{"gene":"TG_220400","gene_symbol":"ADF","run_date":"2026-09-08T11:06:36","timeline":{"discoveries":[{"year":1997,"finding":"Recombinant T. gondii ADF purified from E. coli binds actin monomers and depolymerizes F-actin in vitro; by immunofluorescence and immunoelectron microscopy, ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane.","method":"Biochemical assay with recombinant protein (actin binding/depolymerization); immunofluorescence and immunoelectron microscopy","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro activity assay plus localization by two imaging methods in a single study","pmids":["9274866"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"TgADF acts primarily as a strong actin monomer-sequestering protein (inhibiting TgACT polymerization at very low concentrations) and is only a weak filament-severing protein, due to the absence of key F-actin binding sites conserved in canonical ADF/cofilin family members; restoration of the C-terminal F-actin binding site stabilized filament interaction but reduced net disassembly activity.","method":"Kinetic and steady-state polymerization assays, severing assays, site-directed mutagenesis to restore C-terminal F-actin binding site, in vitro biochemical assays with recombinant TgADF and TgACT","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal in vitro assays (polymerization kinetics, severing, mutagenesis) in a single rigorous study","pmids":["20042603"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Conditional knockout of TgADF in vivo leads to accumulation of actin-rich filaments, reduced gliding speed, aberrant motility patterns, loss of sustained helical gliding, and severe defects in host cell invasion and egress, establishing that TgADF is required for rapid actin filament turnover, gliding motility, and host cell invasion.","method":"Conditional knockout (DiCre or analogous system), immunofluorescence microscopy, electron microscopy, motility and invasion/egress assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple orthogonal phenotypic readouts (filament accumulation by IF and EM, motility, invasion, egress)","pmids":["21346192"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"NMR solution structure of TgADF reveals a conserved ADF/cofilin fold (central mixed β-sheet of six β-strands surrounded by three α-helices and a C-terminal helical turn) lacking the C-terminal helix α4 implicated in F-actin binding; conformational flexibility of G-actin binding surfaces underlies its high G-actin affinity (Kd = 23.81 nM by ITC); PIP2 does not affect TgADF interaction with G-actin.","method":"NMR spectroscopy, 15N-relaxation backbone dynamics, ITC (isothermal titration calorimetry)","journal":"Journal of structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional validation by ITC and dynamics analysis in a single rigorous study","pmids":["21820516"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"T. gondii deoxyribose phosphate aldolase-like protein (TgDPA) physically interacts with TgADF (confirmed by Co-IP and GST pull-down), and addition of TgDPA to TgADF lowers actin polymerization extent and enhances F-actin disassembly, indicating TgDPA enhances TgADF activity on actin filament dynamics in bradyzoites.","method":"Yeast two-hybrid screening, Co-immunoprecipitation, GST pull-down, actin polymerization and disassembly kinetic assays","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays (Co-IP + pull-down) plus functional actin kinetics, single lab","pmids":["20433874"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"Mutation of Lys-68 (a key basic residue in the disassembly site) in TgADF severely impairs organelle segregation but does not affect parasite growth or motility, whereas the equivalent mutation in PfADF1 has no detectable phenotypic effect; biochemical analyses confirmed reduced filament disassembly (but not severing) activity of lysine mutants, indicating that actin filament disassembly is essential for apicomplexan parasite development but dispensable for motility.","method":"Conditional T. gondii ADF-knockout complemented with ADF variants (TgADF Lys-68 mutant, PfADF1, PfADF1 Lys-72 mutant); growth, motility, and organelle segregation assays; biochemical actin depolymerization assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation with defined point mutants plus orthogonal biochemical and cell biological readouts across two apicomplexan species","pmids":["26157165"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"TgADF is a small (118 aa) cytoplasmic protein that maintains the ~98% monomeric actin pool in Toxoplasma gondii primarily through potent G-actin sequestering activity (Kd ~24 nM) and weak filament severing; its conserved ADF/cofilin fold lacks the C-terminal helix required for stable F-actin binding, and this structural feature underpins its sequestering-dominant mechanism; in vivo, TgADF is essential for rapid actin filament turnover, gliding motility, host cell invasion, and egress, while its filament disassembly activity (mediated by Lys-68) is additionally required for organelle segregation during parasite development; its activity can be further enhanced by the bradyzoite-enriched binding partner TgDPA."},"narrative":{"mechanistic_narrative":"TgADF is a small ADF/cofilin-family protein that governs the actin monomer/filament equilibrium in Toxoplasma gondii, maintaining rapid filament turnover required for gliding motility, host cell invasion, and egress [PMID:20042603, PMID:21346192]. It acts predominantly as a potent G-actin sequestering protein that inhibits TgACT polymerization at very low concentrations and is only a weak filament-severing protein, a mechanistic bias that arises from the absence of key F-actin binding sites otherwise conserved in canonical ADF/cofilins; experimentally restoring the C-terminal F-actin binding site stabilizes filament interaction but reduces net disassembly [PMID:20042603]. Its NMR solution structure confirms a conserved ADF/cofilin fold lacking the C-terminal α4 helix implicated in F-actin binding, with conformationally flexible G-actin binding surfaces underlying high G-actin affinity (Kd ~24 nM) [PMID:21820516]. In vivo, conditional depletion causes accumulation of actin-rich filaments and severe motility, invasion, and egress defects [PMID:21346192], and the filament-disassembly activity mediated by Lys-68 is specifically required for organelle segregation during parasite development while being dispensable for motility [PMID:26157165]. TgADF activity is enhanced by the bradyzoite-associated binding partner TgDPA, which lowers actin polymerization and promotes filament disassembly [PMID:20433874].","teleology":[{"year":1997,"claim":"Establishing that T. gondii encodes a functional actin-regulatory protein answered whether the parasite possesses cofilin-like machinery to control its actin pool.","evidence":"In vitro actin binding/depolymerization assays with recombinant protein plus immunofluorescence and immunoelectron microscopy","pmids":["9274866"],"confidence":"Medium","gaps":["Relative contribution of monomer sequestering versus severing not resolved","No structural basis for activity","No in vivo functional test"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Quantitative biochemistry resolved the mechanistic bias of TgADF, showing it is primarily a monomer sequestering protein rather than a severing enzyme, and tied this to the loss of conserved F-actin binding sites.","evidence":"Kinetic/steady-state polymerization and severing assays with recombinant TgADF and TgACT, and mutagenesis restoring the C-terminal F-actin binding site","pmids":["20042603"],"confidence":"High","gaps":["In vivo relevance of sequestering-dominant activity untested here","Structural model of missing F-actin sites not yet determined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Conditional knockout established that TgADF is physiologically required for rapid actin filament turnover and the resulting gliding motility, invasion, and egress.","evidence":"Conditional knockout with immunofluorescence, electron microscopy, and motility/invasion/egress assays","pmids":["21346192"],"confidence":"High","gaps":["Does not separate which biochemical activity (sequestering vs disassembly) drives each phenotype","No structural correlate"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"The NMR structure explained the biochemical bias structurally, showing an ADF/cofilin fold lacking the F-actin-binding α4 helix and flexible surfaces conferring high G-actin affinity.","evidence":"NMR spectroscopy with 15N-relaxation dynamics and ITC affinity measurement","pmids":["21820516"],"confidence":"High","gaps":["No structure of the TgADF–G-actin complex","PIP2 shown not to affect binding but regulatory inputs otherwise unexplored"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Identifying TgDPA as a physical partner that enhances TgADF activity revealed a mechanism for stage-specific modulation of actin dynamics in bradyzoites.","evidence":"Yeast two-hybrid, Co-IP, GST pull-down, and actin polymerization/disassembly kinetics","pmids":["20433874"],"confidence":"Medium","gaps":["In vivo consequence of the TgADF–TgDPA interaction not tested","Structural basis and stoichiometry of the interaction unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Structure-guided complementation dissociated the two activities functionally, showing that Lys-68-dependent filament disassembly is required for organelle segregation but dispensable for motility.","evidence":"Conditional TgADF knockout complemented with defined point mutants (TgADF Lys-68, PfADF1, PfADF1 Lys-72) plus growth, motility, organelle segregation, and biochemical depolymerization assays","pmids":["26157165"],"confidence":"High","gaps":["Molecular link between disassembly activity and organelle segregation unresolved","Divergent phenotype of the equivalent PfADF1 mutation not mechanistically explained"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How TgADF sequestering versus disassembly activities are spatiotemporally coordinated with the actomyosin glideosome, and how TgDPA modulation is integrated in vivo, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of TgADF bound to G-actin or F-actin","In vivo role of TgDPA-mediated enhancement untested","Upstream regulation of TgADF activity unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["TGDPA (T. GONDII DEOXYRIBOSE PHOSPHATE ALDOLASE-LIKE PROTEIN) [#4]","TGACT / ACTIN [#0, #1, #3]"],"other_free_text":["TgADF lacks the C-terminal α4 helix and conserved F-actin binding sites of canonical ADF/cofilins, biasing it toward G-actin sequestering over severing/disassembly [#1, #3].","High G-actin affinity measured by ITC (Kd ~24 nM); PIP2 does not affect G-actin interaction [#3].","Lys-68 defines the disassembly site; its mutation impairs organelle segregation without affecting motility, whereas the equivalent PfADF1 Lys-72 mutation has no detectable phenotype [#5]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"9274866","id":"PMC_9274866","title":"Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii.","date":"1997","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/9274866","citation_count":61,"is_preprint":false},{"pmid":"20042603","id":"PMC_20042603","title":"Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20042603","citation_count":55,"is_preprint":false},{"pmid":"21346192","id":"PMC_21346192","title":"Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii.","date":"2011","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21346192","citation_count":54,"is_preprint":false},{"pmid":"21820516","id":"PMC_21820516","title":"Solution structure and dynamics of ADF from Toxoplasma gondii.","date":"2011","source":"Journal of structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/21820516","citation_count":22,"is_preprint":false},{"pmid":"22248986","id":"PMC_22248986","title":"Toxoplasma gondii: protective immunity against toxoplasmosis with recombinant actin depolymerizing factor protein in BALB/c mice.","date":"2012","source":"Experimental parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/22248986","citation_count":21,"is_preprint":false},{"pmid":"26157165","id":"PMC_26157165","title":"Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.","date":"2015","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/26157165","citation_count":15,"is_preprint":false},{"pmid":"21489695","id":"PMC_21489695","title":"Immune response and protective efficacy against homologous challenge in BALB/c mice vaccinated with DNA vaccine encoding Toxoplasma gondii actin depolymerizing factor gene.","date":"2011","source":"Veterinary parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/21489695","citation_count":15,"is_preprint":false},{"pmid":"20433874","id":"PMC_20433874","title":"Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF) to enhance the actin filament dynamics in the bradyzoite stage.","date":"2010","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/20433874","citation_count":6,"is_preprint":false},{"pmid":"41455416","id":"PMC_41455416","title":"From nucleation to capping: The lifecycle of an actin filament in Toxoplasma gondii gliding.","date":"2025","source":"Veterinary parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/41455416","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6308,"output_tokens":1924,"usd":0.023892,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7156,"output_tokens":3442,"usd":0.060915,"stage2_stop_reason":"end_turn"},"total_usd":0.084807,"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\": 1997,\n      \"finding\": \"Recombinant T. gondii ADF purified from E. coli binds actin monomers and depolymerizes F-actin in vitro; by immunofluorescence and immunoelectron microscopy, ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane.\",\n      \"method\": \"Biochemical assay with recombinant protein (actin binding/depolymerization); immunofluorescence and immunoelectron microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro activity assay plus localization by two imaging methods in a single study\",\n      \"pmids\": [\"9274866\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TgADF acts primarily as a strong actin monomer-sequestering protein (inhibiting TgACT polymerization at very low concentrations) and is only a weak filament-severing protein, due to the absence of key F-actin binding sites conserved in canonical ADF/cofilin family members; restoration of the C-terminal F-actin binding site stabilized filament interaction but reduced net disassembly activity.\",\n      \"method\": \"Kinetic and steady-state polymerization assays, severing assays, site-directed mutagenesis to restore C-terminal F-actin binding site, in vitro biochemical assays with recombinant TgADF and TgACT\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal in vitro assays (polymerization kinetics, severing, mutagenesis) in a single rigorous study\",\n      \"pmids\": [\"20042603\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Conditional knockout of TgADF in vivo leads to accumulation of actin-rich filaments, reduced gliding speed, aberrant motility patterns, loss of sustained helical gliding, and severe defects in host cell invasion and egress, establishing that TgADF is required for rapid actin filament turnover, gliding motility, and host cell invasion.\",\n      \"method\": \"Conditional knockout (DiCre or analogous system), immunofluorescence microscopy, electron microscopy, motility and invasion/egress assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple orthogonal phenotypic readouts (filament accumulation by IF and EM, motility, invasion, egress)\",\n      \"pmids\": [\"21346192\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NMR solution structure of TgADF reveals a conserved ADF/cofilin fold (central mixed β-sheet of six β-strands surrounded by three α-helices and a C-terminal helical turn) lacking the C-terminal helix α4 implicated in F-actin binding; conformational flexibility of G-actin binding surfaces underlies its high G-actin affinity (Kd = 23.81 nM by ITC); PIP2 does not affect TgADF interaction with G-actin.\",\n      \"method\": \"NMR spectroscopy, 15N-relaxation backbone dynamics, ITC (isothermal titration calorimetry)\",\n      \"journal\": \"Journal of structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional validation by ITC and dynamics analysis in a single rigorous study\",\n      \"pmids\": [\"21820516\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"T. gondii deoxyribose phosphate aldolase-like protein (TgDPA) physically interacts with TgADF (confirmed by Co-IP and GST pull-down), and addition of TgDPA to TgADF lowers actin polymerization extent and enhances F-actin disassembly, indicating TgDPA enhances TgADF activity on actin filament dynamics in bradyzoites.\",\n      \"method\": \"Yeast two-hybrid screening, Co-immunoprecipitation, GST pull-down, actin polymerization and disassembly kinetic assays\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays (Co-IP + pull-down) plus functional actin kinetics, single lab\",\n      \"pmids\": [\"20433874\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Mutation of Lys-68 (a key basic residue in the disassembly site) in TgADF severely impairs organelle segregation but does not affect parasite growth or motility, whereas the equivalent mutation in PfADF1 has no detectable phenotypic effect; biochemical analyses confirmed reduced filament disassembly (but not severing) activity of lysine mutants, indicating that actin filament disassembly is essential for apicomplexan parasite development but dispensable for motility.\",\n      \"method\": \"Conditional T. gondii ADF-knockout complemented with ADF variants (TgADF Lys-68 mutant, PfADF1, PfADF1 Lys-72 mutant); growth, motility, and organelle segregation assays; biochemical actin depolymerization assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic complementation with defined point mutants plus orthogonal biochemical and cell biological readouts across two apicomplexan species\",\n      \"pmids\": [\"26157165\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"TgADF is a small (118 aa) cytoplasmic protein that maintains the ~98% monomeric actin pool in Toxoplasma gondii primarily through potent G-actin sequestering activity (Kd ~24 nM) and weak filament severing; its conserved ADF/cofilin fold lacks the C-terminal helix required for stable F-actin binding, and this structural feature underpins its sequestering-dominant mechanism; in vivo, TgADF is essential for rapid actin filament turnover, gliding motility, host cell invasion, and egress, while its filament disassembly activity (mediated by Lys-68) is additionally required for organelle segregation during parasite development; its activity can be further enhanced by the bradyzoite-enriched binding partner TgDPA.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgADF is a small ADF/cofilin-family protein that governs the actin monomer/filament equilibrium in Toxoplasma gondii, maintaining rapid filament turnover required for gliding motility, host cell invasion, and egress [#1, #2]. It acts predominantly as a potent G-actin sequestering protein that inhibits TgACT polymerization at very low concentrations and is only a weak filament-severing protein, a mechanistic bias that arises from the absence of key F-actin binding sites otherwise conserved in canonical ADF/cofilins; experimentally restoring the C-terminal F-actin binding site stabilizes filament interaction but reduces net disassembly [#1]. Its NMR solution structure confirms a conserved ADF/cofilin fold lacking the C-terminal \\u03b14 helix implicated in F-actin binding, with conformationally flexible G-actin binding surfaces underlying high G-actin affinity (Kd ~24 nM) [#3]. In vivo, conditional depletion causes accumulation of actin-rich filaments and severe motility, invasion, and egress defects [#2], and the filament-disassembly activity mediated by Lys-68 is specifically required for organelle segregation during parasite development while being dispensable for motility [#5]. TgADF activity is enhanced by the bradyzoite-associated binding partner TgDPA, which lowers actin polymerization and promotes filament disassembly [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that T. gondii encodes a functional actin-regulatory protein answered whether the parasite possesses cofilin-like machinery to control its actin pool.\",\n      \"evidence\": \"In vitro actin binding/depolymerization assays with recombinant protein plus immunofluorescence and immunoelectron microscopy\",\n      \"pmids\": [\n        \"9274866\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Relative contribution of monomer sequestering versus severing not resolved\",\n        \"No structural basis for activity\",\n        \"No in vivo functional test\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Quantitative biochemistry resolved the mechanistic bias of TgADF, showing it is primarily a monomer sequestering protein rather than a severing enzyme, and tied this to the loss of conserved F-actin binding sites.\",\n      \"evidence\": \"Kinetic/steady-state polymerization and severing assays with recombinant TgADF and TgACT, and mutagenesis restoring the C-terminal F-actin binding site\",\n      \"pmids\": [\n        \"20042603\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo relevance of sequestering-dominant activity untested here\",\n        \"Structural model of missing F-actin sites not yet determined\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Conditional knockout established that TgADF is physiologically required for rapid actin filament turnover and the resulting gliding motility, invasion, and egress.\",\n      \"evidence\": \"Conditional knockout with immunofluorescence, electron microscopy, and motility/invasion/egress assays\",\n      \"pmids\": [\n        \"21346192\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Does not separate which biochemical activity (sequestering vs disassembly) drives each phenotype\",\n        \"No structural correlate\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The NMR structure explained the biochemical bias structurally, showing an ADF/cofilin fold lacking the F-actin-binding \\u03b14 helix and flexible surfaces conferring high G-actin affinity.\",\n      \"evidence\": \"NMR spectroscopy with 15N-relaxation dynamics and ITC affinity measurement\",\n      \"pmids\": [\n        \"21820516\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure of the TgADF\\u2013G-actin complex\",\n        \"PIP2 shown not to affect binding but regulatory inputs otherwise unexplored\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying TgDPA as a physical partner that enhances TgADF activity revealed a mechanism for stage-specific modulation of actin dynamics in bradyzoites.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, GST pull-down, and actin polymerization/disassembly kinetics\",\n      \"pmids\": [\n        \"20433874\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"In vivo consequence of the TgADF\\u2013TgDPA interaction not tested\",\n        \"Structural basis and stoichiometry of the interaction unknown\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Structure-guided complementation dissociated the two activities functionally, showing that Lys-68-dependent filament disassembly is required for organelle segregation but dispensable for motility.\",\n      \"evidence\": \"Conditional TgADF knockout complemented with defined point mutants (TgADF Lys-68, PfADF1, PfADF1 Lys-72) plus growth, motility, organelle segregation, and biochemical depolymerization assays\",\n      \"pmids\": [\n        \"26157165\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular link between disassembly activity and organelle segregation unresolved\",\n        \"Divergent phenotype of the equivalent PfADF1 mutation not mechanistically explained\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        },\n        {\n          \"species\": \"Plasmodium falciparum\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TgADF sequestering versus disassembly activities are spatiotemporally coordinated with the actomyosin glideosome, and how TgDPA modulation is integrated in vivo, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structure of TgADF bound to G-actin or F-actin\",\n        \"In vivo role of TgDPA-mediated enhancement untested\",\n        \"Upstream regulation of TgADF activity unknown\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\n        \"term_id\": \"GO:0003785\",\n        \"supporting_discovery_ids\": [\n          0,\n          1,\n          3\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      },\n      {\n        \"term_id\": \"GO:0051014\",\n        \"supporting_discovery_ids\": [\n          0,\n          1,\n          5\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      },\n      {\n        \"term_id\": \"GO:0030042\",\n        \"supporting_discovery_ids\": [\n          1,\n          5\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0005737\",\n        \"supporting_discovery_ids\": [\n          0\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      },\n      {\n        \"term_id\": \"GO:0009898\",\n        \"supporting_discovery_ids\": [\n          0\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"GO:0030041\",\n        \"supporting_discovery_ids\": [\n          1,\n          2,\n          3\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      },\n      {\n        \"term_id\": \"GO:0071965\",\n        \"supporting_discovery_ids\": [\n          2\n        ],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TgDPA (T. gondii deoxyribose phosphate aldolase-like protein) [#4]\",\n      \"TgACT / actin [#0, #1, #3]\"\n    ],\n    \"other_free_text\": [\n      \"TgADF lacks the C-terminal \\u03b14 helix and conserved F-actin binding sites of canonical ADF/cofilins, biasing it toward G-actin sequestering over severing/disassembly [#1, #3].\",\n      \"High G-actin affinity measured by ITC (Kd ~24 nM); PIP2 does not affect G-actin interaction [#3].\",\n      \"Lys-68 defines the disassembly site; its mutation impairs organelle segregation without affecting motility, whereas the equivalent PfADF1 Lys-72 mutation has no detectable phenotype [#5].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_220400","name":"ADF","product":"actin depolymerizing factor ADF","uniprot_acc":"S8GGZ6","chromosome":"TGME49_chrV","start_pos":352248},{"strain":"GT1","gene_id":"TGGT1_220400","name":null,"product":"actin depolymerizing factor ADF","uniprot_acc":null,"chromosome":"TGGT1_chrV","start_pos":254875},{"strain":"RH88","gene_id":"TGRH88_023730","name":null,"product":"actin depolymerizing factor ADF","uniprot_acc":null,"chromosome":"CM023086","start_pos":326431},{"strain":"VEG","gene_id":"TGVEG_220400","name":null,"product":"actin depolymerizing factor ADF","uniprot_acc":"B9Q2C8","chromosome":"TGVEG_chrV","start_pos":272754}]}