2, D-PLP) centriolar components and stabilizes the connection between these inner and outer components.","method":"Drosophila Cep135/Bld10 mutant analysis, electron tomography, 3D structured illumination microscopy (SIM)","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — electron tomography plus super-resolution microscopy with genetic loss-of-function, rigorous structural analysis","pmids":["22976301"],"is_preprint":false},{"year":2013,"finding":"Human CEP135 directly interacts with hSAS-6 via its carboxyl-terminus and with microtubules via its amino-terminus. CEP135 also interacts with microcephaly protein CPAP via its amino-terminal domain. CEP135 depletion perturbed centriolar localization of CPAP, blocked CPAP-induced centriole elongation, and caused abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. A CEP135 mutant lacking the hSAS-6 interaction had a dominant-negative effect on centriole assembly, establishing CEP135 as a linker between the SAS-6 cartwheel hub and outer MTs.","method":"Co-immunoprecipitation, in vitro binding assays, RNAi depletion, overexpression of domain mutants, immunofluorescence and electron microscopy","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro binding assays with domain mapping, co-IP, functional knockdown and dominant-negative overexpression, multiple orthogonal methods","pmids":["23511974"],"is_preprint":false},{"year":2013,"finding":"Disruption of Cep135 in DT40 chicken cells produces viable cells with a small decrease in centriole numbers, increased monopolar spindles, and an atypical structure in the centriole lumen by electron microscopy. Cep135 loss significantly increases centrosome amplification after S-phase arrest (hydroxyurea treatment), indicating Cep135 inhibits centrosome reduplication during S-phase delay and is required for structural integrity of centrioles.","method":"Targeted gene disruption in DT40 cells, electron microscopy, flow cytometry, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean gene KO with multiple orthogonal readouts (EM, cell cycle analysis, centrosome counting), well-controlled study","pmids":["23864714"],"is_preprint":false},{"year":2014,"finding":"Nek2-mediated multisite phosphorylation of C-Nap1's C-terminal domain perturbs its interaction with Cep135. Interaction between endogenous C-Nap1 and Cep135 is specifically lost in mitosis. Phosphorylation of C-Nap1 leads to loss of oligomerization and centrosome association, and loss of Cep135 binding contributes to centrosome disjunction at mitotic entry.","method":"In vitro kinase assay, co-immunoprecipitation of endogenous proteins across cell cycle stages, phosphomimetic/non-phosphorylatable mutant analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay combined with endogenous co-IP across cell cycle stages and phosphomimetic mutant analysis, multiple orthogonal methods","pmids":["24695856"],"is_preprint":false},{"year":2014,"finding":"The Drosophila ortholog Bld10 (Cep135) is required to establish centrosome asymmetry in neuroblasts by mediating shedding of Polo kinase from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM from the mother centrosome, generating two active MTOCs, causing spindle alignment defects, centrosome segregation errors, and incorrect retention of the older mother centrosome by neuroblasts.","method":"Drosophila bld10 mutant analysis, live imaging, immunofluorescence microscopy","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with defined molecular (Polo/PCM levels) and cellular (spindle alignment, centrosome segregation) phenotypes using multiple imaging approaches","pmids":["24954048"],"is_preprint":false},{"year":2016,"finding":"The N-terminal 158 residues of human CEP135 form a parallel two-stranded coiled-coil structure. This domain binds tubulin, protofilaments, and microtubules in vitro and induces MT bundle formation. A 13-amino-acid segment (residues 96–108) represents the major MT-binding site, containing three lysine residues that contribute to the MT bundling activity.","method":"X-ray crystallography, small-angle X-ray scattering (SAXS), cryo-electron microscopy, fluorescence microscopy, in vitro MT binding/bundling assays, site-directed mutagenesis","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with SAXS and cryo-EM structural validation, in vitro reconstitution of MT binding, and mutagenesis of active residues","pmids":["27477386"],"is_preprint":false},{"year":2015,"finding":"A short splice isoform of CEP135 (CEP135mini) represses centriole duplication by limiting centriolar localization of CEP135full binding partners SAS-6 and CPAP, and pericentriolar localization of γ-tubulin. CEP135mini and CEP135full have distinct and complementary centrosomal localizations during the cell cycle; CEP135mini decreases from centrosomes at anaphase onset, which is proposed to allow new centriole assembly.","method":"Isoform-specific overexpression and knockdown, immunofluorescence microscopy, cell cycle staging","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression and knockdown of specific isoforms with defined molecular readouts (SAS-6, CPAP, γ-tubulin localization), single lab","pmids":["26412126"],"is_preprint":false},{"year":2019,"finding":"The ratio of full-length CEP135 (CEP135full) to CEP135mini is increased in breast cancer cell lines with high centrosome amplification. Inducing expression of CEP135full increases centrosome amplification frequency, multipolar spindles, anaphase-lagging chromosomes, and micronuclei; inducing CEP135mini reduces centrosome number. The differential isoform expression is regulated by alternative polyadenylation. Directed mutations near the CEP135mini alternative polyadenylation signal reduce the CEP135full:mini ratio and decrease centrosome amplification.","method":"Isoform-specific induction in breast cancer cell lines, immunofluorescence for centrosome number and spindle phenotypes, genome editing of polyadenylation signal","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic manipulation of isoform ratio with defined cellular phenotypes, single lab, multiple orthogonal readouts","pmids":["30811267"],"is_preprint":false},{"year":2022,"finding":"CEP135 loss-of-function (CRISPR knockout) in human cells causes compromised PCM recruitment, reduced MTOC function, and premature centrosome splitting with imbalanced PCMs during interphase. However, defective CEP135 KO centrosomes compensate during mitosis to establish balanced spindle poles, allowing unperturbed mitosis and regular cell proliferation. CEP135 was also found to form a complex with centriolar satellite proteins SSX2IP and WDR8 before centrosome assembly.","method":"CRISPR knockout, immunofluorescence microscopy, co-immunoprecipitation","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complete genetic KO with quantitative phenotypic analysis across cell cycle stages and co-IP for complex identification, single lab","pmids":["35406752"],"is_preprint":false},{"year":2022,"finding":"In Chlamydomonas, Bld10p/Cep135 connects cartwheel spokes to triplet microtubules and determines the inter-triplet distance in the centriole, thereby regulating the number of triplet microtubules in a cartwheel-independent manner. Truncated Bld10p in cartwheel-deficient centrioles significantly reduces the inter-triplet distance and frequently generates eight-microtubule centrioles. Immunoelectron microscopy localized hemagglutinin-tagged Bld10p along two lines connecting adjacent triplets, corresponding to crosslinking structures identified by conventional and cryo-EM.","method":"Chlamydomonas mutant analysis, immunoelectron microscopy with HA epitope tagging, conventional and cryo-electron microscopy","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM and immunoEM structural localization combined with genetic mutant analysis demonstrating altered triplet number, multiple orthogonal methods","pmids":["36093892"],"is_preprint":false},{"year":2025,"finding":"LZTS2 negatively regulates centrosomal CEP135 levels. Depletion of LZTS2 increases microtubule nucleation at the centrosome, and this effect is dependent on CEP135 since depletion of LZTS2 partially rescues impaired centrosome microtubule nucleation caused by CEP135 knockdown.","method":"RNAi knockdown of LZTS2 and CEP135, fluorescence microscopy for centrosomal CEP135 levels and microtubule nucleation assays, epistasis analysis","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with quantitative imaging readouts, single lab","pmids":["40521914"],"is_preprint":false},{"year":2025,"finding":"CEP135 is a component of a luminal ring network at the distal centriole that includes C2CD3/SFI1/centrin-2/CEP135/NA14. C2CD3 depletion destabilizes this luminal ring network, placing CEP135 within an architectural scaffold at the distal centriole lumen connected to the distal microtubule cap and appendages.","method":"Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, cryo-electron tomography, RNAi depletion","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-ET and U-ExM provide high-resolution structural evidence placing CEP135 in the luminal ring, but CEP135's specific role within this complex is inferred from C2CD3 depletion rather than direct CEP135 manipulation; preprint","pmids":["bio_10.1101_2025.06.17.660204"],"is_preprint":true},{"year":2025,"finding":"CEP135 interacts with spermatogenic proteins SPATA6 and AKAP3, regulating their expression and stability. Conditional knockout of Cep135 in premeiotic germ cells (Stra8-Cre) causes defects in acrosome formation, flagellum structure, and head-to-tail connections during spermatogenesis, leading to oligoasthenoteratozoospermia and male infertility, without affecting premeiosis.","method":"Conditional knockout (Stra8-Cre × Cep135flox/flox), scanning and transmission electron microscopy, proteomics, co-immunoprecipitation","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined structural phenotypes by EM and co-IP identifying binding partners, single lab","pmids":["40095067"],"is_preprint":false},{"year":2023,"finding":"CEP135 promotes endothelial cell migration by mediating centrosome polarization and microtubule stability. CEP135 siRNA inhibits in vivo angiogenesis. CEP135 affects spindle orientation and mediates cell cycle progression in endothelial cells. A tubulin turbidity assay confirmed CEP135 promotes microtubule stabilization.","method":"siRNA knockdown, tube formation assay, in vivo angiogenesis assay, wound healing and transwell migration assays, tubulin turbidity assay, flow cytometry","journal":"Frontiers in bioscience (Landmark edition)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, primarily phenotypic readouts with limited mechanistic resolution; tubulin turbidity assay provides some biochemical support","pmids":["38062802"],"is_preprint":false}],"current_model":"CEP135 is a conserved coiled-coil centrosomal protein that functions as a structural linker between the SAS-6-based cartwheel hub and the outer triplet microtubules of the centriole; its N-terminal two-stranded coiled-coil domain (residues 96–108, containing key lysines) directly binds and bundles microtubules, while its C-terminus binds hSAS-6, and its N-terminal domain binds CPAP to promote centriole elongation and a separate region interacts with C-NAP1 (whose Nek2-dependent phosphorylation releases it from CEP135 to trigger centrosome disjunction at mitotic entry) and the dynactin subunit p50/dynamitin for MTOC maintenance; a short antagonistic splice isoform (CEP135mini) represses centriole duplication by competing with full-length CEP135 for centriolar recruitment of SAS-6, CPAP, and γ-tubulin, and dysregulation of this isoform balance drives centrosome amplification in cancer cells."},"narrative":{"mechanistic_narrative":"CEP135 is a conserved coiled-coil centriolar protein that acts as a structural linker between the inner SAS-6 cartwheel hub and the outer triplet microtubules, organizing centriole architecture and centrosomal microtubule nucleation throughout the cell cycle [PMID:11781336, PMID:23511974]. Its N-terminal domain forms a parallel two-stranded coiled coil that directly binds tubulin and microtubules and bundles them through a 13-residue lysine-containing site (residues 96–108), while its C-terminus binds hSAS-6 and its N-terminus binds the microcephaly protein CPAP to drive centriole elongation; loss of these interactions yields short centrioles with abnormal triplet number and dominant-negative blockade of centriole assembly [PMID:23511974, PMID:27477386]. Across Drosophila, Tetrahymena, and Chlamydomonas, CEP135/Bld10 connects cartwheel spokes to triplet microtubules, sets inter-triplet spacing and triplet number, and stabilizes basal bodies against mechanical force [PMID:23115304, PMID:22976301, PMID:36093892]. CEP135 additionally serves as a centriolar platform for C-NAP1, whose Nek2-dependent phosphorylation releases it from CEP135 to trigger centrosome disjunction at mitotic entry, and binds the dynactin subunit p50/dynamitin to support PCM and γ-tubulin retention and MTOC function [PMID:14983524, PMID:18851962, PMID:24695856]. A short antagonistic splice isoform, CEP135mini, represses centriole duplication by competing for centriolar recruitment of SAS-6, CPAP, and γ-tubulin, and an increased CEP135full:mini ratio—set by alternative polyadenylation—drives centrosome amplification and mitotic errors in breast cancer cells [PMID:26412126, PMID:30811267]. Conditional loss in germ cells causes acrosome, flagellar, and head-to-tail connection defects leading to male infertility, linking CEP135 to spermatogenesis [PMID:40095067].","teleology":[{"year":2000,"claim":"Establishing whether Cep135 is itself a structural building block, EM of overexpressed protein showed it self-assembles into filamentous polymers, framing it as an intrinsic structural component rather than a passive marker.","evidence":"Tagged full-length and truncated construct expression in CHO/Sf9 cells with electron microscopy","pmids":["10842375"],"confidence":"Medium","gaps":["Polymers were seen on overexpression, not in endogenous centrioles","No partner or microtubule-linkage role yet defined"]},{"year":2002,"claim":"To define the cellular role of the 135-kDa centrosomal protein, RNAi and deletion-construct overexpression established that Cep135 organizes centrosomal microtubules and carries three independent centrosome-targeting domains.","evidence":"Monoclonal antibody identification, RNAi, deletion overexpression, immunofluorescence","pmids":["11781336"],"confidence":"High","gaps":["Molecular partners unknown","No structural mechanism for microtubule organization"]},{"year":2004,"claim":"Identifying a functional partner, a yeast two-hybrid screen mapped a direct Cep135 C-terminus–p50/dynamitin interaction required for centrosomal targeting of p50 and retention of γ-tubulin and pericentrin.","evidence":"Yeast two-hybrid, co-IP, domain-mapped immunostaining, RNAi in CHO cells","pmids":["14983524"],"confidence":"High","gaps":["Link between dynactin recruitment and centriole structure unresolved","No in vitro reconstitution"]},{"year":2008,"claim":"Addressing centrosome cohesion, depletion and mutant studies showed CEP135 is a platform required for centriolar localization of C-NAP1, with loss causing premature centrosome splitting.","evidence":"RNAi, mutant overexpression, immunofluorescence","pmids":["18851962"],"confidence":"Medium","gaps":["Direct CEP135–C-NAP1 binding not yet biochemically mapped","Regulation of the interaction unknown"]},{"year":2012,"claim":"Cross-species genetics defined the conserved structural role: CEP135/Bld10 nucleates and stabilizes central-pair and triplet microtubules, links inner and outer centriolar components, and maintains basal body integrity against ciliary force.","evidence":"Drosophila and Tetrahymena genetics, electron tomography, SIM, EM, MT-binding assays","pmids":["22898782","23115304","22976301"],"confidence":"High","gaps":["Human counterpart of these structural roles not yet shown","Molecular determinants of inner-outer connection unmapped"]},{"year":2013,"claim":"Defining the core human mechanism, in vitro binding and dominant-negative assays established CEP135 as the linker bridging the hSAS-6 cartwheel hub to outer microtubules and to CPAP-driven centriole elongation; KO studies showed it restrains centrosome reduplication and preserves centriole structure.","evidence":"Co-IP, in vitro binding, RNAi, domain-mutant overexpression, EM; gene disruption in DT40 cells","pmids":["23511974","23864714"],"confidence":"High","gaps":["Atomic basis of microtubule binding not yet resolved","How linker function integrates with reduplication control unclear"]},{"year":2014,"claim":"Mechanism of cohesion control and asymmetry was clarified: Nek2 multisite phosphorylation of C-Nap1 disrupts its mitotic binding to Cep135 to drive disjunction, and Drosophila Bld10 mediates Polo shedding to establish centrosome asymmetry.","evidence":"In vitro kinase assays, endogenous co-IP across cell cycle, phosphomutants; Drosophila mutant live imaging","pmids":["24695856","24954048"],"confidence":"High","gaps":["Whether Polo shedding role is conserved in humans untested","Phosphosite-level control of CEP135 itself unknown"]},{"year":2015,"claim":"Discovery of an antagonistic regulatory layer: a short CEP135mini isoform with distinct cell-cycle localization represses centriole duplication by limiting recruitment of SAS-6, CPAP, and γ-tubulin.","evidence":"Isoform-specific overexpression/knockdown, immunofluorescence, cell-cycle staging","pmids":["26412126"],"confidence":"Medium","gaps":["Direct competition mechanism not biochemically dissected","Single-lab finding"]},{"year":2016,"claim":"Structural work resolved the microtubule-binding mechanism: the N-terminal 158 residues form a parallel two-stranded coiled coil that binds and bundles microtubules via a lysine-rich 13-residue site.","evidence":"X-ray crystallography, SAXS, cryo-EM, in vitro MT binding/bundling, mutagenesis","pmids":["27477386"],"confidence":"High","gaps":["Structure of SAS-6 and CPAP binding regions not solved","Full-length architecture unknown"]},{"year":2019,"claim":"Linking isoform balance to disease, manipulating the CEP135full:mini ratio (set by alternative polyadenylation) demonstrated that excess full-length drives centrosome amplification and mitotic errors in breast cancer cells.","evidence":"Isoform induction, centrosome/spindle phenotyping, polyadenylation-signal genome editing","pmids":["30811267"],"confidence":"Medium","gaps":["Causal role in tumorigenesis in vivo untested","Upstream control of polyadenylation choice unknown"]},{"year":2022,"claim":"CRISPR KO and complex identification refined the picture: CEP135 supports PCM recruitment and MTOC function and forms a complex with satellite proteins SSX2IP and WDR8, though mitotic compensation permits viable proliferation.","evidence":"CRISPR KO, immunofluorescence, co-IP","pmids":["35406752"],"confidence":"Medium","gaps":["Nature of mitotic compensation undefined","Functional role of SSX2IP/WDR8 complex unmapped"]},{"year":2022,"claim":"Cryo-EM and immunoEM in Chlamydomonas showed Bld10p/Cep135 crosslinks adjacent triplets and sets inter-triplet distance, controlling triplet number independently of the cartwheel.","evidence":"Chlamydomonas mutants, immunoEM with HA tagging, conventional and cryo-EM","pmids":["36093892"],"confidence":"High","gaps":["Human conservation of inter-triplet spacing role not directly shown","Binding partners on the triplet side unidentified"]},{"year":2025,"claim":"Newer studies extended CEP135 function to upstream regulation (LZTS2 limits centrosomal CEP135 and MT nucleation), a distal luminal ring scaffold, and a tissue role in spermatogenesis via SPATA6/AKAP3.","evidence":"RNAi epistasis with imaging; U-ExM and cryo-ET; conditional germ-cell KO with EM, proteomics, co-IP","pmids":["40521914","40095067","bio_10.1101_2025.06.17.660204"],"confidence":"Medium","gaps":["CEP135's direct role in the luminal ring inferred from C2CD3 depletion, not CEP135 manipulation (preprint)","Mechanism of LZTS2 regulation of CEP135 unknown","How germ-cell partners connect to centriolar structural role unresolved"]},{"year":null,"claim":"How CEP135's distinct binding interfaces (SAS-6, CPAP, C-NAP1, p50, satellite and luminal-ring partners) are spatially and temporally coordinated on a single molecule to build, link, and maintain the centriole remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structure integrating multiple interaction domains","Order of assembly and competition among partners undefined","In vivo disease relevance of isoform imbalance untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4,7,11,19]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,6,15]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,3,7]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,2,7,14]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[4,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,11]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[7,12,15]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8,9,10]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[18]}],"complexes":["CEP135–SSX2IP–WDR8 complex","distal centriole luminal ring (C2CD3/SFI1/centrin-2/CEP135/NA14)"],"partners":["SASS6","CPAP","CEP250","DCTN2","SSX2IP","WDR8","SPATA6","AKAP3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q66GS9","full_name":"Centrosomal protein of 135 kDa","aliases":["Centrosomal protein 4"],"length_aa":1140,"mass_kda":133.5,"function":"Centrosomal microtubule-binding protein involved in centriole biogenesis (PubMed:27477386). Acts as a scaffolding protein during early centriole biogenesis. Required for the targeting of centriole satellite proteins to centrosomes such as of PCM1, SSX2IP and CEP290 and recruitment of WRAP73 to centrioles. Also required for centriole-centriole cohesion during interphase by acting as a platform protein for CEP250 at the centriole. Required for the recruitment of CEP295 to the proximal end of new-born centrioles at the centriolar microtubule wall during early S phase in a PLK4-dependent manner (PubMed:27185865)","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole","url":"https://www.uniprot.org/uniprotkb/Q66GS9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CEP135","classification":"Not Classified","n_dependent_lines":161,"n_total_lines":1208,"dependency_fraction":0.13327814569536423},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000174799","cell_line_id":"CID000202","localizations":[{"compartment":"centrosome","grade":3}],"interactors":[{"gene":"ATP6V1D","stoichiometry":0.2},{"gene":"NME2;NME1-NME2;NME1","stoichiometry":0.2},{"gene":"MAPRE1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000202","total_profiled":1310},"omim":[{"mim_id":"620217","title":"CENTROSOMAL PROTEIN, 44-KD; CEP44","url":"https://www.omim.org/entry/620217"},{"mim_id":"617728","title":"CENTROSOMAL PROTEIN, 295-KD; CEP295","url":"https://www.omim.org/entry/617728"},{"mim_id":"614673","title":"MICROCEPHALY 8, PRIMARY, AUTOSOMAL RECESSIVE; MCPH8","url":"https://www.omim.org/entry/614673"},{"mim_id":"613447","title":"SPINDLE- AND CENTRIOLE-ASSOCIATED PROTEIN 1; SPICE1","url":"https://www.omim.org/entry/613447"},{"mim_id":"613446","title":"CENTROSOMAL PROTEIN, 120-KD; CEP120","url":"https://www.omim.org/entry/613446"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mid piece","reliability":"Approved"},{"location":"Principal piece","reliability":"Approved"},{"location":"Acrosome","reliability":"Additional"},{"location":"Annulus","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CEP135"},"hgnc":{"alias_symbol":["FLJ13621"],"prev_symbol":["KIAA0635","CEP4"]},"alphafold":{"accession":"Q66GS9","domains":[{"cath_id":"-","chopping":"2-139","consensus_level":"medium","plddt":83.1703,"start":2,"end":139},{"cath_id":"-","chopping":"1051-1090","consensus_level":"medium","plddt":56.4405,"start":1051,"end":1090},{"cath_id":"1.20.5","chopping":"264-427","consensus_level":"medium","plddt":81.9886,"start":264,"end":427}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q66GS9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q66GS9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q66GS9-F1-predicted_aligned_error_v6.png","plddt_mean":76.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CEP135","jax_strain_url":"https://www.jax.org/strain/search?query=CEP135"},"sequence":{"accession":"Q66GS9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q66GS9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q66GS9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q66GS9"}},"corpus_meta":[{"pmid":"23511974","id":"PMC_23511974","title":"Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly.","date":"2013","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/23511974","citation_count":166,"is_preprint":false},{"pmid":"22521416","id":"PMC_22521416","title":"A truncating mutation of CEP135 causes primary microcephaly and disturbed centrosomal function.","date":"2012","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22521416","citation_count":138,"is_preprint":false},{"pmid":"11781336","id":"PMC_11781336","title":"Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.","date":"2002","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11781336","citation_count":119,"is_preprint":false},{"pmid":"28866084","id":"PMC_28866084","title":"A homozygous CEP135 mutation is associated with multiple morphological abnormalities of the sperm flagella 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Appendages","date":"2025-06-18","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.17.660204","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.28.620588","title":"Interactions of N- and C-terminal parts of Ana1 permitting centriole duplication but not elongation","date":"2024-10-31","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.28.620588","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.26.661826","title":"Interactome Analysis of the CC2D1A Scaffold Reveals Novel Neuronal Interactions and a Postsynaptic Role","date":"2025-06-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.26.661826","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16837,"output_tokens":5520,"usd":0.066656,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13892,"output_tokens":4467,"usd":0.090567,"stage2_stop_reason":"end_turn"},"total_usd":0.157223,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"Cep135 is a 135-kDa coiled-coil centrosomal protein that localizes to the centrosome throughout the cell cycle, independent of the microtubule network. It distributes in association with electron-dense pericentriolar material. Three independent centrosome-targeting domains were identified by deletion construct analysis. Overexpression or RNAi suppression both caused disorganization of interphase and mitotic spindle microtubules, establishing a role in centrosomal microtubule organization.\",\n      \"method\": \"Monoclonal antibody identification, deletion construct overexpression, RNA interference, immunofluorescence microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (RNAi knockdown, overexpression of deletion constructs, immunofluorescence localization), replicated across conditions in a single rigorous study\",\n      \"pmids\": [\"11781336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Overexpressed Cep135 assembles into filamentous polymers and whorl-like particles composed of ~7 nm parallel dense lines both at the centrosome and in the cytoplasm, with the polymer architecture dependent on specific domains of the protein, indicating Cep135 is a structural self-assembling component of the centrosome.\",\n      \"method\": \"Transient transfection of HA/GFP-tagged full-length and truncated constructs in CHO cells and baculovirus expression in Sf9 cells, electron microscopy\",\n      \"journal\": \"Microscopy research and technique\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct structural visualization by EM with domain-specific constructs, single lab\",\n      \"pmids\": [\"10842375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cep135 directly interacts with the p50 dynactin subunit (dynamitin) via the C-terminal sequence of Cep135 binding the central domain of p50. This interaction is required for centrosomal targeting of p50; exogenous p50 lacking the Cep135-binding domain failed to localize to the centrosome. Altered levels of either protein displaced the other as well as γ-tubulin and pericentrin, leading to microtubule disorganization.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, immunostaining of co-expressed binding domains in CHO cells, RNAi, overexpression\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid discovery confirmed by co-IP and domain-mapping with functional consequences (centrosome targeting, microtubule organization), multiple orthogonal methods\",\n      \"pmids\": [\"14983524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CEP135 acts as a platform protein for C-NAP1 at the centriole. Depletion of CEP135 caused premature centrosome splitting accompanied by specific reduction of centrosomal C-NAP1 levels. Ectopic expression of CEP135 mutant proteins also caused centrosome splitting with reduced centrosomal C-NAP1, establishing CEP135 as required for C-NAP1 centriolar localization.\",\n      \"method\": \"RNAi knockdown, overexpression of CEP135 mutants, immunofluorescence microscopy\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi and dominant-negative overexpression with defined phenotypic readout, single lab\",\n      \"pmids\": [\"18851962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CEP135/Bld10 can bind and stabilize microtubules and is required for the early steps of central microtubule pair formation in Drosophila flagella. Assembly of the central MT pair begins prior to meiotic divisions with nucleation of a singlet MT within the basal body, and BLD10/CEP135 is essential for this early nucleation step.\",\n      \"method\": \"Drosophila genetic analysis, electron microscopy of spermatogenesis, MT binding/stabilization assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with defined ultrastructural phenotype plus biochemical MT binding assay, multiple orthogonal methods\",\n      \"pmids\": [\"22898782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In Tetrahymena, Bld10/Cep135 is an outer cartwheel domain protein that stabilizes basal bodies to resist the forces generated by ciliary beating. Bld10 promotes stability of the A- and C-tubules of the triplet microtubules and proper positioning of the triplet MT blades. In bld10Δ cells, ciliary beating forces promote basal body disassembly, revealing a role in basal body maintenance distinct from its assembly role.\",\n      \"method\": \"Tetrahymena genetic deletion, electron microscopy, live-cell analysis of basal body dynamics\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined ultrastructural and functional phenotypes, multiple orthogonal methods (EM, live imaging)\",\n      \"pmids\": [\"23115304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In Drosophila, Cep135/Bld10 is not essential for cartwheel formation or establishing the ninefold symmetry of centrioles. However, absence of Cep135/Bld10 leads to increased centriole width and progressive cartwheel disassembly over time. Cep135/Bld10 localizes between inner (SAS-6, Ana2) and outer (Asl, DSpd-2, D-PLP) centriolar components and stabilizes the connection between these inner and outer components.\",\n      \"method\": \"Drosophila Cep135/Bld10 mutant analysis, electron tomography, 3D structured illumination microscopy (SIM)\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — electron tomography plus super-resolution microscopy with genetic loss-of-function, rigorous structural analysis\",\n      \"pmids\": [\"22976301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human CEP135 directly interacts with hSAS-6 via its carboxyl-terminus and with microtubules via its amino-terminus. CEP135 also interacts with microcephaly protein CPAP via its amino-terminal domain. CEP135 depletion perturbed centriolar localization of CPAP, blocked CPAP-induced centriole elongation, and caused abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. A CEP135 mutant lacking the hSAS-6 interaction had a dominant-negative effect on centriole assembly, establishing CEP135 as a linker between the SAS-6 cartwheel hub and outer MTs.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, RNAi depletion, overexpression of domain mutants, immunofluorescence and electron microscopy\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro binding assays with domain mapping, co-IP, functional knockdown and dominant-negative overexpression, multiple orthogonal methods\",\n      \"pmids\": [\"23511974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Disruption of Cep135 in DT40 chicken cells produces viable cells with a small decrease in centriole numbers, increased monopolar spindles, and an atypical structure in the centriole lumen by electron microscopy. Cep135 loss significantly increases centrosome amplification after S-phase arrest (hydroxyurea treatment), indicating Cep135 inhibits centrosome reduplication during S-phase delay and is required for structural integrity of centrioles.\",\n      \"method\": \"Targeted gene disruption in DT40 cells, electron microscopy, flow cytometry, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean gene KO with multiple orthogonal readouts (EM, cell cycle analysis, centrosome counting), well-controlled study\",\n      \"pmids\": [\"23864714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Nek2-mediated multisite phosphorylation of C-Nap1's C-terminal domain perturbs its interaction with Cep135. Interaction between endogenous C-Nap1 and Cep135 is specifically lost in mitosis. Phosphorylation of C-Nap1 leads to loss of oligomerization and centrosome association, and loss of Cep135 binding contributes to centrosome disjunction at mitotic entry.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation of endogenous proteins across cell cycle stages, phosphomimetic/non-phosphorylatable mutant analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay combined with endogenous co-IP across cell cycle stages and phosphomimetic mutant analysis, multiple orthogonal methods\",\n      \"pmids\": [\"24695856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The Drosophila ortholog Bld10 (Cep135) is required to establish centrosome asymmetry in neuroblasts by mediating shedding of Polo kinase from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM from the mother centrosome, generating two active MTOCs, causing spindle alignment defects, centrosome segregation errors, and incorrect retention of the older mother centrosome by neuroblasts.\",\n      \"method\": \"Drosophila bld10 mutant analysis, live imaging, immunofluorescence microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with defined molecular (Polo/PCM levels) and cellular (spindle alignment, centrosome segregation) phenotypes using multiple imaging approaches\",\n      \"pmids\": [\"24954048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The N-terminal 158 residues of human CEP135 form a parallel two-stranded coiled-coil structure. This domain binds tubulin, protofilaments, and microtubules in vitro and induces MT bundle formation. A 13-amino-acid segment (residues 96–108) represents the major MT-binding site, containing three lysine residues that contribute to the MT bundling activity.\",\n      \"method\": \"X-ray crystallography, small-angle X-ray scattering (SAXS), cryo-electron microscopy, fluorescence microscopy, in vitro MT binding/bundling assays, site-directed mutagenesis\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with SAXS and cryo-EM structural validation, in vitro reconstitution of MT binding, and mutagenesis of active residues\",\n      \"pmids\": [\"27477386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A short splice isoform of CEP135 (CEP135mini) represses centriole duplication by limiting centriolar localization of CEP135full binding partners SAS-6 and CPAP, and pericentriolar localization of γ-tubulin. CEP135mini and CEP135full have distinct and complementary centrosomal localizations during the cell cycle; CEP135mini decreases from centrosomes at anaphase onset, which is proposed to allow new centriole assembly.\",\n      \"method\": \"Isoform-specific overexpression and knockdown, immunofluorescence microscopy, cell cycle staging\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression and knockdown of specific isoforms with defined molecular readouts (SAS-6, CPAP, γ-tubulin localization), single lab\",\n      \"pmids\": [\"26412126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The ratio of full-length CEP135 (CEP135full) to CEP135mini is increased in breast cancer cell lines with high centrosome amplification. Inducing expression of CEP135full increases centrosome amplification frequency, multipolar spindles, anaphase-lagging chromosomes, and micronuclei; inducing CEP135mini reduces centrosome number. The differential isoform expression is regulated by alternative polyadenylation. Directed mutations near the CEP135mini alternative polyadenylation signal reduce the CEP135full:mini ratio and decrease centrosome amplification.\",\n      \"method\": \"Isoform-specific induction in breast cancer cell lines, immunofluorescence for centrosome number and spindle phenotypes, genome editing of polyadenylation signal\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic manipulation of isoform ratio with defined cellular phenotypes, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"30811267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CEP135 loss-of-function (CRISPR knockout) in human cells causes compromised PCM recruitment, reduced MTOC function, and premature centrosome splitting with imbalanced PCMs during interphase. However, defective CEP135 KO centrosomes compensate during mitosis to establish balanced spindle poles, allowing unperturbed mitosis and regular cell proliferation. CEP135 was also found to form a complex with centriolar satellite proteins SSX2IP and WDR8 before centrosome assembly.\",\n      \"method\": \"CRISPR knockout, immunofluorescence microscopy, co-immunoprecipitation\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complete genetic KO with quantitative phenotypic analysis across cell cycle stages and co-IP for complex identification, single lab\",\n      \"pmids\": [\"35406752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Chlamydomonas, Bld10p/Cep135 connects cartwheel spokes to triplet microtubules and determines the inter-triplet distance in the centriole, thereby regulating the number of triplet microtubules in a cartwheel-independent manner. Truncated Bld10p in cartwheel-deficient centrioles significantly reduces the inter-triplet distance and frequently generates eight-microtubule centrioles. Immunoelectron microscopy localized hemagglutinin-tagged Bld10p along two lines connecting adjacent triplets, corresponding to crosslinking structures identified by conventional and cryo-EM.\",\n      \"method\": \"Chlamydomonas mutant analysis, immunoelectron microscopy with HA epitope tagging, conventional and cryo-electron microscopy\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM and immunoEM structural localization combined with genetic mutant analysis demonstrating altered triplet number, multiple orthogonal methods\",\n      \"pmids\": [\"36093892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LZTS2 negatively regulates centrosomal CEP135 levels. Depletion of LZTS2 increases microtubule nucleation at the centrosome, and this effect is dependent on CEP135 since depletion of LZTS2 partially rescues impaired centrosome microtubule nucleation caused by CEP135 knockdown.\",\n      \"method\": \"RNAi knockdown of LZTS2 and CEP135, fluorescence microscopy for centrosomal CEP135 levels and microtubule nucleation assays, epistasis analysis\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with quantitative imaging readouts, single lab\",\n      \"pmids\": [\"40521914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CEP135 is a component of a luminal ring network at the distal centriole that includes C2CD3/SFI1/centrin-2/CEP135/NA14. C2CD3 depletion destabilizes this luminal ring network, placing CEP135 within an architectural scaffold at the distal centriole lumen connected to the distal microtubule cap and appendages.\",\n      \"method\": \"Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, cryo-electron tomography, RNAi depletion\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-ET and U-ExM provide high-resolution structural evidence placing CEP135 in the luminal ring, but CEP135's specific role within this complex is inferred from C2CD3 depletion rather than direct CEP135 manipulation; preprint\",\n      \"pmids\": [\"bio_10.1101_2025.06.17.660204\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CEP135 interacts with spermatogenic proteins SPATA6 and AKAP3, regulating their expression and stability. Conditional knockout of Cep135 in premeiotic germ cells (Stra8-Cre) causes defects in acrosome formation, flagellum structure, and head-to-tail connections during spermatogenesis, leading to oligoasthenoteratozoospermia and male infertility, without affecting premeiosis.\",\n      \"method\": \"Conditional knockout (Stra8-Cre × Cep135flox/flox), scanning and transmission electron microscopy, proteomics, co-immunoprecipitation\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined structural phenotypes by EM and co-IP identifying binding partners, single lab\",\n      \"pmids\": [\"40095067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CEP135 promotes endothelial cell migration by mediating centrosome polarization and microtubule stability. CEP135 siRNA inhibits in vivo angiogenesis. CEP135 affects spindle orientation and mediates cell cycle progression in endothelial cells. A tubulin turbidity assay confirmed CEP135 promotes microtubule stabilization.\",\n      \"method\": \"siRNA knockdown, tube formation assay, in vivo angiogenesis assay, wound healing and transwell migration assays, tubulin turbidity assay, flow cytometry\",\n      \"journal\": \"Frontiers in bioscience (Landmark edition)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, primarily phenotypic readouts with limited mechanistic resolution; tubulin turbidity assay provides some biochemical support\",\n      \"pmids\": [\"38062802\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CEP135 is a conserved coiled-coil centrosomal protein that functions as a structural linker between the SAS-6-based cartwheel hub and the outer triplet microtubules of the centriole; its N-terminal two-stranded coiled-coil domain (residues 96–108, containing key lysines) directly binds and bundles microtubules, while its C-terminus binds hSAS-6, and its N-terminal domain binds CPAP to promote centriole elongation and a separate region interacts with C-NAP1 (whose Nek2-dependent phosphorylation releases it from CEP135 to trigger centrosome disjunction at mitotic entry) and the dynactin subunit p50/dynamitin for MTOC maintenance; a short antagonistic splice isoform (CEP135mini) represses centriole duplication by competing with full-length CEP135 for centriolar recruitment of SAS-6, CPAP, and γ-tubulin, and dysregulation of this isoform balance drives centrosome amplification in cancer cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CEP135 is a conserved coiled-coil centriolar protein that acts as a structural linker between the inner SAS-6 cartwheel hub and the outer triplet microtubules, organizing centriole architecture and centrosomal microtubule nucleation throughout the cell cycle [#0, #7]. Its N-terminal domain forms a parallel two-stranded coiled coil that directly binds tubulin and microtubules and bundles them through a 13-residue lysine-containing site (residues 96–108), while its C-terminus binds hSAS-6 and its N-terminus binds the microcephaly protein CPAP to drive centriole elongation; loss of these interactions yields short centrioles with abnormal triplet number and dominant-negative blockade of centriole assembly [#7, #11]. Across Drosophila, Tetrahymena, and Chlamydomonas, CEP135/Bld10 connects cartwheel spokes to triplet microtubules, sets inter-triplet spacing and triplet number, and stabilizes basal bodies against mechanical force [#5, #6, #15]. CEP135 additionally serves as a centriolar platform for C-NAP1, whose Nek2-dependent phosphorylation releases it from CEP135 to trigger centrosome disjunction at mitotic entry, and binds the dynactin subunit p50/dynamitin to support PCM and γ-tubulin retention and MTOC function [#2, #3, #9]. A short antagonistic splice isoform, CEP135mini, represses centriole duplication by competing for centriolar recruitment of SAS-6, CPAP, and γ-tubulin, and an increased CEP135full:mini ratio—set by alternative polyadenylation—drives centrosome amplification and mitotic errors in breast cancer cells [#12, #13]. Conditional loss in germ cells causes acrosome, flagellar, and head-to-tail connection defects leading to male infertility, linking CEP135 to spermatogenesis [#18].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing whether Cep135 is itself a structural building block, EM of overexpressed protein showed it self-assembles into filamentous polymers, framing it as an intrinsic structural component rather than a passive marker.\",\n      \"evidence\": \"Tagged full-length and truncated construct expression in CHO/Sf9 cells with electron microscopy\",\n      \"pmids\": [\"10842375\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Polymers were seen on overexpression, not in endogenous centrioles\", \"No partner or microtubule-linkage role yet defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"To define the cellular role of the 135-kDa centrosomal protein, RNAi and deletion-construct overexpression established that Cep135 organizes centrosomal microtubules and carries three independent centrosome-targeting domains.\",\n      \"evidence\": \"Monoclonal antibody identification, RNAi, deletion overexpression, immunofluorescence\",\n      \"pmids\": [\"11781336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners unknown\", \"No structural mechanism for microtubule organization\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identifying a functional partner, a yeast two-hybrid screen mapped a direct Cep135 C-terminus–p50/dynamitin interaction required for centrosomal targeting of p50 and retention of γ-tubulin and pericentrin.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, domain-mapped immunostaining, RNAi in CHO cells\",\n      \"pmids\": [\"14983524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link between dynactin recruitment and centriole structure unresolved\", \"No in vitro reconstitution\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Addressing centrosome cohesion, depletion and mutant studies showed CEP135 is a platform required for centriolar localization of C-NAP1, with loss causing premature centrosome splitting.\",\n      \"evidence\": \"RNAi, mutant overexpression, immunofluorescence\",\n      \"pmids\": [\"18851962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CEP135–C-NAP1 binding not yet biochemically mapped\", \"Regulation of the interaction unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Cross-species genetics defined the conserved structural role: CEP135/Bld10 nucleates and stabilizes central-pair and triplet microtubules, links inner and outer centriolar components, and maintains basal body integrity against ciliary force.\",\n      \"evidence\": \"Drosophila and Tetrahymena genetics, electron tomography, SIM, EM, MT-binding assays\",\n      \"pmids\": [\"22898782\", \"23115304\", \"22976301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human counterpart of these structural roles not yet shown\", \"Molecular determinants of inner-outer connection unmapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining the core human mechanism, in vitro binding and dominant-negative assays established CEP135 as the linker bridging the hSAS-6 cartwheel hub to outer microtubules and to CPAP-driven centriole elongation; KO studies showed it restrains centrosome reduplication and preserves centriole structure.\",\n      \"evidence\": \"Co-IP, in vitro binding, RNAi, domain-mutant overexpression, EM; gene disruption in DT40 cells\",\n      \"pmids\": [\"23511974\", \"23864714\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic basis of microtubule binding not yet resolved\", \"How linker function integrates with reduplication control unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mechanism of cohesion control and asymmetry was clarified: Nek2 multisite phosphorylation of C-Nap1 disrupts its mitotic binding to Cep135 to drive disjunction, and Drosophila Bld10 mediates Polo shedding to establish centrosome asymmetry.\",\n      \"evidence\": \"In vitro kinase assays, endogenous co-IP across cell cycle, phosphomutants; Drosophila mutant live imaging\",\n      \"pmids\": [\"24695856\", \"24954048\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Polo shedding role is conserved in humans untested\", \"Phosphosite-level control of CEP135 itself unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery of an antagonistic regulatory layer: a short CEP135mini isoform with distinct cell-cycle localization represses centriole duplication by limiting recruitment of SAS-6, CPAP, and γ-tubulin.\",\n      \"evidence\": \"Isoform-specific overexpression/knockdown, immunofluorescence, cell-cycle staging\",\n      \"pmids\": [\"26412126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct competition mechanism not biochemically dissected\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Structural work resolved the microtubule-binding mechanism: the N-terminal 158 residues form a parallel two-stranded coiled coil that binds and bundles microtubules via a lysine-rich 13-residue site.\",\n      \"evidence\": \"X-ray crystallography, SAXS, cryo-EM, in vitro MT binding/bundling, mutagenesis\",\n      \"pmids\": [\"27477386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of SAS-6 and CPAP binding regions not solved\", \"Full-length architecture unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linking isoform balance to disease, manipulating the CEP135full:mini ratio (set by alternative polyadenylation) demonstrated that excess full-length drives centrosome amplification and mitotic errors in breast cancer cells.\",\n      \"evidence\": \"Isoform induction, centrosome/spindle phenotyping, polyadenylation-signal genome editing\",\n      \"pmids\": [\"30811267\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role in tumorigenesis in vivo untested\", \"Upstream control of polyadenylation choice unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CRISPR KO and complex identification refined the picture: CEP135 supports PCM recruitment and MTOC function and forms a complex with satellite proteins SSX2IP and WDR8, though mitotic compensation permits viable proliferation.\",\n      \"evidence\": \"CRISPR KO, immunofluorescence, co-IP\",\n      \"pmids\": [\"35406752\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nature of mitotic compensation undefined\", \"Functional role of SSX2IP/WDR8 complex unmapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cryo-EM and immunoEM in Chlamydomonas showed Bld10p/Cep135 crosslinks adjacent triplets and sets inter-triplet distance, controlling triplet number independently of the cartwheel.\",\n      \"evidence\": \"Chlamydomonas mutants, immunoEM with HA tagging, conventional and cryo-EM\",\n      \"pmids\": [\"36093892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human conservation of inter-triplet spacing role not directly shown\", \"Binding partners on the triplet side unidentified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Newer studies extended CEP135 function to upstream regulation (LZTS2 limits centrosomal CEP135 and MT nucleation), a distal luminal ring scaffold, and a tissue role in spermatogenesis via SPATA6/AKAP3.\",\n      \"evidence\": \"RNAi epistasis with imaging; U-ExM and cryo-ET; conditional germ-cell KO with EM, proteomics, co-IP\",\n      \"pmids\": [\"40521914\", \"40095067\", \"bio_10.1101_2025.06.17.660204\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CEP135's direct role in the luminal ring inferred from C2CD3 depletion, not CEP135 manipulation (preprint)\", \"Mechanism of LZTS2 regulation of CEP135 unknown\", \"How germ-cell partners connect to centriolar structural role unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CEP135's distinct binding interfaces (SAS-6, CPAP, C-NAP1, p50, satellite and luminal-ring partners) are spatially and temporally coordinated on a single molecule to build, link, and maintain the centriole remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure integrating multiple interaction domains\", \"Order of assembly and competition among partners undefined\", \"In vivo disease relevance of isoform imbalance untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 7, 11, 19]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 6, 15]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 3, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 2, 7, 14]},\n      {\"term_id\": \"GO:0005813\", \"supporting_discovery_ids\": [0, 6, 7, 15]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [7, 12, 15]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8, 9, 10]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"complexes\": [\n      \"CEP135–SSX2IP–WDR8 complex\",\n      \"distal centriole luminal ring (C2CD3/SFI1/centrin-2/CEP135/NA14)\"\n    ],\n    \"partners\": [\n      \"SASS6\",\n      \"CPAP\",\n      \"CEP250\",\n      \"DCTN2\",\n      \"SSX2IP\",\n      \"WDR8\",\n      \"SPATA6\",\n      \"AKAP3\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}