{"gene":"TG_257680","gene_symbol":"","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2002,"finding":"TgMLC1 is a novel myosin light chain that binds TgMyoA; the TgMyoA-TgMLC1 motor complex is a fast, single-headed, plus-end-directed motor moving in unitary steps of 5.3 nm at 5.2 µm/s toward the plus end of actin filaments, as established by microneedle-laser trap and sliding velocity assays.","method":"In vitro motility assay, microneedle-laser trap, biochemical characterization of purified motor complex","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted motor complex, in vitro motility assay with mechanical measurements, multiple orthogonal methods","pmids":["11980712"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"TgMLC1 is a component of a four-protein complex with TgMyoA, TgGAP45, and TgGAP50 that localizes to the inner membrane complex (IMC); this complex is anchored to the IMC via TgGAP50, an integral membrane glycoprotein; assembly occurs in two stages and is spatially controlled.","method":"Co-immunoprecipitation, subcellular fractionation, localization by immunofluorescence/electron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, fractionation, localization, replicated in subsequent papers","pmids":["15123738"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"GAP45 phosphorylation at Ser163 and Ser167 controls the final assembly step of the myosin XIV motor complex: phospho-mimetic (Glu) mutations at these sites prevent association of the MyoA-MLC1-GAP45 soluble complex with membrane-anchored GAP50, while phospho-null (Ala) mutations do not affect complex assembly.","method":"Mass spectrometry identification of phosphorylation sites, site-directed mutagenesis (phospho-mimetic and phospho-null), co-immunoprecipitation","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of identified phosphorylation sites combined with Co-IP, single lab but multiple orthogonal methods","pmids":["19047362"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"TgMLC1 (MTIP) associates with Rab11A, a vesicular trafficking GTPase involved in IMC maturation and plasma membrane delivery during daughter cell formation; this interaction links the glideosome motor to Rab11A-mediated vesicular traffic required for completion of cytokinesis.","method":"Co-immunoprecipitation, conditional dominant-negative expression, live imaging","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP association shown, functional link via dominant-negative, single lab","pmids":["19165333"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgMLC1 is a component of the glideosome with GAP40, GAP45, GAP50, and MyoA; GAP45 C-terminal region recruits MLC1-MyoA to the IMC, while GAP45 N-terminal acylation and coiled-coil domain preserves pellicle integrity during invasion.","method":"Conditional knockdown, co-immunoprecipitation, complementation assays, immunofluorescence","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, genetic complementation, conditional KD with phenotypic readout, replicated across labs","pmids":["20951968"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Treatment with a small-molecule inhibitor induces a post-translational modification of TgMLC1 mapped to the peptide Val46-Arg59; myosin motor complexes containing modified TgMLC1 show significantly decreased in vitro motor activity, demonstrating that PTMs on TgMLC1 can modulate Class XIV myosin mechanical activity.","method":"In vitro motility assay with motor complexes from treated vs. control parasites, peptide mass mapping, 2D gel electrophoresis","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro motility assay with purified complexes, PTM mapping, functional consequence demonstrated, single lab","pmids":["20084115"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"SILAC-based quantitative phosphoproteomics identified calcium-dependent phosphorylation of multiple residues on MLC1 in live tachyzoites upon Ca²⁺-mobilizing drug stimulation; phosphorylation of these sites correlates with invasion motor activity.","method":"SILAC quantitative LC-MS/MS, ³²P labeling, 2D gel electrophoresis","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — quantitative MS identifies Ca²⁺-dependent phosphorylation sites with correlation to activity, single lab, correlative link to activity","pmids":["21980283"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgMLC1 (as regulatory light chain) complexed with TgMyoA heavy chain moves actin at ~1.5 µm/s in an in vitro motility assay; addition of the essential light chain TgELC1 to the complex more than doubles actin sliding speed to ~3.4 µm/s, indicating MLC1 and ELC1 together stabilize the lever arm for fast motility. Co-expression with the T. gondii-specific co-chaperone TgUNC is required for functional motor assembly.","method":"Baculovirus/Sf9 heterologous expression, in vitro actin gliding assay, co-chaperone co-expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted motor complex, in vitro motility assay, direct quantitative comparison of light chain combinations, single lab","pmids":["25231988"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Conditional knockdown of MLC1 results in parasites with significantly reduced gliding motility and host cell invasion efficiency, but mutant parasites are not completely blocked and remain invasive, indicating MLC1 is important but not strictly essential for motility.","method":"Conditional knockout (Tet-regulatable), gliding motility assay, invasion assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with quantitative phenotypic readout, single lab, two orthogonal functional assays","pmids":["24632839"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Phospho-mimetic and phospho-null mutations at all mapped TgMLC1 phosphorylation sites do not impact glideosome assembly or parasite motility, indicating TgMLC1 phosphorylation is not critical for glideosome function.","method":"Site-directed mutagenesis of phosphorylation sites, allelic replacement, motility assays, co-immunoprecipitation","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-mutant allelic replacement with motility and assembly readouts, single lab; this is a negative finding","pmids":["24779470"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgMLC1 is identified as a direct and covalent target of the small-molecule inhibitor tachypleginA-2; the compound forms a covalent adduct (225.118 Da) on C58 of TgMLC1; the C58S knock-in mutation renders parasites less sensitive to the compound in motility assays, establishing C58 as the functionally relevant binding site.","method":"Click chemistry labeling, mass spectrometry adduct mapping, site-directed mutagenesis (C58S knock-in), 3D motility assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — covalent target identification by click chemistry + MS, mutagenesis knock-in confirms site, functional assay, single lab but multiple orthogonal methods","pmids":["24892871"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"MLC1 occupies one of two adjacent light chain binding sites on the MyoA neck (IQ motifs); molecular simulations indicate Ca²⁺ promotes intermolecular contacts between ELC1/ELC2 and the adjacent MLC1 to stabilize the lever arm; point mutations predicted to ablate ELC-MLC1 interface interactions reduce gliding quality, displacement, and speed.","method":"Molecular dynamics simulation, site-directed mutagenesis, gliding motility assay, conditional knockdown","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutagenesis combined with simulations and motility assays; simulation-based structural inference not fully experimentally validated, single lab","pmids":["26374117"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Crystal structure of MLC1 at 1.85 Å resolution in complex with the MyoA neck peptide reveals a bilobed architecture with MLC1 clamping around the helical MyoA peptide (residues 775–818); isothermal titration calorimetry measured a stable ~10 nM Kd for MLC1–MyoA peptide binding; prebinding of MLC1 to the MyoA neck enhances ELC1 affinity for the neck 8-fold, supporting a cooperative staged assembly model.","method":"X-ray crystallography (1.85 Å), hydrogen-deuterium exchange mass spectrometry, isothermal titration calorimetry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, HDX-MS, ITC; multiple orthogonal biophysical methods in a single rigorous study","pmids":["28972141"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Conditional knockdown of MLC1 results in parasites that can still initiate 3D movement at wildtype speeds but are virtually unable to attach to collagen-coated substrates under flow conditions, demonstrating that MLC1 (and the actomyosin system) is specifically required for substrate attachment rather than solely for force generation during motility.","method":"Conditional knockout (mlc1 cKO), 3D motility assay, substrate attachment assay under flow conditions","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with specific attachment assay readout, single lab, two orthogonal assay types","pmids":["28100223"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"TgMLC1 is palmitoylated on two N-terminal cysteine residues, C8 and C11; mutations blocking palmitoylation completely abrogate binding of TgMLC1 to TgGAP45, yet these palmitoylation-deficient mutant parasites show little impact on gliding motility, challenging the model that MLC1-GAP45 interaction is essential for force transduction.","method":"Site-directed mutagenesis (C8/C11 mutations), palmitoylation assay, co-immunoprecipitation, motility assays","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of palmitoylation sites, biochemical confirmation of interaction loss, functional motility assays, single lab but multiple orthogonal methods","pmids":["34011689"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgMLC1 is a regulatory light chain that binds the IQ motif region (residues 775–818) of the class XIV myosin TgMyoA with ~10 nM affinity (crystal structure resolved at 1.85 Å), forming a core glideosome motor complex with ELC1/2, GAP45, and the IMC-anchored GAP50; MLC1 is palmitoylated on C8/C11 (mediating GAP45 binding) and phosphorylated in a Ca²⁺-dependent manner (neither modification being strictly essential for motility), and covalent modification of C58 by small-molecule inhibitors decreases motor activity; functionally, MLC1 contributes to fast myosin lever-arm mechanics (its absence halving actin sliding speed), parasite substrate attachment, IMC maturation during cytokinesis, and efficient—though not absolutely essential—gliding motility and host cell invasion."},"narrative":{"mechanistic_narrative":"TgMLC1 is a regulatory light chain of the Toxoplasma gondii class XIV myosin TgMyoA and a core component of the glideosome, the pellicle-anchored actomyosin motor that powers gliding motility, substrate attachment, and host cell invasion [PMID:11980712, PMID:15123738]. It clamps around the helical MyoA neck (IQ-motif region, residues 775–818) with a bilobed architecture and ~10 nM affinity, and its prebinding cooperatively enhances recruitment of the essential light chain ELC1 to the same neck, supporting a staged lever-arm assembly [PMID:28972141]. Within the assembled motor, MLC1 together with ELC1 stabilizes the lever arm to enable fast actin sliding, with the two light chains more than doubling actin gliding speed relative to MyoA alone [PMID:25231988]. The MLC1-MyoA complex assembles into a four-protein glideosome with GAP45 and the IMC-anchored integral membrane glycoprotein GAP50, GAP45 bridging the soluble MyoA-MLC1 module to the inner membrane complex [PMID:15123738, PMID:20951968]. MLC1 is palmitoylated on N-terminal cysteines C8 and C11, a modification required for MLC1-GAP45 binding but largely dispensable for gliding [PMID:34011689], and covalent modification of C58 by the small-molecule inhibitor tachypleginA-2 reduces motor activity [PMID:24892871, PMID:20084115]. Conditional depletion of MLC1 halves motor speed and impairs but does not abolish gliding and invasion, with a specific requirement for substrate attachment under flow rather than for movement initiation per se [PMID:24632839, PMID:28100223].","teleology":[{"year":2002,"claim":"Establishing that TgMLC1 is a bona fide myosin light chain answered whether Toxoplasma possesses a defined fast motor complex, showing MyoA-MLC1 is a single-headed, plus-end-directed motor with measurable mechanics.","evidence":"In vitro motility assay and microneedle-laser trap on purified motor complex","pmids":["11980712"],"confidence":"High","gaps":["Did not resolve MLC1's structural mode of binding to MyoA","Role of additional light chains not yet defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"claim":"Defining the glideosome as a four-protein IMC-localized complex answered how the soluble motor is physically coupled to the parasite pellicle, identifying GAP50 as the membrane anchor and a spatially staged assembly.","evidence":"Reciprocal co-immunoprecipitation, subcellular fractionation, and IF/EM localization","pmids":["15123738"],"confidence":"High","gaps":["Molecular triggers controlling assembly stages not defined","MLC1's individual contribution to anchoring not isolated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Linking MLC1 to the vesicular GTPase Rab11A extended its role beyond force generation, connecting the motor to IMC maturation and membrane delivery during daughter cell formation.","evidence":"Co-immunoprecipitation, conditional dominant-negative expression, live imaging","pmids":["19165333"],"confidence":"Medium","gaps":["Direct vs. indirect nature of MLC1-Rab11A association not resolved","Single-lab Co-IP association"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Mapping GAP45 as the recruiter of the MLC1-MyoA module to the IMC clarified how the motor is targeted and how pellicle integrity is preserved during invasion.","evidence":"Conditional knockdown, reciprocal Co-IP, and complementation assays","pmids":["20951968"],"confidence":"High","gaps":["Exact MLC1 residues contacting GAP45 not defined here","Distinction between structural anchoring and force transduction unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Demonstrating that a small-molecule-induced PTM on MLC1 (Val46-Arg59 region) lowers motor activity showed that MLC1 covalent modification can directly modulate class XIV myosin mechanics.","evidence":"In vitro motility assay on complexes from treated parasites, peptide mass mapping","pmids":["20084115"],"confidence":"High","gaps":["Precise modified residue not pinpointed in this study","Physiological equivalent of the induced PTM unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Identifying Ca2+-dependent phosphorylation of multiple MLC1 residues addressed whether calcium signaling regulates the motor, correlating these marks with invasion-associated motor activity.","evidence":"SILAC quantitative LC-MS/MS and 32P labeling in Ca2+-stimulated tachyzoites","pmids":["21980283"],"confidence":"Medium","gaps":["Link to activity is correlative","Kinase(s) responsible not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Reconstituting the motor from heterologous subunits established that MLC1 and ELC1 together stabilize the lever arm for fast motility and that the TgUNC co-chaperone is needed for functional assembly.","evidence":"Baculovirus/Sf9 expression, in vitro actin gliding assay with defined light chain combinations","pmids":["25231988"],"confidence":"High","gaps":["Structural basis of the ELC1-MLC1 cooperativity not resolved here","In vivo relevance of the speed increment not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Conditional depletion showed MLC1 is important but not strictly essential, defining a quantitative rather than absolute requirement for gliding and invasion.","evidence":"Tet-regulatable conditional knockout with gliding and invasion assays","pmids":["24632839"],"confidence":"Medium","gaps":["Residual motility source not defined","Single-lab phenotype"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Testing all mapped phospho-mutants established that MLC1 phosphorylation is dispensable for glideosome assembly and motility, dissociating the Ca2+-dependent marks from essential motor function.","evidence":"Site-directed phospho-mimetic/phospho-null allelic replacement with motility and Co-IP readouts","pmids":["24779470"],"confidence":"Medium","gaps":["Negative result; subtle or combinatorial phospho-roles not excluded","Does not address non-motility phenotypes"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Identifying MLC1 C58 as the covalent target of tachypleginA-2 pinpointed a functionally relevant, druggable residue whose modification impairs motility.","evidence":"Click chemistry, MS adduct mapping, C58S knock-in, 3D motility assay","pmids":["24892871"],"confidence":"High","gaps":["Structural mechanism by which C58 modification impairs the lever arm not resolved","Single-lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Placing MLC1 at one of two adjacent IQ light chain sites and modeling a Ca2+-promoted ELC-MLC1 interface addressed how the light chains cooperate to stabilize the lever arm, with interface mutations degrading gliding.","evidence":"Molecular dynamics simulation, interface mutagenesis, gliding assay, conditional knockdown","pmids":["26374117"],"confidence":"Medium","gaps":["Interface is simulation-inferred, not directly resolved","Direct Ca2+-binding role not experimentally confirmed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"The 1.85 A crystal structure of MLC1 on the MyoA neck resolved its bilobed clamping architecture, quantified ~10 nM binding, and showed MLC1 prebinding boosts ELC1 affinity 8-fold, providing the structural basis for staged cooperative assembly.","evidence":"X-ray crystallography, HDX-MS, and isothermal titration calorimetry","pmids":["28972141"],"confidence":"High","gaps":["Full-length assembled motor structure with ELC1/2 not captured","Conformational changes during force generation not visualized"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Conditional depletion under flow revealed MLC1 is specifically required for substrate attachment, not for initiating 3D movement, refining its functional role within the actomyosin system.","evidence":"mlc1 conditional KO with 3D motility and collagen-substrate attachment assays under flow","pmids":["28100223"],"confidence":"Medium","gaps":["Molecular link between the motor and adhesin traction not defined","Single-lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"Mapping palmitoylation to C8/C11 and showing it is required for MLC1-GAP45 binding yet dispensable for gliding challenged the assumption that the MLC1-GAP45 link is essential for force transduction.","evidence":"C8/C11 mutagenesis, palmitoylation and Co-IP assays, motility assays","pmids":["34011689"],"confidence":"High","gaps":["How the motor remains functional without the GAP45 link not resolved","Alternative anchoring route not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How MLC1-dependent lever-arm mechanics are physically coupled to surface adhesins to produce substrate traction, and how the motor stays functional when its GAP45 anchoring is lost, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the fully assembled, membrane-anchored glideosome","Mechanism connecting motor force to attachment not defined","Kinase and functional consequence of Ca2+-dependent phosphorylation unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Glideosome motor complex (MyoA-MLC1-GAP45-GAP50, with GAP40)","MyoA-MLC1-ELC1 tripartite motor module"],"partners":["TGMYOA (MYOSIN A HEAVY CHAIN, IQ-MOTIF NECK, ~10 NM)","TGELC1 (ESSENTIAL LIGHT CHAIN)","TGELC2","TGGAP45","TGGAP50","TGGAP40","RAB11A","TGUNC (CO-CHAPERONE REQUIRED FOR ASSEMBLY)"],"other_free_text":["Palmitoylated on N-terminal cysteines C8 and C11; palmitoylation required for GAP45 binding but dispensable for gliding [#14]","Covalent modification of C58 by tachypleginA-2 reduces motor activity; C58S confers resistance [#10]","Ca2+-dependent phosphorylation on multiple sites correlating with invasion motor activity, but phospho-mutants have no motility/assembly defect [#6, #9]","MLC1 loss halves actin sliding speed; ELC1 addition more than doubles speed of the MyoA-MLC1 complex [#7]","MLC1 prebinding to the MyoA neck enhances ELC1 affinity ~8-fold, supporting staged cooperative assembly [#12]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"20951968","id":"PMC_20951968","title":"Functional dissection of the apicomplexan glideosome molecular architecture.","date":"2010","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/20951968","citation_count":234,"is_preprint":false},{"pmid":"15123738","id":"PMC_15123738","title":"Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii.","date":"2004","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15123738","citation_count":214,"is_preprint":false},{"pmid":"11980712","id":"PMC_11980712","title":"Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.","date":"2002","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11980712","citation_count":197,"is_preprint":false},{"pmid":"19165333","id":"PMC_19165333","title":"Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis.","date":"2009","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/19165333","citation_count":95,"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":"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":"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":"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":"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":"24779470","id":"PMC_24779470","title":"Assessment of phosphorylation in Toxoplasma glideosome assembly and function.","date":"2014","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/24779470","citation_count":25,"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":"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":"30130043","id":"PMC_30130043","title":"[Research Advances on Gliding-associated Proteins of Toxoplasma gondii].","date":"2016","source":"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/30130043","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10743,"output_tokens":4021,"usd":0.046272,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10329,"output_tokens":5230,"usd":0.091198,"stage2_stop_reason":"end_turn"},"total_usd":0.13747,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"TgMLC1 is a novel myosin light chain that binds TgMyoA; the TgMyoA-TgMLC1 motor complex is a fast, single-headed, plus-end-directed motor moving in unitary steps of 5.3 nm at 5.2 µm/s toward the plus end of actin filaments, as established by microneedle-laser trap and sliding velocity assays.\",\n      \"method\": \"In vitro motility assay, microneedle-laser trap, biochemical characterization of purified motor complex\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted motor complex, in vitro motility assay with mechanical measurements, multiple orthogonal methods\",\n      \"pmids\": [\"11980712\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TgMLC1 is a component of a four-protein complex with TgMyoA, TgGAP45, and TgGAP50 that localizes to the inner membrane complex (IMC); this complex is anchored to the IMC via TgGAP50, an integral membrane glycoprotein; assembly occurs in two stages and is spatially controlled.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, localization by immunofluorescence/electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, fractionation, localization, replicated in subsequent papers\",\n      \"pmids\": [\"15123738\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GAP45 phosphorylation at Ser163 and Ser167 controls the final assembly step of the myosin XIV motor complex: phospho-mimetic (Glu) mutations at these sites prevent association of the MyoA-MLC1-GAP45 soluble complex with membrane-anchored GAP50, while phospho-null (Ala) mutations do not affect complex assembly.\",\n      \"method\": \"Mass spectrometry identification of phosphorylation sites, site-directed mutagenesis (phospho-mimetic and phospho-null), co-immunoprecipitation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of identified phosphorylation sites combined with Co-IP, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"19047362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TgMLC1 (MTIP) associates with Rab11A, a vesicular trafficking GTPase involved in IMC maturation and plasma membrane delivery during daughter cell formation; this interaction links the glideosome motor to Rab11A-mediated vesicular traffic required for completion of cytokinesis.\",\n      \"method\": \"Co-immunoprecipitation, conditional dominant-negative expression, live imaging\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP association shown, functional link via dominant-negative, single lab\",\n      \"pmids\": [\"19165333\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgMLC1 is a component of the glideosome with GAP40, GAP45, GAP50, and MyoA; GAP45 C-terminal region recruits MLC1-MyoA to the IMC, while GAP45 N-terminal acylation and coiled-coil domain preserves pellicle integrity during invasion.\",\n      \"method\": \"Conditional knockdown, co-immunoprecipitation, complementation assays, immunofluorescence\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, genetic complementation, conditional KD with phenotypic readout, replicated across labs\",\n      \"pmids\": [\"20951968\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Treatment with a small-molecule inhibitor induces a post-translational modification of TgMLC1 mapped to the peptide Val46-Arg59; myosin motor complexes containing modified TgMLC1 show significantly decreased in vitro motor activity, demonstrating that PTMs on TgMLC1 can modulate Class XIV myosin mechanical activity.\",\n      \"method\": \"In vitro motility assay with motor complexes from treated vs. control parasites, peptide mass mapping, 2D gel electrophoresis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro motility assay with purified complexes, PTM mapping, functional consequence demonstrated, single lab\",\n      \"pmids\": [\"20084115\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SILAC-based quantitative phosphoproteomics identified calcium-dependent phosphorylation of multiple residues on MLC1 in live tachyzoites upon Ca²⁺-mobilizing drug stimulation; phosphorylation of these sites correlates with invasion motor activity.\",\n      \"method\": \"SILAC quantitative LC-MS/MS, ³²P labeling, 2D gel electrophoresis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — quantitative MS identifies Ca²⁺-dependent phosphorylation sites with correlation to activity, single lab, correlative link to activity\",\n      \"pmids\": [\"21980283\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMLC1 (as regulatory light chain) complexed with TgMyoA heavy chain moves actin at ~1.5 µm/s in an in vitro motility assay; addition of the essential light chain TgELC1 to the complex more than doubles actin sliding speed to ~3.4 µm/s, indicating MLC1 and ELC1 together stabilize the lever arm for fast motility. Co-expression with the T. gondii-specific co-chaperone TgUNC is required for functional motor assembly.\",\n      \"method\": \"Baculovirus/Sf9 heterologous expression, in vitro actin gliding assay, co-chaperone co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted motor complex, in vitro motility assay, direct quantitative comparison of light chain combinations, single lab\",\n      \"pmids\": [\"25231988\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Conditional knockdown of MLC1 results in parasites with significantly reduced gliding motility and host cell invasion efficiency, but mutant parasites are not completely blocked and remain invasive, indicating MLC1 is important but not strictly essential for motility.\",\n      \"method\": \"Conditional knockout (Tet-regulatable), gliding motility assay, invasion assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with quantitative phenotypic readout, single lab, two orthogonal functional assays\",\n      \"pmids\": [\"24632839\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Phospho-mimetic and phospho-null mutations at all mapped TgMLC1 phosphorylation sites do not impact glideosome assembly or parasite motility, indicating TgMLC1 phosphorylation is not critical for glideosome function.\",\n      \"method\": \"Site-directed mutagenesis of phosphorylation sites, allelic replacement, motility assays, co-immunoprecipitation\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-mutant allelic replacement with motility and assembly readouts, single lab; this is a negative finding\",\n      \"pmids\": [\"24779470\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMLC1 is identified as a direct and covalent target of the small-molecule inhibitor tachypleginA-2; the compound forms a covalent adduct (225.118 Da) on C58 of TgMLC1; the C58S knock-in mutation renders parasites less sensitive to the compound in motility assays, establishing C58 as the functionally relevant binding site.\",\n      \"method\": \"Click chemistry labeling, mass spectrometry adduct mapping, site-directed mutagenesis (C58S knock-in), 3D motility assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — covalent target identification by click chemistry + MS, mutagenesis knock-in confirms site, functional assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24892871\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MLC1 occupies one of two adjacent light chain binding sites on the MyoA neck (IQ motifs); molecular simulations indicate Ca²⁺ promotes intermolecular contacts between ELC1/ELC2 and the adjacent MLC1 to stabilize the lever arm; point mutations predicted to ablate ELC-MLC1 interface interactions reduce gliding quality, displacement, and speed.\",\n      \"method\": \"Molecular dynamics simulation, site-directed mutagenesis, gliding motility assay, conditional knockdown\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutagenesis combined with simulations and motility assays; simulation-based structural inference not fully experimentally validated, single lab\",\n      \"pmids\": [\"26374117\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of MLC1 at 1.85 Å resolution in complex with the MyoA neck peptide reveals a bilobed architecture with MLC1 clamping around the helical MyoA peptide (residues 775–818); isothermal titration calorimetry measured a stable ~10 nM Kd for MLC1–MyoA peptide binding; prebinding of MLC1 to the MyoA neck enhances ELC1 affinity for the neck 8-fold, supporting a cooperative staged assembly model.\",\n      \"method\": \"X-ray crystallography (1.85 Å), hydrogen-deuterium exchange mass spectrometry, isothermal titration calorimetry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, HDX-MS, ITC; multiple orthogonal biophysical methods in a single rigorous study\",\n      \"pmids\": [\"28972141\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Conditional knockdown of MLC1 results in parasites that can still initiate 3D movement at wildtype speeds but are virtually unable to attach to collagen-coated substrates under flow conditions, demonstrating that MLC1 (and the actomyosin system) is specifically required for substrate attachment rather than solely for force generation during motility.\",\n      \"method\": \"Conditional knockout (mlc1 cKO), 3D motility assay, substrate attachment assay under flow conditions\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with specific attachment assay readout, single lab, two orthogonal assay types\",\n      \"pmids\": [\"28100223\"],\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; mutations blocking palmitoylation completely abrogate binding of TgMLC1 to TgGAP45, yet these palmitoylation-deficient mutant parasites show little impact on gliding motility, challenging the model that MLC1-GAP45 interaction is essential for force transduction.\",\n      \"method\": \"Site-directed mutagenesis (C8/C11 mutations), palmitoylation assay, co-immunoprecipitation, motility assays\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of palmitoylation sites, biochemical confirmation of interaction loss, functional motility assays, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34011689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgMLC1 is a regulatory light chain that binds the IQ motif region (residues 775–818) of the class XIV myosin TgMyoA with ~10 nM affinity (crystal structure resolved at 1.85 Å), forming a core glideosome motor complex with ELC1/2, GAP45, and the IMC-anchored GAP50; MLC1 is palmitoylated on C8/C11 (mediating GAP45 binding) and phosphorylated in a Ca²⁺-dependent manner (neither modification being strictly essential for motility), and covalent modification of C58 by small-molecule inhibitors decreases motor activity; functionally, MLC1 contributes to fast myosin lever-arm mechanics (its absence halving actin sliding speed), parasite substrate attachment, IMC maturation during cytokinesis, and efficient—though not absolutely essential—gliding motility and host cell invasion.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TgMLC1 is a regulatory light chain of the Toxoplasma gondii class XIV myosin TgMyoA and a core component of the glideosome, the pellicle-anchored actomyosin motor that powers gliding motility, substrate attachment, and host cell invasion [#0, #1]. It clamps around the helical MyoA neck (IQ-motif region, residues 775\\u2013818) with a bilobed architecture and ~10 nM affinity, and its prebinding cooperatively enhances recruitment of the essential light chain ELC1 to the same neck, supporting a staged lever-arm assembly [#12]. Within the assembled motor, MLC1 together with ELC1 stabilizes the lever arm to enable fast actin sliding, with the two light chains more than doubling actin gliding speed relative to MyoA alone [#7]. The MLC1-MyoA complex assembles into a four-protein glideosome with GAP45 and the IMC-anchored integral membrane glycoprotein GAP50, GAP45 bridging the soluble MyoA-MLC1 module to the inner membrane complex [#1, #4]. MLC1 is palmitoylated on N-terminal cysteines C8 and C11, a modification required for MLC1-GAP45 binding but largely dispensable for gliding [#14], and covalent modification of C58 by the small-molecule inhibitor tachypleginA-2 reduces motor activity [#10, #5]. Conditional depletion of MLC1 halves motor speed and impairs but does not abolish gliding and invasion, with a specific requirement for substrate attachment under flow rather than for movement initiation per se [#8, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that TgMLC1 is a bona fide myosin light chain answered whether Toxoplasma possesses a defined fast motor complex, showing MyoA-MLC1 is a single-headed, plus-end-directed motor with measurable mechanics.\",\n      \"evidence\": \"In vitro motility assay and microneedle-laser trap on purified motor complex\",\n      \"pmids\": [\"11980712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve MLC1's structural mode of binding to MyoA\", \"Role of additional light chains not yet defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defining the glideosome as a four-protein IMC-localized complex answered how the soluble motor is physically coupled to the parasite pellicle, identifying GAP50 as the membrane anchor and a spatially staged assembly.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, subcellular fractionation, and IF/EM localization\",\n      \"pmids\": [\"15123738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular triggers controlling assembly stages not defined\", \"MLC1's individual contribution to anchoring not isolated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linking MLC1 to the vesicular GTPase Rab11A extended its role beyond force generation, connecting the motor to IMC maturation and membrane delivery during daughter cell formation.\",\n      \"evidence\": \"Co-immunoprecipitation, conditional dominant-negative expression, live imaging\",\n      \"pmids\": [\"19165333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect nature of MLC1-Rab11A association not resolved\", \"Single-lab Co-IP association\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping GAP45 as the recruiter of the MLC1-MyoA module to the IMC clarified how the motor is targeted and how pellicle integrity is preserved during invasion.\",\n      \"evidence\": \"Conditional knockdown, reciprocal Co-IP, and complementation assays\",\n      \"pmids\": [\"20951968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact MLC1 residues contacting GAP45 not defined here\", \"Distinction between structural anchoring and force transduction unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that a small-molecule-induced PTM on MLC1 (Val46-Arg59 region) lowers motor activity showed that MLC1 covalent modification can directly modulate class XIV myosin mechanics.\",\n      \"evidence\": \"In vitro motility assay on complexes from treated parasites, peptide mass mapping\",\n      \"pmids\": [\"20084115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise modified residue not pinpointed in this study\", \"Physiological equivalent of the induced PTM unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identifying Ca2+-dependent phosphorylation of multiple MLC1 residues addressed whether calcium signaling regulates the motor, correlating these marks with invasion-associated motor activity.\",\n      \"evidence\": \"SILAC quantitative LC-MS/MS and 32P labeling in Ca2+-stimulated tachyzoites\",\n      \"pmids\": [\"21980283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link to activity is correlative\", \"Kinase(s) responsible not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Reconstituting the motor from heterologous subunits established that MLC1 and ELC1 together stabilize the lever arm for fast motility and that the TgUNC co-chaperone is needed for functional assembly.\",\n      \"evidence\": \"Baculovirus/Sf9 expression, in vitro actin gliding assay with defined light chain combinations\",\n      \"pmids\": [\"25231988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the ELC1-MLC1 cooperativity not resolved here\", \"In vivo relevance of the speed increment not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Conditional depletion showed MLC1 is important but not strictly essential, defining a quantitative rather than absolute requirement for gliding and invasion.\",\n      \"evidence\": \"Tet-regulatable conditional knockout with gliding and invasion assays\",\n      \"pmids\": [\"24632839\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Residual motility source not defined\", \"Single-lab phenotype\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Testing all mapped phospho-mutants established that MLC1 phosphorylation is dispensable for glideosome assembly and motility, dissociating the Ca2+-dependent marks from essential motor function.\",\n      \"evidence\": \"Site-directed phospho-mimetic/phospho-null allelic replacement with motility and Co-IP readouts\",\n      \"pmids\": [\"24779470\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result; subtle or combinatorial phospho-roles not excluded\", \"Does not address non-motility phenotypes\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying MLC1 C58 as the covalent target of tachypleginA-2 pinpointed a functionally relevant, druggable residue whose modification impairs motility.\",\n      \"evidence\": \"Click chemistry, MS adduct mapping, C58S knock-in, 3D motility assay\",\n      \"pmids\": [\"24892871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism by which C58 modification impairs the lever arm not resolved\", \"Single-lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placing MLC1 at one of two adjacent IQ light chain sites and modeling a Ca2+-promoted ELC-MLC1 interface addressed how the light chains cooperate to stabilize the lever arm, with interface mutations degrading gliding.\",\n      \"evidence\": \"Molecular dynamics simulation, interface mutagenesis, gliding assay, conditional knockdown\",\n      \"pmids\": [\"26374117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interface is simulation-inferred, not directly resolved\", \"Direct Ca2+-binding role not experimentally confirmed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The 1.85 A crystal structure of MLC1 on the MyoA neck resolved its bilobed clamping architecture, quantified ~10 nM binding, and showed MLC1 prebinding boosts ELC1 affinity 8-fold, providing the structural basis for staged cooperative assembly.\",\n      \"evidence\": \"X-ray crystallography, HDX-MS, and isothermal titration calorimetry\",\n      \"pmids\": [\"28972141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length assembled motor structure with ELC1/2 not captured\", \"Conformational changes during force generation not visualized\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Conditional depletion under flow revealed MLC1 is specifically required for substrate attachment, not for initiating 3D movement, refining its functional role within the actomyosin system.\",\n      \"evidence\": \"mlc1 conditional KO with 3D motility and collagen-substrate attachment assays under flow\",\n      \"pmids\": [\"28100223\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between the motor and adhesin traction not defined\", \"Single-lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping palmitoylation to C8/C11 and showing it is required for MLC1-GAP45 binding yet dispensable for gliding challenged the assumption that the MLC1-GAP45 link is essential for force transduction.\",\n      \"evidence\": \"C8/C11 mutagenesis, palmitoylation and Co-IP assays, motility assays\",\n      \"pmids\": [\"34011689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the motor remains functional without the GAP45 link not resolved\", \"Alternative anchoring route not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MLC1-dependent lever-arm mechanics are physically coupled to surface adhesins to produce substrate traction, and how the motor stays functional when its GAP45 anchoring is lost, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the fully assembled, membrane-anchored glideosome\", \"Mechanism connecting motor force to attachment not defined\", \"Kinase and functional consequence of Ca2+-dependent phosphorylation unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0000146\", \"supporting_discovery_ids\": [0, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0017022\", \"supporting_discovery_ids\": [0, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0032027\", \"supporting_discovery_ids\": [7, 11, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0033111\", \"supporting_discovery_ids\": [1, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016460\", \"supporting_discovery_ids\": [0, 1, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0071965\", \"supporting_discovery_ids\": [8, 11, 13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [8, 13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000281\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Glideosome motor complex (MyoA-MLC1-GAP45-GAP50, with GAP40)\",\n      \"MyoA-MLC1-ELC1 tripartite motor module\"\n    ],\n    \"partners\": [\n      \"TgMyoA (myosin A heavy chain, IQ-motif neck, ~10 nM)\",\n      \"TgELC1 (essential light chain)\",\n      \"TgELC2\",\n      \"TgGAP45\",\n      \"TgGAP50\",\n      \"TgGAP40\",\n      \"Rab11A\",\n      \"TgUNC (co-chaperone required for assembly)\"\n    ],\n    \"other_free_text\": [\n      \"Palmitoylated on N-terminal cysteines C8 and C11; palmitoylation required for GAP45 binding but dispensable for gliding [#14]\",\n      \"Covalent modification of C58 by tachypleginA-2 reduces motor activity; C58S confers resistance [#10]\",\n      \"Ca2+-dependent phosphorylation on multiple sites correlating with invasion motor activity, but phospho-mutants have no motility/assembly defect [#6, #9]\",\n      \"MLC1 loss halves actin sliding speed; ELC1 addition more than doubles speed of the MyoA-MLC1 complex [#7]\",\n      \"MLC1 prebinding to the MyoA neck enhances ELC1 affinity ~8-fold, supporting staged cooperative assembly [#12]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_257680","name":null,"product":"myosin light chain MLC1","uniprot_acc":"A0A125YYD4","chromosome":"TGME49_chrVIIb","start_pos":3909108},{"strain":"GT1","gene_id":"TGGT1_257680","name":null,"product":"myosin light chain MLC1","uniprot_acc":null,"chromosome":"TGGT1_chrVIIb","start_pos":3843366},{"strain":"RH88","gene_id":"TGRH88_072250","name":null,"product":"myosin light chain MLC1","uniprot_acc":null,"chromosome":"CM023089","start_pos":3944959},{"strain":"VEG","gene_id":"TGVEG_257680","name":null,"product":"myosin light chain MLC1","uniprot_acc":"A0A0F7V1A1","chromosome":"TGVEG_chrVIIb","start_pos":3928516}]}