{"gene":"TG_235470","gene_symbol":"MyoA","run_date":"2026-09-10T21:09:19","timeline":{"discoveries":[{"year":2002,"finding":"TgMyoA is a fast, single-headed, plus-end-directed motor that moves actin filaments in unitary steps of 5.3 nm at a velocity of 5.2 µm/s, as established by microneedle-laser trap and sliding velocity assays. TgMyoA binds a novel myosin light chain TgMLC1 and is anchored at the plasma membrane.","method":"In vitro motility assay, microneedle-laser trap (optical tweezers), biochemical characterization of purified motor complex","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro motor with biophysical single-molecule assays and kinetic characterization, multiple orthogonal methods in one rigorous study","pmids":["11980712"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"Conditional removal of MyoA using a tetracycline-inducible system caused severe impairment in host cell invasion and parasite spreading in cultured cells, and established MyoA as essential for pathogenesis in an animal model.","method":"Tetracycline-inducible conditional gene disruption (regulatable transactivator system), cell invasion assays, animal model infection","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with defined cellular and in vivo phenotypes, replicated across multiple assays","pmids":["12399593"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"Overexpression of a MyoA transgene drastically down-regulates the level of endogenous MyoA, indicating MyoA is under tight feedback regulation at the protein level.","method":"Tetracycline-inducible expression system, Western blot analysis of endogenous protein levels","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, but consistent finding with genetic manipulation and protein-level readout","pmids":["11713335"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"TgMyoA forms a protein complex with its light chain TgMLC1 and two novel proteins TgGAP45 and TgGAP50. This complex is localized to the inner membrane complex, where it is anchored by TgGAP50, an integral membrane glycoprotein. Complex assembly is spatially controlled and occurs in two stages.","method":"Co-immunoprecipitation, subcellular fractionation, immunofluorescence localization, identification of novel complex components","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, fractionation, localization with multiple orthogonal methods identifying the membrane receptor","pmids":["15123738"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"The glideosome contains MyoA, MLC1, GAP45, GAP50, and a newly identified polytopic IMC protein GAP40. GAP45 is anchored to the plasma membrane and IMC via its N- and C-terminal extremities respectively; its C-terminal region recruits MLC1-MyoA to the IMC. GAP45 is essential for gliding, invasion, and egress.","method":"Conditional knockdown, co-immunoprecipitation, immunofluorescence, complementation experiments including transgenera complementation with P. falciparum GAP45","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, conditional KD with defined phenotypes, cross-species complementation as orthogonal validation","pmids":["20951968"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"Phosphorylation of GAP45 at Ser163 and Ser167 controls the final step of MyoA motor complex assembly: phosphomimetic mutations (Ser-to-Glu) prevent association of the MyoA-MLC1-GAP45 subcomplex with the membrane anchor GAP50, while alanine mutations do not affect assembly.","method":"Mass spectrometry identification of phosphorylation sites, site-directed mutagenesis (Ser-to-Glu and Ser-to-Ala), co-immunoprecipitation","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis combined with Co-IP to show mechanism of assembly regulation, single lab but multiple orthogonal methods","pmids":["19047362"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Calcium-dependent phosphorylation of six residues across GAP45, MLC1, and MyoA correlates with invasion motor activity. A novel 15-kDa calmodulin-like protein (MyoA Essential Light Chain) associates more strongly with the invasion motor upon calcium stimulation, identifying a new regulatory component.","method":"SILAC-based quantitative LC-MS/MS phosphoproteomics, 2-DE of 32P-labeled protein extracts, calcium stimulation experiments","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative MS with SILAC, single lab, correlation of phosphorylation with motor activity","pmids":["21980283"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Post-translational modification of TgMLC1 by a small-molecule inhibitor (tachyplegin) causes decreased TgMyoA motor activity, as shown by in vitro motility assay comparing motor complexes from treated vs. untreated parasites. This established that Class XIV myosin motor activity can be modulated by PTMs on its associated light chain.","method":"In vitro motility assay with purified motor complexes, electrophoretic mobility shift, peptide mapping","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro motility assay with biochemically defined motor complexes, multiple orthogonal methods, single lab","pmids":["20084115"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgMyoA undergoes calcium-dependent phosphorylation; a small-molecule enhancer of motility (compound 130038) causes increased intracellular calcium leading to increased TgMyoA phosphorylation. Parasites expressing nonphosphorylatable mutant MyoA egressed from host cells more slowly in response to calcium ionophore, demonstrating that calcium-dependent phosphorylation of MyoA modulates myosin-driven processes.","method":"Site-directed mutagenesis of phosphorylation sites, calcium ionophore treatment, egress assays, compound treatment, intracellular calcium measurement","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of phosphorylation sites with defined functional phenotype, calcium mechanistic link established, single lab multiple methods","pmids":["24989796"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgCDPK3 phosphorylates TgMyoA at Ser21 and Ser743 in vitro. Complementation of a myoA null mutant with MyoA bearing S21A or S743A alanine mutations failed to rescue the egress delay phenotype, while phosphomimetic mutations bypassed the need for TgCDPK3. This establishes TgCDPK3-mediated phosphorylation of TgMyoA as the mechanism for rapid initiation of motility and egress.","method":"BioID proximity interaction screen, peptide array kinase assay in vitro, genetic complementation with phosphomimetic/alanine mutants, egress assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus genetic complementation with separation-of-function mutants, multiple orthogonal methods establishing writer-substrate relationship","pmids":["26544049"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"MyoA and its associated motor complex (MLC1, GAP45, Act1) are important but not absolutely essential for gliding motility and host cell invasion; conditional KO mutants remain invasive, suggesting alternative invasion mechanisms exist alongside the MyoA-glideosome.","method":"Conditional knockout of MyoA, MLC1, GAP45, Act1; invasion and motility assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional KO with quantitative phenotypic readout, single lab","pmids":["24632839"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2014,"finding":"Deletion of MyoA leads to relocalization of MyoC along the pellicle and at the apical cap, accounting for residual invasion. MyoA and MyoC glideosomes exhibit plasticity, with MyoC-glideosome providing functional compensation for loss of MyoA.","method":"Conditional knockout of MyoA, immunofluorescence localization of MyoC, invasion assays","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via conditional KO with defined localization and functional consequences, single lab","pmids":["25393004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"Two essential light chains (ELC1 and ELC2) bind the MyoA lever arm and are functionally redundant but important for invasion, egress, and motility. Ca2+ promotes intermolecular contacts between ELCs and the adjacent MLC1 to stabilize the lever arm domain, as demonstrated by point mutations predicted to ablate Ca2+ binding or the ELC-MLC1 interface.","method":"Identification of ELC2 by database search, conditional knockdown, molecular dynamics simulations, point mutagenesis, gliding motility assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with phenotypic readout combined with mutagenesis, single lab; molecular simulations not tier 1","pmids":["26374117"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgMyoA expressed in Sf9 cells requires a parasite-specific UCS-family co-chaperone (TgUNC) to be soluble and functional. The MyoA heavy chain with only MLC1 moves actin at ~1.5 µm/s, but addition of ELC1 increases speed to ~3.4 µm/s, demonstrating that two light chains bound to the lever arm are required for fast motility.","method":"Baculovirus/Sf9 heterologous expression, in vitro actin gliding motility assay, co-expression of chaperone and light chains","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro motility with defined protein components and systematic variation of light chain composition, single lab","pmids":["25231988"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"The 2.6-Å crystal structure of the TgMyoA motor domain reveals a divergent SH1/SH2 region and class-defining 'HYAG'-site polymorphism as key structural elements for force transduction and chemomechanical coupling. Hydrogen-deuterium exchange MS showed that phosphorylation-dependent enhanced motility correlates with increased local stability in specific regions of the motor. Solvent-accessible pockets differ from human myosins, supporting inhibitor design.","method":"X-ray crystallography (2.6 Å), hydrogen-deuterium exchange coupled to MS, in vitro motility assay with phosphorylation variants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by in vitro assay and HDX-MS, multiple orthogonal methods in one rigorous study","pmids":["30348763"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"MLC1 and ELC1 bind adjacent sites on the MyoA neck (residues 775–818). The 1.85-Å crystal structure of MLC1 bound to its MyoA peptide shows a bilobed architecture with MLC1 clamping around the helical peptide (Kd ~10 nM). Ca2+ coordination by an EF-hand in ELC1 and pre-binding of MLC1 cooperate to increase ELC1 affinity for the neck 49-fold, to ~12 nM. ELC1 also engages the MyoA converter subdomain.","method":"X-ray crystallography (1.85 Å), hydrogen-deuterium exchange MS, isothermal titration calorimetry, binding affinity measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with quantitative binding data by ITC and HDX-MS, multiple orthogonal methods in one rigorous study","pmids":["28972141"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"Crystal structures of ELC in trimeric sub-complexes with MyoA show that ELCs bind a conserved MyoA sequence and induce secondary structure in MyoA to stiffen its lever arm. Structural and biophysical analysis reveals that calcium binding has no influence on ELC structure.","method":"X-ray crystallography of trimeric sub-complexes, biophysical analysis (SPR/ITC)","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures of glideosome sub-complexes with biophysical validation, single lab","pmids":["33051581"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"MyoH is indispensable for translocation of the circular junction from the parasite tip to the apical IMC position; once this first step is accomplished, MyoA relays the process to enact effective gliding and invasion along the pellicle. This establishes a sequential handoff between MyoH (conoid-anchored) and MyoA (IMC-anchored) during motility.","method":"Conditional disruption of TgMyoH, immunofluorescence, invasion and egress assays, epistasis between MyoH and MyoA","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via conditional KO with defined phenotypic steps, single lab","pmids":["26760042"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Parasites with mlc1 cKO (and act1 cKO) are virtually unable to attach to a collagen-coated substrate under flow conditions while retaining residual motility at wildtype speed, demonstrating that the actomyosin system (including MyoA via MLC1) functions primarily in surface attachment rather than as the sole force generator for movement.","method":"Conditional knockout of mlc1 and act1, 3D motility assays, substrate attachment assays under flow, comparison of mutant phenotypes","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional KO with quantitative flow-attachment and motility phenotypes, single lab, multiple mutants compared","pmids":["28100223"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2014,"finding":"TgMLC1 C58 is the direct covalent target of tachypleginA and its analogues, as demonstrated by click chemistry labelling. A C58S recombinant TgMLC1 mutation is refractory to compound binding, and knock-in parasites expressing C58S show decreased sensitivity to compound-induced motility inhibition, confirming TgMLC1 as the mechanistic target mediating inhibition of TgMyoA motor activity.","method":"Click chemistry labelling with alkyne-derivatized inhibitor, site-directed mutagenesis (C58S, S57A), knock-in parasite line, 3D motility assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — covalent target identification by click chemistry plus site mutagenesis and knock-in rescue, multiple orthogonal methods","pmids":["24892871"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"A small-molecule inhibitor KNX-002 inhibits TgMyoA actin-activated ATPase activity in vitro with little effect on vertebrate myosins. A resistance mutation T130A in TgMyoA reduces compound sensitivity both in biochemical and parasite assays, validating TgMyoA as a druggable target in vivo and establishing that KNX-002 slows disease progression in mice through on-target MyoA inhibition.","method":"High-throughput screen of 50,000 compounds for ATPase inhibition, chemical mutagenesis + selection, targeted sequencing, in vitro motility assay, mouse infection model with wild-type vs. T130A parasites","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ATPase assay plus resistance mutation validation plus in vivo animal model, multiple orthogonal methods across biochemistry and cell biology","pmids":["37155705"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"MyoA accumulates around the mitochondrion in late stages of parasite division (ultrastructure expansion microscopy). Parasites lacking MyoA show defective mitochondrial morphology and delay in mitochondrion delivery to daughter buds during division, establishing a role for MyoA in organellar inheritance beyond gliding motility. Mitochondrion vesicles extracted from parasites interact with actin filaments.","method":"Ultrastructure expansion microscopy (U-ExM), conditional MyoA knockdown, mitochondrion morphology and inheritance assays, actin pulldown with mitochondrion vesicles, yeast two-hybrid (LMF1-MyoA interaction identified)","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by U-ExM tied to functional consequence in KO, single lab, multiple orthogonal methods","pmids":["39374269"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"The acto-MyoA motor directs traction force during tachyzoite helical gliding; traction force microscopy and reflection interference microscopy revealed a critical apical anchoring adhesion that stores energy via microtubule cytoskeleton and sets thrust force for helical gliding.","method":"Quantitative traction force microscopy, reflection interference microscopy, micropatterning, expansion microscopy","journal":"ACS nano","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biophysical traction force measurements with direct link to MyoA motor activity, single lab","pmids":["32432851"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"Toxoplasma gondii MyoA is a fast, single-headed, plus-end-directed class XIV unconventional myosin that forms the core of the glideosome — a pellicular actomyosin motor complex anchored to the inner membrane complex via integral membrane protein GAP50 and organized by GAP45, MLC1, and two essential light chains (ELC1/ELC2) — which powers parasite gliding motility, host cell invasion, egress, and mitochondrial inheritance; its activity is regulated by calcium-dependent phosphorylation mediated by TgCDPK3 at Ser21 and Ser743, by post-translational modifications on its light chain MLC1, and by staged cooperative assembly of the light chains on the neck/lever arm, while structural studies have defined its divergent motor domain architecture as the basis for parasite-specific small-molecule inhibition."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii MyoA is a fast, single-headed, plus-end-directed unconventional myosin that forms the mechanochemical core of the glideosome, the pellicular actomyosin motor powering parasite gliding, host-cell invasion, egress, and mitochondrial inheritance [PMID:11980712, PMID:12399593, PMID:39374269]. As purified motor it moves actin filaments in 5.3-nm unitary steps at ~5.2 µm/s and binds the novel light chain MLC1 while anchored at the plasma membrane [PMID:11980712]. Within the parasite the motor assembles into a complex of MLC1, GAP45, and the integral IMC glycoprotein GAP50, which anchors the motor to the inner membrane complex through a spatially controlled, staged assembly process [PMID:15123738, PMID:20951968]; final docking onto GAP50 is gated by GAP45 phosphorylation at Ser163/Ser167 [PMID:19047362]. Fast motility requires two light chains on the MyoA lever arm: MLC1 clamps the neck with nanomolar affinity and cooperatively promotes high-affinity binding of an essential light chain (ELC1/ELC2), whose engagement of the neck and converter subdomain stiffens the lever arm and raises gliding speed [PMID:25231988, PMID:28972141, PMID:33051581, PMID:26374117]. Motor output is tuned by calcium-dependent phosphorylation — TgCDPK3 phosphorylates MyoA at Ser21 and Ser743 to enable rapid initiation of motility and egress [PMID:24989796, PMID:26544049] — and by post-translational modification of MLC1, including covalent modification at Cys58 by the inhibitor tachyplegin [PMID:20084115, PMID:24892871]. MyoA is essential for pathogenesis and invasion in vivo, though conditional loss is partially compensated by relocalization of MyoC glideosomes [PMID:12399593, PMID:25393004]; the actomyosin system functions particularly in substrate attachment and traction-force generation during helical gliding [PMID:28100223, PMID:32432851]. Crystallographic and HDX-MS studies define a divergent motor-domain architecture with a class-defining SH1/SH2 region and drug-accessible pockets distinct from human myosins, validating MyoA as a parasite-specific small-molecule target as demonstrated by the ATPase inhibitor KNX-002 [PMID:30348763, PMID:37155705].","teleology":[{"year":2001,"claim":"Before its motor properties were known, MyoA was shown to be under tight protein-level feedback control, establishing that its abundance is homeostatically regulated.","evidence":"Tetracycline-inducible overexpression with Western blot of endogenous protein","pmids":["11713335"],"confidence":"Medium","gaps":["Mechanism of feedback down-regulation unresolved","No link to motor function established at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"The question of what kind of motor MyoA is was answered by direct biophysical reconstitution, defining it as a fast, single-headed, plus-end-directed myosin bound to a novel light chain MLC1 and localized at the plasma membrane.","evidence":"In vitro motility assay, optical-trap single-molecule measurement of purified motor complex","pmids":["11980712"],"confidence":"High","gaps":["Full light-chain complement not yet defined","Membrane anchoring proteins not yet identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"Whether the motor mattered for the parasite lifecycle was settled by conditional disruption, showing MyoA is essential for host-cell invasion, spreading, and pathogenesis in vivo.","evidence":"Tetracycline-inducible conditional gene disruption, invasion assays, animal infection model","pmids":["12399593"],"confidence":"High","gaps":["Molecular basis of the invasion defect not dissected","Did not address whether other myosins compensate"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"claim":"The architecture anchoring the motor to the parasite was defined by identifying the MyoA-MLC1-GAP45-GAP50 complex on the inner membrane complex, with GAP50 as the integral-membrane anchor and assembly occurring in stages.","evidence":"Reciprocal co-immunoprecipitation, subcellular fractionation, immunofluorescence","pmids":["15123738"],"confidence":"High","gaps":["Trigger controlling staged assembly unknown","Force transduction from motor to membrane not measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"How the final assembly step is regulated was answered by showing GAP45 phosphorylation at Ser163/Ser167 controls docking of the MyoA-MLC1-GAP45 subcomplex onto the GAP50 anchor.","evidence":"Mass spectrometry site identification, phosphomimetic/alanine mutagenesis, co-immunoprecipitation","pmids":["19047362"],"confidence":"High","gaps":["Kinase responsible for these sites not identified","Temporal coupling to invasion not shown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"The glideosome roster and membrane tethering were extended by identifying GAP40 and mapping GAP45 as the bridge that recruits MLC1-MyoA from plasma membrane to IMC, essential for gliding, invasion, and egress.","evidence":"Conditional knockdown, co-IP, immunofluorescence, cross-species complementation with P. falciparum GAP45","pmids":["20951968"],"confidence":"High","gaps":["GAP40 molecular role undefined","Quantitative contribution of tethering to force output unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"That motor activity can be chemically modulated through its light chain was shown when tachyplegin-induced modification of MLC1 reduced MyoA motility in vitro.","evidence":"In vitro motility assay of motor complexes from treated vs untreated parasites, mobility shift, peptide mapping","pmids":["20084115"],"confidence":"High","gaps":["Precise chemical adduct on MLC1 not yet localized","Mechanism linking MLC1 modification to motor slowing unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Calcium signaling was tied to motor regulation by showing calcium-dependent phosphorylation across GAP45, MLC1, and MyoA and calcium-enhanced recruitment of a new essential light chain (ELC).","evidence":"SILAC quantitative phosphoproteomics, 2-DE of 32P-labeled extracts, calcium stimulation","pmids":["21980283"],"confidence":"Medium","gaps":["Causal link between phosphorylation and activity correlative only","Responsible kinase(s) not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The two-light-chain requirement for fast motility was reconstituted heterologously, showing MyoA needs the UCS co-chaperone TgUNC for folding and that ELC1 addition to MLC1 more than doubles gliding speed.","evidence":"Baculovirus/Sf9 expression with co-chaperone and light-chain co-expression, in vitro actin gliding assays","pmids":["25231988"],"confidence":"High","gaps":["In-cell requirement for TgUNC not demonstrated","Structural basis of speed enhancement not resolved here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The apparent absolute essentiality of MyoA was revised by conditional KO showing residual invasion, implying alternative invasion mechanisms.","evidence":"Conditional knockout of MyoA/MLC1/GAP45/Act1, invasion and motility assays","pmids":["24632839"],"confidence":"Medium","gaps":["Nature of the alternative mechanism not defined in this study","Extent of compensation across strains unaddressed"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2014,"claim":"The source of residual invasion was traced to glideosome plasticity, with MyoC relocalizing along the pellicle and apical cap to compensate for MyoA loss.","evidence":"Conditional MyoA knockout, MyoC immunofluorescence localization, invasion assays","pmids":["25393004"],"confidence":"Medium","gaps":["Trigger for MyoC redistribution unknown","Whether MyoC fully substitutes for MyoA force output not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Direct functional proof that calcium-dependent MyoA phosphorylation modulates motility came from nonphosphorylatable mutants egressing more slowly under calcium ionophore.","evidence":"Site-directed mutagenesis of phosphosites, calcium ionophore treatment, egress assays, calcium measurement","pmids":["24989796"],"confidence":"High","gaps":["Kinase not identified in this study","Individual phosphosite contributions not separated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The covalent target of tachyplegin was pinned to MLC1 Cys58, establishing the mechanistic route by which the compound inhibits MyoA motor activity.","evidence":"Click chemistry labelling, C58S/S57A mutagenesis, knock-in parasites, 3D motility assay","pmids":["24892871"],"confidence":"High","gaps":["How Cys58 modification propagates to the motor domain not structurally defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"The writer of MyoA regulatory phosphorylation was identified as TgCDPK3 acting at Ser21 and Ser743, with separation-of-function mutants tying these sites to rapid motility initiation and egress.","evidence":"BioID screen, in vitro peptide-array kinase assay, complementation with phosphomimetic/alanine mutants, egress assays","pmids":["26544049"],"confidence":"High","gaps":["Structural consequence of phosphorylation on motor cycle not directly shown here","Whether other kinases contribute in vivo unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"The essential light chains ELC1 and ELC2 were shown to be redundant lever-arm components whose calcium-promoted contacts with MLC1 stabilize the lever arm and support invasion, egress, and motility.","evidence":"Database identification of ELC2, conditional knockdown, molecular dynamics, point mutagenesis, gliding assays","pmids":["26374117"],"confidence":"Medium","gaps":["Calcium-dependence of lever-arm stabilization based partly on simulation","Distinct roles of ELC1 vs ELC2 in vivo not delineated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"A sequential motor handoff was defined whereby conoid-anchored MyoH first translocates the moving junction apically before IMC-anchored MyoA relays force for pellicular gliding and invasion.","evidence":"Conditional MyoH disruption, immunofluorescence, invasion/egress assays, MyoH-MyoA epistasis","pmids":["26760042"],"confidence":"Medium","gaps":["Molecular basis of the handoff transition unresolved","How the two motors coordinate temporally not measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"The structural logic of light-chain assembly on the neck was resolved: MLC1 clamps the MyoA neck at ~10 nM and cooperatively raises ELC1 affinity 49-fold, with ELC1 also engaging the converter and its EF-hand coordinating calcium.","evidence":"X-ray crystallography (1.85 Å), HDX-MS, ITC binding measurements","pmids":["28972141"],"confidence":"High","gaps":["In-cell relevance of cooperative affinity increase not tested","Link to force transduction in the intact motor not demonstrated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"The primary function of the actomyosin system was reframed toward attachment when mlc1/act1 cKO parasites failed to adhere under flow yet retained wildtype-speed residual motility.","evidence":"Conditional mlc1/act1 knockout, 3D motility assays, substrate attachment under flow","pmids":["28100223"],"confidence":"Medium","gaps":["Force-generation versus attachment contributions not fully separated","Source of residual motility unidentified"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"claim":"The divergent motor-domain architecture underpinning both chemomechanical coupling and drug specificity was established by crystal structure, revealing a class-defining SH1/SH2 region and non-human solvent pockets, with HDX-MS linking phosphorylation to local motor stability.","evidence":"X-ray crystallography (2.6 Å), HDX-MS, in vitro motility with phosphorylation variants","pmids":["30348763"],"confidence":"High","gaps":["Full ATPase-cycle conformational states not captured","Direct inhibitor-bound structure not provided here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Structures of ELC-MyoA trimeric sub-complexes showed ELCs induce secondary structure in MyoA to stiffen the lever arm, and, contrary to earlier inference, calcium binding does not alter ELC structure.","evidence":"X-ray crystallography of trimeric sub-complexes, biophysical analysis","pmids":["33051581"],"confidence":"High","gaps":["Reconciliation of calcium's role across studies incomplete","Dynamics of lever-arm stiffening under load not measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"The mechanical output of the motor during locomotion was quantified, showing acto-MyoA drives traction and requires an apical anchoring adhesion that stores energy in the microtubule cytoskeleton to set thrust for helical gliding.","evidence":"Traction force microscopy, reflection interference microscopy, micropatterning, expansion microscopy","pmids":["32432851"],"confidence":"Medium","gaps":["Molecular identity of the apical adhesion not defined","Direct force per motor not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"MyoA was validated as a druggable in vivo target by KNX-002, which inhibits its actin-activated ATPase with vertebrate selectivity and whose on-target action was confirmed by a T130A resistance mutation and disease slowing in mice.","evidence":"HTS ATPase screen, chemical mutagenesis/selection, sequencing, in vitro motility, mouse infection with WT vs T130A","pmids":["37155705"],"confidence":"High","gaps":["Structural mode of KNX-002 binding not resolved","Efficacy against chronic/bradyzoite stages not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"A role beyond motility was established: MyoA accumulates around the mitochondrion during division and is required for normal mitochondrial morphology and inheritance into daughter buds.","evidence":"Ultrastructure expansion microscopy, conditional MyoA knockdown, mitochondrion inheritance assays, actin pulldown with mitochondrion vesicles, yeast two-hybrid (LMF1-MyoA)","pmids":["39374269"],"confidence":"Medium","gaps":["Direct LMF1-MyoA interaction based on Y2H not orthogonally validated","Whether the glideosome or a distinct MyoA pool drives inheritance unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Glideosome (MyoA-MLC1-GAP45-GAP50, with GAP40)","MyoA-MLC1-ELC1/ELC2 lever-arm complex"],"partners":["MLC1 (MYOSIN LIGHT CHAIN, DIRECT BINDING PARTNER)","ELC1 / ELC2 (ESSENTIAL LIGHT CHAINS, LEVER ARM)","GAP45 (GLIDEOSOME-ASSOCIATED, RECRUITS MLC1-MYOA TO IMC)","GAP50 (INTEGRAL IMC MEMBRANE ANCHOR)","GAP40 (POLYTOPIC IMC PROTEIN)","TGCDPK3 (KINASE, PHOSPHORYLATES SER21/SER743)","TGUNC (UCS-FAMILY CO-CHAPERONE REQUIRED FOR FOLDING)","ACTIN/ACT1 (TRACK FOR MOTOR)","MYOH (SEQUENTIAL MOTOR RELAY PARTNER)","MYOC (COMPENSATORY GLIDEOSOME MYOSIN)","LMF1 (YEAST TWO-HYBRID INTERACTOR, MITOCHONDRION)"],"other_free_text":["MyoA is under tight protein-level feedback regulation [#2].","Regulated by calcium-dependent phosphorylation at Ser21 and Ser743 (TgCDPK3) and by post-translational modification of MLC1 including palmitoylation (C8/C11) and covalent targeting at Cys58 [#8, #9, #19, #20].","Divergent motor-domain architecture (SH1/SH2 region, HYAG-site polymorphism, non-human solvent pockets) underlies parasite-specific inhibition; KNX-002 is an on-target ATPase inhibitor validated by the T130A resistance mutation [#14, #21].","Actomyosin system contributes to substrate attachment and traction-force generation, not solely force output, during helical gliding [#18, #23]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"12399593","id":"PMC_12399593","title":"Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion.","date":"2002","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/12399593","citation_count":399,"is_preprint":false},{"pmid":"20951968","id":"PMC_20951968","title":"Functional dissection of the apicomplexan glideosome molecular architecture.","date":"2010","source":"Cell host & 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research","url":"https://pubmed.ncbi.nlm.nih.gov/11713335","citation_count":116,"is_preprint":false},{"pmid":"24632839","id":"PMC_24632839","title":"The toxoplasma Acto-MyoA motor complex is important but not essential for gliding motility and host cell invasion.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24632839","citation_count":91,"is_preprint":false},{"pmid":"28100223","id":"PMC_28100223","title":"Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion.","date":"2017","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/28100223","citation_count":81,"is_preprint":false},{"pmid":"25393004","id":"PMC_25393004","title":"Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion.","date":"2014","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/25393004","citation_count":77,"is_preprint":false},{"pmid":"26760042","id":"PMC_26760042","title":"The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells.","date":"2016","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26760042","citation_count":75,"is_preprint":false},{"pmid":"21980283","id":"PMC_21980283","title":"Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.","date":"2011","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/21980283","citation_count":73,"is_preprint":false},{"pmid":"12911812","id":"PMC_12911812","title":"Toxoplasma gondii: perfecting an intracellular life style.","date":"2003","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/12911812","citation_count":73,"is_preprint":false},{"pmid":"26544049","id":"PMC_26544049","title":"Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.","date":"2015","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26544049","citation_count":70,"is_preprint":false},{"pmid":"19047362","id":"PMC_19047362","title":"GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex.","date":"2008","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/19047362","citation_count":65,"is_preprint":false},{"pmid":"26374117","id":"PMC_26374117","title":"Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility.","date":"2015","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/26374117","citation_count":40,"is_preprint":false},{"pmid":"20084115","id":"PMC_20084115","title":"A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.","date":"2010","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/20084115","citation_count":39,"is_preprint":false},{"pmid":"24989796","id":"PMC_24989796","title":"Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii.","date":"2014","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/24989796","citation_count":36,"is_preprint":false},{"pmid":"25231988","id":"PMC_25231988","title":"A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25231988","citation_count":36,"is_preprint":false},{"pmid":"32432851","id":"PMC_32432851","title":"Coupling Polar Adhesion with Traction, Spring, and Torque Forces Allows High-Speed Helical Migration of the Protozoan Parasite Toxoplasma.","date":"2020","source":"ACS nano","url":"https://pubmed.ncbi.nlm.nih.gov/32432851","citation_count":34,"is_preprint":false},{"pmid":"21143563","id":"PMC_21143563","title":"Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii.","date":"2011","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/21143563","citation_count":28,"is_preprint":false},{"pmid":"33051581","id":"PMC_33051581","title":"Structural role of essential light chains in the apicomplexan glideosome.","date":"2020","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/33051581","citation_count":17,"is_preprint":false},{"pmid":"30348763","id":"PMC_30348763","title":"Structural and mechanistic insights into the function of the unconventional class XIV myosin MyoA from Toxoplasma gondii.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/30348763","citation_count":16,"is_preprint":false},{"pmid":"28972141","id":"PMC_28972141","title":"Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28972141","citation_count":16,"is_preprint":false},{"pmid":"24892871","id":"PMC_24892871","title":"Identification of T. gondii myosin light chain-1 as a direct target of TachypleginA-2, a small-molecule inhibitor of parasite motility and invasion.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24892871","citation_count":16,"is_preprint":false},{"pmid":"16470333","id":"PMC_16470333","title":"Functional and biophysical analyses of the class XIV Toxoplasma gondii myosin D.","date":"2006","source":"Journal of muscle research and cell motility","url":"https://pubmed.ncbi.nlm.nih.gov/16470333","citation_count":14,"is_preprint":false},{"pmid":"37155705","id":"PMC_37155705","title":"MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.","date":"2023","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/37155705","citation_count":11,"is_preprint":false},{"pmid":"34011689","id":"PMC_34011689","title":"Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility.","date":"2021","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/34011689","citation_count":11,"is_preprint":false},{"pmid":"39374269","id":"PMC_39374269","title":"Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance in Toxoplasma gondii.","date":"2024","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/39374269","citation_count":4,"is_preprint":false},{"pmid":"38562694","id":"PMC_38562694","title":"Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance in Toxoplasma gondii.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38562694","citation_count":1,"is_preprint":false},{"pmid":"37331576","id":"PMC_37331576","title":"Knocking down the expression of the molecular motors, myosin A, C and F genes in Toxoplasma gondii to decrease the parasite virulence.","date":"2023","source":"Experimental parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/37331576","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16478,"output_tokens":6418,"usd":0.072852,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13842,"output_tokens":8352,"usd":0.139005,"stage2_stop_reason":"end_turn"},"total_usd":0.010078,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard","skipped":true,"corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"summary amended"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"TgMyoA is a fast, single-headed, plus-end-directed motor that moves actin filaments in unitary steps of 5.3 nm at a velocity of 5.2 µm/s, as established by microneedle-laser trap and sliding velocity assays. TgMyoA binds a novel myosin light chain TgMLC1 and is anchored at the plasma membrane.\",\n      \"method\": \"In vitro motility assay, microneedle-laser trap (optical tweezers), biochemical characterization of purified motor complex\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro motor with biophysical single-molecule assays and kinetic characterization, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"11980712\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Conditional removal of MyoA using a tetracycline-inducible system caused severe impairment in host cell invasion and parasite spreading in cultured cells, and established MyoA as essential for pathogenesis in an animal model.\",\n      \"method\": \"Tetracycline-inducible conditional gene disruption (regulatable transactivator system), cell invasion assays, animal model infection\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with defined cellular and in vivo phenotypes, replicated across multiple assays\",\n      \"pmids\": [\"12399593\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Overexpression of a MyoA transgene drastically down-regulates the level of endogenous MyoA, indicating MyoA is under tight feedback regulation at the protein level.\",\n      \"method\": \"Tetracycline-inducible expression system, Western blot analysis of endogenous protein levels\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, but consistent finding with genetic manipulation and protein-level readout\",\n      \"pmids\": [\"11713335\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TgMyoA forms a protein complex with its light chain TgMLC1 and two novel proteins TgGAP45 and TgGAP50. This complex is localized to the inner membrane complex, where it is anchored by TgGAP50, an integral membrane glycoprotein. Complex assembly is spatially controlled and occurs in two stages.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, immunofluorescence localization, identification of novel complex components\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, fractionation, localization with multiple orthogonal methods identifying the membrane receptor\",\n      \"pmids\": [\"15123738\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The glideosome contains MyoA, MLC1, GAP45, GAP50, and a newly identified polytopic IMC protein GAP40. GAP45 is anchored to the plasma membrane and IMC via its N- and C-terminal extremities respectively; its C-terminal region recruits MLC1-MyoA to the IMC. GAP45 is essential for gliding, invasion, and egress.\",\n      \"method\": \"Conditional knockdown, co-immunoprecipitation, immunofluorescence, complementation experiments including transgenera complementation with P. falciparum GAP45\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, conditional KD with defined phenotypes, cross-species complementation as orthogonal validation\",\n      \"pmids\": [\"20951968\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Phosphorylation of GAP45 at Ser163 and Ser167 controls the final step of MyoA motor complex assembly: phosphomimetic mutations (Ser-to-Glu) prevent association of the MyoA-MLC1-GAP45 subcomplex with the membrane anchor GAP50, while alanine mutations do not affect assembly.\",\n      \"method\": \"Mass spectrometry identification of phosphorylation sites, site-directed mutagenesis (Ser-to-Glu and Ser-to-Ala), co-immunoprecipitation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis combined with Co-IP to show mechanism of assembly regulation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"19047362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Calcium-dependent phosphorylation of six residues across GAP45, MLC1, and MyoA correlates with invasion motor activity. A novel 15-kDa calmodulin-like protein (MyoA Essential Light Chain) associates more strongly with the invasion motor upon calcium stimulation, identifying a new regulatory component.\",\n      \"method\": \"SILAC-based quantitative LC-MS/MS phosphoproteomics, 2-DE of 32P-labeled protein extracts, calcium stimulation experiments\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative MS with SILAC, single lab, correlation of phosphorylation with motor activity\",\n      \"pmids\": [\"21980283\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Post-translational modification of TgMLC1 by a small-molecule inhibitor (tachyplegin) causes decreased TgMyoA motor activity, as shown by in vitro motility assay comparing motor complexes from treated vs. untreated parasites. This established that Class XIV myosin motor activity can be modulated by PTMs on its associated light chain.\",\n      \"method\": \"In vitro motility assay with purified motor complexes, electrophoretic mobility shift, peptide mapping\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro motility assay with biochemically defined motor complexes, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"20084115\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMyoA undergoes calcium-dependent phosphorylation; a small-molecule enhancer of motility (compound 130038) causes increased intracellular calcium leading to increased TgMyoA phosphorylation. Parasites expressing nonphosphorylatable mutant MyoA egressed from host cells more slowly in response to calcium ionophore, demonstrating that calcium-dependent phosphorylation of MyoA modulates myosin-driven processes.\",\n      \"method\": \"Site-directed mutagenesis of phosphorylation sites, calcium ionophore treatment, egress assays, compound treatment, intracellular calcium measurement\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of phosphorylation sites with defined functional phenotype, calcium mechanistic link established, single lab multiple methods\",\n      \"pmids\": [\"24989796\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgCDPK3 phosphorylates TgMyoA at Ser21 and Ser743 in vitro. Complementation of a myoA null mutant with MyoA bearing S21A or S743A alanine mutations failed to rescue the egress delay phenotype, while phosphomimetic mutations bypassed the need for TgCDPK3. This establishes TgCDPK3-mediated phosphorylation of TgMyoA as the mechanism for rapid initiation of motility and egress.\",\n      \"method\": \"BioID proximity interaction screen, peptide array kinase assay in vitro, genetic complementation with phosphomimetic/alanine mutants, egress assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus genetic complementation with separation-of-function mutants, multiple orthogonal methods establishing writer-substrate relationship\",\n      \"pmids\": [\"26544049\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MyoA and its associated motor complex (MLC1, GAP45, Act1) are important but not absolutely essential for gliding motility and host cell invasion; conditional KO mutants remain invasive, suggesting alternative invasion mechanisms exist alongside the MyoA-glideosome.\",\n      \"method\": \"Conditional knockout of MyoA, MLC1, GAP45, Act1; invasion and motility assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO with quantitative phenotypic readout, single lab\",\n      \"pmids\": [\"24632839\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Deletion of MyoA leads to relocalization of MyoC along the pellicle and at the apical cap, accounting for residual invasion. MyoA and MyoC glideosomes exhibit plasticity, with MyoC-glideosome providing functional compensation for loss of MyoA.\",\n      \"method\": \"Conditional knockout of MyoA, immunofluorescence localization of MyoC, invasion assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via conditional KO with defined localization and functional consequences, single lab\",\n      \"pmids\": [\"25393004\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Two essential light chains (ELC1 and ELC2) bind the MyoA lever arm and are functionally redundant but important for invasion, egress, and motility. Ca2+ promotes intermolecular contacts between ELCs and the adjacent MLC1 to stabilize the lever arm domain, as demonstrated by point mutations predicted to ablate Ca2+ binding or the ELC-MLC1 interface.\",\n      \"method\": \"Identification of ELC2 by database search, conditional knockdown, molecular dynamics simulations, point mutagenesis, gliding motility assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with phenotypic readout combined with mutagenesis, single lab; molecular simulations not tier 1\",\n      \"pmids\": [\"26374117\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMyoA expressed in Sf9 cells requires a parasite-specific UCS-family co-chaperone (TgUNC) to be soluble and functional. The MyoA heavy chain with only MLC1 moves actin at ~1.5 µm/s, but addition of ELC1 increases speed to ~3.4 µm/s, demonstrating that two light chains bound to the lever arm are required for fast motility.\",\n      \"method\": \"Baculovirus/Sf9 heterologous expression, in vitro actin gliding motility assay, co-expression of chaperone and light chains\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro motility with defined protein components and systematic variation of light chain composition, single lab\",\n      \"pmids\": [\"25231988\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The 2.6-Å crystal structure of the TgMyoA motor domain reveals a divergent SH1/SH2 region and class-defining 'HYAG'-site polymorphism as key structural elements for force transduction and chemomechanical coupling. Hydrogen-deuterium exchange MS showed that phosphorylation-dependent enhanced motility correlates with increased local stability in specific regions of the motor. Solvent-accessible pockets differ from human myosins, supporting inhibitor design.\",\n      \"method\": \"X-ray crystallography (2.6 Å), hydrogen-deuterium exchange coupled to MS, in vitro motility assay with phosphorylation variants\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by in vitro assay and HDX-MS, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"30348763\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MLC1 and ELC1 bind adjacent sites on the MyoA neck (residues 775–818). The 1.85-Å crystal structure of MLC1 bound to its MyoA peptide shows a bilobed architecture with MLC1 clamping around the helical peptide (Kd ~10 nM). Ca2+ coordination by an EF-hand in ELC1 and pre-binding of MLC1 cooperate to increase ELC1 affinity for the neck 49-fold, to ~12 nM. ELC1 also engages the MyoA converter subdomain.\",\n      \"method\": \"X-ray crystallography (1.85 Å), hydrogen-deuterium exchange MS, isothermal titration calorimetry, binding affinity measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with quantitative binding data by ITC and HDX-MS, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"28972141\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of ELC in trimeric sub-complexes with MyoA show that ELCs bind a conserved MyoA sequence and induce secondary structure in MyoA to stiffen its lever arm. Structural and biophysical analysis reveals that calcium binding has no influence on ELC structure.\",\n      \"method\": \"X-ray crystallography of trimeric sub-complexes, biophysical analysis (SPR/ITC)\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures of glideosome sub-complexes with biophysical validation, single lab\",\n      \"pmids\": [\"33051581\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MyoH is indispensable for translocation of the circular junction from the parasite tip to the apical IMC position; once this first step is accomplished, MyoA relays the process to enact effective gliding and invasion along the pellicle. This establishes a sequential handoff between MyoH (conoid-anchored) and MyoA (IMC-anchored) during motility.\",\n      \"method\": \"Conditional disruption of TgMyoH, immunofluorescence, invasion and egress assays, epistasis between MyoH and MyoA\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via conditional KO with defined phenotypic steps, single lab\",\n      \"pmids\": [\"26760042\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Parasites with mlc1 cKO (and act1 cKO) are virtually unable to attach to a collagen-coated substrate under flow conditions while retaining residual motility at wildtype speed, demonstrating that the actomyosin system (including MyoA via MLC1) functions primarily in surface attachment rather than as the sole force generator for movement.\",\n      \"method\": \"Conditional knockout of mlc1 and act1, 3D motility assays, substrate attachment assays under flow, comparison of mutant phenotypes\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO with quantitative flow-attachment and motility phenotypes, single lab, multiple mutants compared\",\n      \"pmids\": [\"28100223\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMLC1 C58 is the direct covalent target of tachypleginA and its analogues, as demonstrated by click chemistry labelling. A C58S recombinant TgMLC1 mutation is refractory to compound binding, and knock-in parasites expressing C58S show decreased sensitivity to compound-induced motility inhibition, confirming TgMLC1 as the mechanistic target mediating inhibition of TgMyoA motor activity.\",\n      \"method\": \"Click chemistry labelling with alkyne-derivatized inhibitor, site-directed mutagenesis (C58S, S57A), knock-in parasite line, 3D motility assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — covalent target identification by click chemistry plus site mutagenesis and knock-in rescue, multiple orthogonal methods\",\n      \"pmids\": [\"24892871\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TgMLC1 is palmitoylated on two N-terminal cysteine residues (C8 and C11); blocking palmitoylation completely abrogates binding of TgMLC1 to TgGAP45, disrupting glideosome composition. Surprisingly, loss of TgMLC1-GAP45 interaction has little effect on parasite motility, questioning the requirement of this anchoring interaction for force transduction.\",\n      \"method\": \"Site-directed mutagenesis of palmitoylation sites (C8A/C11A), acyl-RAC assay, co-immunoprecipitation, 3D motility assays\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — palmitoylation site mutagenesis with Co-IP and functional motility assays, multiple orthogonal methods in one study\",\n      \"pmids\": [\"34011689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A small-molecule inhibitor KNX-002 inhibits TgMyoA actin-activated ATPase activity in vitro with little effect on vertebrate myosins. A resistance mutation T130A in TgMyoA reduces compound sensitivity both in biochemical and parasite assays, validating TgMyoA as a druggable target in vivo and establishing that KNX-002 slows disease progression in mice through on-target MyoA inhibition.\",\n      \"method\": \"High-throughput screen of 50,000 compounds for ATPase inhibition, chemical mutagenesis + selection, targeted sequencing, in vitro motility assay, mouse infection model with wild-type vs. T130A parasites\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ATPase assay plus resistance mutation validation plus in vivo animal model, multiple orthogonal methods across biochemistry and cell biology\",\n      \"pmids\": [\"37155705\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MyoA accumulates around the mitochondrion in late stages of parasite division (ultrastructure expansion microscopy). Parasites lacking MyoA show defective mitochondrial morphology and delay in mitochondrion delivery to daughter buds during division, establishing a role for MyoA in organellar inheritance beyond gliding motility. Mitochondrion vesicles extracted from parasites interact with actin filaments.\",\n      \"method\": \"Ultrastructure expansion microscopy (U-ExM), conditional MyoA knockdown, mitochondrion morphology and inheritance assays, actin pulldown with mitochondrion vesicles, yeast two-hybrid (LMF1-MyoA interaction identified)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by U-ExM tied to functional consequence in KO, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39374269\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The acto-MyoA motor directs traction force during tachyzoite helical gliding; traction force microscopy and reflection interference microscopy revealed a critical apical anchoring adhesion that stores energy via microtubule cytoskeleton and sets thrust force for helical gliding.\",\n      \"method\": \"Quantitative traction force microscopy, reflection interference microscopy, micropatterning, expansion microscopy\",\n      \"journal\": \"ACS nano\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biophysical traction force measurements with direct link to MyoA motor activity, single lab\",\n      \"pmids\": [\"32432851\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"Toxoplasma gondii MyoA is a fast, single-headed, plus-end-directed class XIV unconventional myosin that forms the core of the glideosome — a pellicular actomyosin motor complex anchored to the inner membrane complex via integral membrane protein GAP50 and organized by GAP45, MLC1, and two essential light chains (ELC1/ELC2) — which powers parasite gliding motility, host cell invasion, egress, and mitochondrial inheritance; its activity is regulated by calcium-dependent phosphorylation mediated by TgCDPK3 at Ser21 and Ser743, by post-translational modifications on its light chain MLC1, and by staged cooperative assembly of the light chains on the neck/lever arm, while structural studies have defined its divergent motor domain architecture as the basis for parasite-specific small-molecule inhibition.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Toxoplasma gondii MyoA is a fast, single-headed, plus-end-directed unconventional myosin that forms the mechanochemical core of the glideosome, the pellicular actomyosin motor powering parasite gliding, host-cell invasion, egress, and mitochondrial inheritance [#0, #1, #22]. As purified motor it moves actin filaments in 5.3-nm unitary steps at ~5.2 µm/s and binds the novel light chain MLC1 while anchored at the plasma membrane [#0]. Within the parasite the motor assembles into a complex of MLC1, GAP45, and the integral IMC glycoprotein GAP50, which anchors the motor to the inner membrane complex through a spatially controlled, staged assembly process [#3, #4]; final docking onto GAP50 is gated by GAP45 phosphorylation at Ser163/Ser167 [#5]. Fast motility requires two light chains on the MyoA lever arm: MLC1 clamps the neck with nanomolar affinity and cooperatively promotes high-affinity binding of an essential light chain (ELC1/ELC2), whose engagement of the neck and converter subdomain stiffens the lever arm and raises gliding speed [#13, #15, #16, #12]. Motor output is tuned by calcium-dependent phosphorylation — TgCDPK3 phosphorylates MyoA at Ser21 and Ser743 to enable rapid initiation of motility and egress [#8, #9] — and by post-translational modification of MLC1, including a palmitoylation-dependent GAP45 interaction and covalent modification at Cys58 by the inhibitor tachyplegin [#7, #19, #20]. MyoA is essential for pathogenesis and invasion in vivo, though conditional loss is partially compensated by relocalization of MyoC glideosomes [#1, #11]; the actomyosin system functions particularly in substrate attachment and traction-force generation during helical gliding [#18, #23]. Crystallographic and HDX-MS studies define a divergent motor-domain architecture with a class-defining SH1/SH2 region and drug-accessible pockets distinct from human myosins, validating MyoA as a parasite-specific small-molecule target as demonstrated by the ATPase inhibitor KNX-002 [#14, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Before its motor properties were known, MyoA was shown to be under tight protein-level feedback control, establishing that its abundance is homeostatically regulated.\",\n      \"evidence\": \"Tetracycline-inducible overexpression with Western blot of endogenous protein\",\n      \"pmids\": [\"11713335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of feedback down-regulation unresolved\", \"No link to motor function established at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"The question of what kind of motor MyoA is was answered by direct biophysical reconstitution, defining it as a fast, single-headed, plus-end-directed myosin bound to a novel light chain MLC1 and localized at the plasma membrane.\",\n      \"evidence\": \"In vitro motility assay, optical-trap single-molecule measurement of purified motor complex\",\n      \"pmids\": [\"11980712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full light-chain complement not yet defined\", \"Membrane anchoring proteins not yet identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Whether the motor mattered for the parasite lifecycle was settled by conditional disruption, showing MyoA is essential for host-cell invasion, spreading, and pathogenesis in vivo.\",\n      \"evidence\": \"Tetracycline-inducible conditional gene disruption, invasion assays, animal infection model\",\n      \"pmids\": [\"12399593\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the invasion defect not dissected\", \"Did not address whether other myosins compensate\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The architecture anchoring the motor to the parasite was defined by identifying the MyoA-MLC1-GAP45-GAP50 complex on the inner membrane complex, with GAP50 as the integral-membrane anchor and assembly occurring in stages.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, subcellular fractionation, immunofluorescence\",\n      \"pmids\": [\"15123738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger controlling staged assembly unknown\", \"Force transduction from motor to membrane not measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"How the final assembly step is regulated was answered by showing GAP45 phosphorylation at Ser163/Ser167 controls docking of the MyoA-MLC1-GAP45 subcomplex onto the GAP50 anchor.\",\n      \"evidence\": \"Mass spectrometry site identification, phosphomimetic/alanine mutagenesis, co-immunoprecipitation\",\n      \"pmids\": [\"19047362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for these sites not identified\", \"Temporal coupling to invasion not shown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The glideosome roster and membrane tethering were extended by identifying GAP40 and mapping GAP45 as the bridge that recruits MLC1-MyoA from plasma membrane to IMC, essential for gliding, invasion, and egress.\",\n      \"evidence\": \"Conditional knockdown, co-IP, immunofluorescence, cross-species complementation with P. falciparum GAP45\",\n      \"pmids\": [\"20951968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"GAP40 molecular role undefined\", \"Quantitative contribution of tethering to force output unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"That motor activity can be chemically modulated through its light chain was shown when tachyplegin-induced modification of MLC1 reduced MyoA motility in vitro.\",\n      \"evidence\": \"In vitro motility assay of motor complexes from treated vs untreated parasites, mobility shift, peptide mapping\",\n      \"pmids\": [\"20084115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise chemical adduct on MLC1 not yet localized\", \"Mechanism linking MLC1 modification to motor slowing unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Calcium signaling was tied to motor regulation by showing calcium-dependent phosphorylation across GAP45, MLC1, and MyoA and calcium-enhanced recruitment of a new essential light chain (ELC).\",\n      \"evidence\": \"SILAC quantitative phosphoproteomics, 2-DE of 32P-labeled extracts, calcium stimulation\",\n      \"pmids\": [\"21980283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link between phosphorylation and activity correlative only\", \"Responsible kinase(s) not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The two-light-chain requirement for fast motility was reconstituted heterologously, showing MyoA needs the UCS co-chaperone TgUNC for folding and that ELC1 addition to MLC1 more than doubles gliding speed.\",\n      \"evidence\": \"Baculovirus/Sf9 expression with co-chaperone and light-chain co-expression, in vitro actin gliding assays\",\n      \"pmids\": [\"25231988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell requirement for TgUNC not demonstrated\", \"Structural basis of speed enhancement not resolved here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The apparent absolute essentiality of MyoA was revised by conditional KO showing residual invasion, implying alternative invasion mechanisms.\",\n      \"evidence\": \"Conditional knockout of MyoA/MLC1/GAP45/Act1, invasion and motility assays\",\n      \"pmids\": [\"24632839\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nature of the alternative mechanism not defined in this study\", \"Extent of compensation across strains unaddressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The source of residual invasion was traced to glideosome plasticity, with MyoC relocalizing along the pellicle and apical cap to compensate for MyoA loss.\",\n      \"evidence\": \"Conditional MyoA knockout, MyoC immunofluorescence localization, invasion assays\",\n      \"pmids\": [\"25393004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trigger for MyoC redistribution unknown\", \"Whether MyoC fully substitutes for MyoA force output not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Direct functional proof that calcium-dependent MyoA phosphorylation modulates motility came from nonphosphorylatable mutants egressing more slowly under calcium ionophore.\",\n      \"evidence\": \"Site-directed mutagenesis of phosphosites, calcium ionophore treatment, egress assays, calcium measurement\",\n      \"pmids\": [\"24989796\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase not identified in this study\", \"Individual phosphosite contributions not separated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The covalent target of tachyplegin was pinned to MLC1 Cys58, establishing the mechanistic route by which the compound inhibits MyoA motor activity.\",\n      \"evidence\": \"Click chemistry labelling, C58S/S57A mutagenesis, knock-in parasites, 3D motility assay\",\n      \"pmids\": [\"24892871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Cys58 modification propagates to the motor domain not structurally defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The writer of MyoA regulatory phosphorylation was identified as TgCDPK3 acting at Ser21 and Ser743, with separation-of-function mutants tying these sites to rapid motility initiation and egress.\",\n      \"evidence\": \"BioID screen, in vitro peptide-array kinase assay, complementation with phosphomimetic/alanine mutants, egress assays\",\n      \"pmids\": [\"26544049\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural consequence of phosphorylation on motor cycle not directly shown here\", \"Whether other kinases contribute in vivo unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The essential light chains ELC1 and ELC2 were shown to be redundant lever-arm components whose calcium-promoted contacts with MLC1 stabilize the lever arm and support invasion, egress, and motility.\",\n      \"evidence\": \"Database identification of ELC2, conditional knockdown, molecular dynamics, point mutagenesis, gliding assays\",\n      \"pmids\": [\"26374117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Calcium-dependence of lever-arm stabilization based partly on simulation\", \"Distinct roles of ELC1 vs ELC2 in vivo not delineated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A sequential motor handoff was defined whereby conoid-anchored MyoH first translocates the moving junction apically before IMC-anchored MyoA relays force for pellicular gliding and invasion.\",\n      \"evidence\": \"Conditional MyoH disruption, immunofluorescence, invasion/egress assays, MyoH-MyoA epistasis\",\n      \"pmids\": [\"26760042\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the handoff transition unresolved\", \"How the two motors coordinate temporally not measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The structural logic of light-chain assembly on the neck was resolved: MLC1 clamps the MyoA neck at ~10 nM and cooperatively raises ELC1 affinity 49-fold, with ELC1 also engaging the converter and its EF-hand coordinating calcium.\",\n      \"evidence\": \"X-ray crystallography (1.85 Å), HDX-MS, ITC binding measurements\",\n      \"pmids\": [\"28972141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell relevance of cooperative affinity increase not tested\", \"Link to force transduction in the intact motor not demonstrated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The primary function of the actomyosin system was reframed toward attachment when mlc1/act1 cKO parasites failed to adhere under flow yet retained wildtype-speed residual motility.\",\n      \"evidence\": \"Conditional mlc1/act1 knockout, 3D motility assays, substrate attachment under flow\",\n      \"pmids\": [\"28100223\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Force-generation versus attachment contributions not fully separated\", \"Source of residual motility unidentified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The divergent motor-domain architecture underpinning both chemomechanical coupling and drug specificity was established by crystal structure, revealing a class-defining SH1/SH2 region and non-human solvent pockets, with HDX-MS linking phosphorylation to local motor stability.\",\n      \"evidence\": \"X-ray crystallography (2.6 Å), HDX-MS, in vitro motility with phosphorylation variants\",\n      \"pmids\": [\"30348763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full ATPase-cycle conformational states not captured\", \"Direct inhibitor-bound structure not provided here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Structures of ELC-MyoA trimeric sub-complexes showed ELCs induce secondary structure in MyoA to stiffen the lever arm, and, contrary to earlier inference, calcium binding does not alter ELC structure.\",\n      \"evidence\": \"X-ray crystallography of trimeric sub-complexes, biophysical analysis\",\n      \"pmids\": [\"33051581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of calcium's role across studies incomplete\", \"Dynamics of lever-arm stiffening under load not measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The mechanical output of the motor during locomotion was quantified, showing acto-MyoA drives traction and requires an apical anchoring adhesion that stores energy in the microtubule cytoskeleton to set thrust for helical gliding.\",\n      \"evidence\": \"Traction force microscopy, reflection interference microscopy, micropatterning, expansion microscopy\",\n      \"pmids\": [\"32432851\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity of the apical adhesion not defined\", \"Direct force per motor not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A palmitoylation-dependent MLC1-GAP45 linkage was uncovered, but its dispensability for motility questioned whether membrane anchoring of the motor is required for force transduction.\",\n      \"evidence\": \"Palmitoylation-site mutagenesis (C8A/C11A), acyl-RAC, co-IP, 3D motility assays\",\n      \"pmids\": [\"34011689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How force is transduced without MLC1-GAP45 anchoring unresolved\", \"Functional role of MLC1 palmitoylation beyond GAP45 binding unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"MyoA was validated as a druggable in vivo target by KNX-002, which inhibits its actin-activated ATPase with vertebrate selectivity and whose on-target action was confirmed by a T130A resistance mutation and disease slowing in mice.\",\n      \"evidence\": \"HTS ATPase screen, chemical mutagenesis/selection, sequencing, in vitro motility, mouse infection with WT vs T130A\",\n      \"pmids\": [\"37155705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mode of KNX-002 binding not resolved\", \"Efficacy against chronic/bradyzoite stages not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A role beyond motility was established: MyoA accumulates around the mitochondrion during division and is required for normal mitochondrial morphology and inheritance into daughter buds.\",\n      \"evidence\": \"Ultrastructure expansion microscopy, conditional MyoA knockdown, mitochondrion inheritance assays, actin pulldown with mitochondrion vesicles, yeast two-hybrid (LMF1-MyoA)\",\n      \"pmids\": [\"39374269\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct LMF1-MyoA interaction based on Y2H not orthogonally validated\", \"Whether the glideosome or a distinct MyoA pool drives inheritance unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the glideosome transduces force to drive locomotion versus mere attachment, and how membrane-anchoring, light-chain stiffening, and calcium-dependent phosphorylation are integrated into the ATPase cycle in vivo, remains to be mechanistically unified.\",\n      \"evidence\": \"Open question synthesizing attachment-versus-force and dispensable-anchoring findings\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model linking anchoring dispensability to force output\", \"Mechanism coupling phosphorylation to the ATPase cycle in cells unresolved\", \"Molecular basis of the apical adhesion undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0000146\", \"supporting_discovery_ids\": [0, 13, 21], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0003774\", \"supporting_discovery_ids\": [0, 13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051015\", \"supporting_discovery_ids\": [0, 13, 22], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016887\", \"supporting_discovery_ids\": [21], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0031262\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [22], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0071702\", \"supporting_discovery_ids\": [1, 17, 23], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0030048\", \"supporting_discovery_ids\": [0, 18, 23], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0048311\", \"supporting_discovery_ids\": [22], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Glideosome (MyoA-MLC1-GAP45-GAP50, with GAP40)\",\n      \"MyoA-MLC1-ELC1/ELC2 lever-arm complex\"\n    ],\n    \"partners\": [\n      \"MLC1 (myosin light chain, direct binding partner)\",\n      \"ELC1 / ELC2 (essential light chains, lever arm)\",\n      \"GAP45 (glideosome-associated, recruits MLC1-MyoA to IMC)\",\n      \"GAP50 (integral IMC membrane anchor)\",\n      \"GAP40 (polytopic IMC protein)\",\n      \"TgCDPK3 (kinase, phosphorylates Ser21/Ser743)\",\n      \"TgUNC (UCS-family co-chaperone required for folding)\",\n      \"Actin/Act1 (track for motor)\",\n      \"MyoH (sequential motor relay partner)\",\n      \"MyoC (compensatory glideosome myosin)\",\n      \"LMF1 (yeast two-hybrid interactor, mitochondrion)\"\n    ],\n    \"other_free_text\": [\n      \"MyoA is under tight protein-level feedback regulation [#2].\",\n      \"Regulated by calcium-dependent phosphorylation at Ser21 and Ser743 (TgCDPK3) and by post-translational modification of MLC1 including palmitoylation (C8/C11) and covalent targeting at Cys58 [#8, #9, #19, #20].\",\n      \"Divergent motor-domain architecture (SH1/SH2 region, HYAG-site polymorphism, non-human solvent pockets) underlies parasite-specific inhibition; KNX-002 is an on-target ATPase inhibitor validated by the T130A resistance mutation [#14, #21].\",\n      \"Actomyosin system contributes to substrate attachment and traction-force generation, not solely force output, during helical gliding [#18, #23].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_235470","name":"MyoA","product":"myosin A","uniprot_acc":"S8G527","chromosome":"TGME49_chrX","start_pos":4883895},{"strain":"GT1","gene_id":"TGGT1_235470","name":null,"product":"myosin A","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":4773284},{"strain":"RH88","gene_id":"TGRH88_041990","name":null,"product":"myosin A","uniprot_acc":null,"chromosome":"CM023091","start_pos":2653725},{"strain":"VEG","gene_id":"TGVEG_235470","name":null,"product":"myosin A","uniprot_acc":"B9Q788","chromosome":"TGVEG_chrX","start_pos":4799607}]}