{"gene":"TG_227650","gene_symbol":"EB1","run_date":"2026-09-08T11:06:46","timeline":{"discoveries":[{"year":2015,"finding":"TgEB1 is a microtubule plus end-binding protein that, due to a C-terminal nuclear localization signal (NLS), resides in the nucleoplasm during interphase and associates with spindle microtubules during mitosis; it also associates with subpellicular microtubules at the growing end of daughter buds toward the completion of karyokinesis.","method":"Live imaging, endogenous tagging, identification of C-terminal NLS domain","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional domain identification (NLS) and cell-cycle-stage correlation, single lab with multiple orthogonal approaches","pmids":["26466679"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"Depletion of TgEB1 results in escalated disintegration of kinetochore clustering, establishing that TgEB1 is required for mitotic integrity and accurate chromosome segregation.","method":"Conditional depletion / knockdown of TgEB1 with kinetochore localization readout","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (kinetochore clustering defect), single lab with multiple supporting methods","pmids":["26466679"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"Overexpression of a high-MT-affinity TgEB1 mutant promotes formation of overstabilized microtubule bundles, causing avulsion of tightly clustered kinetochores, demonstrating that TgEB1's MT-bundling activity must be precisely regulated for proper mitosis.","method":"Overexpression of gain-of-function TgEB1 mutant with microtubule and kinetochore phenotype analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — active-site/affinity mutant overexpression with defined structural and functional phenotype, single lab, multiple readouts","pmids":["26466679"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgEB1's microtubule association operates on the same principles in Toxoplasma and in a heterologous system (Xenopus), indicating its MT-binding mechanism is conserved.","method":"Heterologous expression in Xenopus system with MT-binding assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, heterologous system experiment supporting conserved mechanism, no independent replication","pmids":["26466679"],"is_preprint":false,"attribution":[{"species":"Xenopus","strain":""}]},{"year":2022,"finding":"In the absence of the inner core centrosomal protein TgCep250L1, TgEB1 fails to localize at the mitotic spindle and unsegregated nuclei accumulate, placing TgEB1 spindle localization downstream of the inner core of the centrosome.","method":"Conditional degradation of TgCep250L1, localization of TgEB1 by fluorescence microscopy, ultrastructure expansion microscopy","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — epistasis via conditional knockdown with direct localization readout, single lab, single study","pmids":["36069445"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgEB1 is a nuclear localization signal-bearing microtubule plus end-binding protein that shuttles from the nucleoplasm during interphase to the intranuclear spindle during mitosis—a process dependent on the inner core of the centrosome (via TgCep250L1)—where it bundles spindle microtubules to maintain kinetochore clustering and ensure accurate chromosome segregation; excessive MT-bundling activity (gain-of-function mutant) is sufficient to avulse kinetochores, underscoring the need for precise regulation of its MT-affinity."},"narrative":{"mechanistic_narrative":"TgEB1 is a microtubule plus end-binding protein that governs mitotic spindle integrity and accurate chromosome segregation in Toxoplasma gondii [PMID:26466679]. A C-terminal nuclear localization signal confines it to the nucleoplasm during interphase, and upon mitosis it redistributes onto the intranuclear spindle microtubules and later associates with subpellicular microtubules at the growing end of daughter buds during karyokinesis [PMID:26466679]. Its essential mitotic role is to bundle spindle microtubules so that kinetochore clustering is maintained: depletion causes disintegration of kinetochore clustering [PMID:26466679], while overexpression of a high-MT-affinity mutant overstabilizes microtubule bundles and avulses clustered kinetochores, showing that MT-bundling activity must be tightly tuned [PMID:26466679]. Recruitment of TgEB1 to the spindle is downstream of the centrosome inner core, as loss of TgCep250L1 abolishes spindle localization and yields unsegregated nuclei [PMID:36069445]. The MT-binding mechanism is conserved, operating on the same principles when expressed heterologously in Xenopus [PMID:26466679].","teleology":[{"year":2015,"claim":"Establishing where and when a microtubule plus-end binder acts across the cell cycle showed that TgEB1 is spatially partitioned by an NLS, tying its activity to specific mitotic structures.","evidence":"Live imaging and endogenous tagging with identification of a C-terminal NLS in Toxoplasma","pmids":["26466679"],"confidence":"High","gaps":["Does not identify the plus-end recognition motif or MT-binding partners","Mechanism of NLS-driven nucleoplasmic-to-spindle switch during mitosis not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Whether TgEB1 is functionally required for mitosis was answered by depletion, which disrupted kinetochore clustering and defined its role in accurate chromosome segregation.","evidence":"Conditional depletion of TgEB1 with kinetochore-clustering readout in Toxoplasma","pmids":["26466679"],"confidence":"High","gaps":["Does not establish whether the defect is direct MT-bundling loss or an indirect spindle collapse","No quantification of segregation error rate or downstream viability"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Whether MT-affinity is a regulated parameter was tested with a gain-of-function mutant, showing that excessive bundling activity is itself pathological by avulsing kinetochores.","evidence":"Overexpression of a high-MT-affinity TgEB1 mutant with microtubule and kinetochore phenotype analysis in Toxoplasma","pmids":["26466679"],"confidence":"High","gaps":["Does not identify the physiological regulator that tunes MT-affinity in wild-type cells","Structural basis of the affinity-increasing mutation not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Whether the MT-binding mechanism is species-specific or conserved was addressed by heterologous expression, indicating a conserved plus-end binding principle.","evidence":"Heterologous expression in a Xenopus system with MT-binding assay","pmids":["26466679"],"confidence":"Medium","gaps":["Single heterologous experiment, not independently replicated","Does not test whether the mitotic bundling function is conserved, only MT association"],"attribution":[{"species":"Xenopus","strain":""}]},{"year":2022,"claim":"What positions TgEB1 on the spindle was answered by centrosome inner-core epistasis, placing TgEB1 spindle recruitment downstream of TgCep250L1.","evidence":"Conditional degradation of TgCep250L1 with TgEB1 localization by fluorescence and ultrastructure expansion microscopy in Toxoplasma","pmids":["36069445"],"confidence":"Medium","gaps":["Does not establish whether recruitment is direct or via intermediate spindle-nucleating factors","Single lab, single study without reciprocal validation"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How TgEB1's MT-bundling affinity is physiologically tuned during the cell cycle, and the molecular link between the centrosome inner core and TgEB1 spindle loading, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No identified regulator of TgEB1 MT-affinity","No direct biochemical partner connecting TgCep250L1 to TgEB1","No structural model of the MT-binding interface"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["TGCEP250L1 (CENTROSOME INNER CORE; REQUIRED UPSTREAM FOR TGEB1 SPINDLE LOCALIZATION)"],"other_free_text":["Bears a C-terminal nuclear localization signal that partitions it to the nucleoplasm in interphase and permits spindle association during mitosis [#0].","Required for kinetochore clustering integrity; depletion disintegrates kinetochore clustering [#1].","MT-affinity must be precisely regulated: a gain-of-function high-affinity mutant overstabilizes MT bundles and avulses kinetochores [#2]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"26466679","id":"PMC_26466679","title":"Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation.","date":"2015","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/26466679","citation_count":34,"is_preprint":false},{"pmid":"36069445","id":"PMC_36069445","title":"Separate To Operate: the Centriole-Free Inner Core of the Centrosome Regulates the Assembly of the Intranuclear Spindle in Toxoplasma gondii.","date":"2022","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/36069445","citation_count":17,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":3763,"output_tokens":1261,"usd":0.015102,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6269,"output_tokens":2690,"usd":0.049298,"stage2_stop_reason":"end_turn"},"total_usd":0.0644,"stage1_batch_id":"msgbatch_01WokvBYTB6JLnYrpD4rXnyV","stage2_batch_id":"msgbatch_01M7hsQDvz6AqVC9DLVjQzzc","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"TgEB1 is a microtubule plus end-binding protein that, due to a C-terminal nuclear localization signal (NLS), resides in the nucleoplasm during interphase and associates with spindle microtubules during mitosis; it also associates with subpellicular microtubules at the growing end of daughter buds toward the completion of karyokinesis.\",\n      \"method\": \"Live imaging, endogenous tagging, identification of C-terminal NLS domain\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional domain identification (NLS) and cell-cycle-stage correlation, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"26466679\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Depletion of TgEB1 results in escalated disintegration of kinetochore clustering, establishing that TgEB1 is required for mitotic integrity and accurate chromosome segregation.\",\n      \"method\": \"Conditional depletion / knockdown of TgEB1 with kinetochore localization readout\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (kinetochore clustering defect), single lab with multiple supporting methods\",\n      \"pmids\": [\"26466679\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Overexpression of a high-MT-affinity TgEB1 mutant promotes formation of overstabilized microtubule bundles, causing avulsion of tightly clustered kinetochores, demonstrating that TgEB1's MT-bundling activity must be precisely regulated for proper mitosis.\",\n      \"method\": \"Overexpression of gain-of-function TgEB1 mutant with microtubule and kinetochore phenotype analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — active-site/affinity mutant overexpression with defined structural and functional phenotype, single lab, multiple readouts\",\n      \"pmids\": [\"26466679\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgEB1's microtubule association operates on the same principles in Toxoplasma and in a heterologous system (Xenopus), indicating its MT-binding mechanism is conserved.\",\n      \"method\": \"Heterologous expression in Xenopus system with MT-binding assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, heterologous system experiment supporting conserved mechanism, no independent replication\",\n      \"pmids\": [\"26466679\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Xenopus\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In the absence of the inner core centrosomal protein TgCep250L1, TgEB1 fails to localize at the mitotic spindle and unsegregated nuclei accumulate, placing TgEB1 spindle localization downstream of the inner core of the centrosome.\",\n      \"method\": \"Conditional degradation of TgCep250L1, localization of TgEB1 by fluorescence microscopy, ultrastructure expansion microscopy\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — epistasis via conditional knockdown with direct localization readout, single lab, single study\",\n      \"pmids\": [\"36069445\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgEB1 is a nuclear localization signal-bearing microtubule plus end-binding protein that shuttles from the nucleoplasm during interphase to the intranuclear spindle during mitosis—a process dependent on the inner core of the centrosome (via TgCep250L1)—where it bundles spindle microtubules to maintain kinetochore clustering and ensure accurate chromosome segregation; excessive MT-bundling activity (gain-of-function mutant) is sufficient to avulse kinetochores, underscoring the need for precise regulation of its MT-affinity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgEB1 is a microtubule plus end-binding protein that governs mitotic spindle integrity and accurate chromosome segregation in Toxoplasma gondii [#0, #1]. A C-terminal nuclear localization signal confines it to the nucleoplasm during interphase, and upon mitosis it redistributes onto the intranuclear spindle microtubules and later associates with subpellicular microtubules at the growing end of daughter buds during karyokinesis [#0]. Its essential mitotic role is to bundle spindle microtubules so that kinetochore clustering is maintained: depletion causes disintegration of kinetochore clustering [#1], while overexpression of a high-MT-affinity mutant overstabilizes microtubule bundles and avulses clustered kinetochores, showing that MT-bundling activity must be tightly tuned [#2]. Recruitment of TgEB1 to the spindle is downstream of the centrosome inner core, as loss of TgCep250L1 abolishes spindle localization and yields unsegregated nuclei [#4]. The MT-binding mechanism is conserved, operating on the same principles when expressed heterologously in Xenopus [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Establishing where and when a microtubule plus-end binder acts across the cell cycle showed that TgEB1 is spatially partitioned by an NLS, tying its activity to specific mitotic structures.\",\n      \"evidence\": \"Live imaging and endogenous tagging with identification of a C-terminal NLS in Toxoplasma\",\n      \"pmids\": [\"26466679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the plus-end recognition motif or MT-binding partners\", \"Mechanism of NLS-driven nucleoplasmic-to-spindle switch during mitosis not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Whether TgEB1 is functionally required for mitosis was answered by depletion, which disrupted kinetochore clustering and defined its role in accurate chromosome segregation.\",\n      \"evidence\": \"Conditional depletion of TgEB1 with kinetochore-clustering readout in Toxoplasma\",\n      \"pmids\": [\"26466679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not establish whether the defect is direct MT-bundling loss or an indirect spindle collapse\", \"No quantification of segregation error rate or downstream viability\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Whether MT-affinity is a regulated parameter was tested with a gain-of-function mutant, showing that excessive bundling activity is itself pathological by avulsing kinetochores.\",\n      \"evidence\": \"Overexpression of a high-MT-affinity TgEB1 mutant with microtubule and kinetochore phenotype analysis in Toxoplasma\",\n      \"pmids\": [\"26466679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the physiological regulator that tunes MT-affinity in wild-type cells\", \"Structural basis of the affinity-increasing mutation not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Whether the MT-binding mechanism is species-specific or conserved was addressed by heterologous expression, indicating a conserved plus-end binding principle.\",\n      \"evidence\": \"Heterologous expression in a Xenopus system with MT-binding assay\",\n      \"pmids\": [\"26466679\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single heterologous experiment, not independently replicated\", \"Does not test whether the mitotic bundling function is conserved, only MT association\"],\n      \"attribution\": [{\"species\": \"Xenopus\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"What positions TgEB1 on the spindle was answered by centrosome inner-core epistasis, placing TgEB1 spindle recruitment downstream of TgCep250L1.\",\n      \"evidence\": \"Conditional degradation of TgCep250L1 with TgEB1 localization by fluorescence and ultrastructure expansion microscopy in Toxoplasma\",\n      \"pmids\": [\"36069445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not establish whether recruitment is direct or via intermediate spindle-nucleating factors\", \"Single lab, single study without reciprocal validation\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TgEB1's MT-bundling affinity is physiologically tuned during the cell cycle, and the molecular link between the centrosome inner core and TgEB1 spindle loading, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No identified regulator of TgEB1 MT-affinity\", \"No direct biochemical partner connecting TgCep250L1 to TgEB1\", \"No structural model of the MT-binding interface\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008017\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Xenopus\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051010\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051011\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005876\", \"supporting_discovery_ids\": [0, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0035371\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007059\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000278\", \"supporting_discovery_ids\": [1, 2, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TgCep250L1 (centrosome inner core; required upstream for TgEB1 spindle localization)\"],\n    \"other_free_text\": [\n      \"Bears a C-terminal nuclear localization signal that partitions it to the nucleoplasm in interphase and permits spindle association during mitosis [#0].\",\n      \"Required for kinetochore clustering integrity; depletion disintegrates kinetochore clustering [#1].\",\n      \"MT-affinity must be precisely regulated: a gain-of-function high-affinity mutant overstabilizes MT bundles and avulses kinetochores [#2].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_227650","name":"EB1","product":"microtubule-associated protein EB1","uniprot_acc":"S8G6B6","chromosome":"TGME49_chrX","start_pos":861567},{"strain":"GT1","gene_id":"TGGT1_227650","name":null,"product":"putative microtubule-associated protein RP/EB family","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":771838},{"strain":"RH88","gene_id":"TGRH88_047070","name":null,"product":"putative microtubule-associated protein RP/EB family","uniprot_acc":null,"chromosome":"CM023091","start_pos":6707933},{"strain":"VEG","gene_id":"TGVEG_227650","name":null,"product":"putative microtubule-associated protein RP/EB family","uniprot_acc":"B9PMZ7","chromosome":"TGVEG_chrX","start_pos":751752}]}