{"gene":"TG_201780","gene_symbol":"MIC2","run_date":"2026-09-07T12:57:46+00:00","timeline":{"discoveries":[{"year":1997,"finding":"MIC2 is sequentially released from micronemes upon host cell binding at the tight attachment zone between the parasite and host cell, prior to rhoptry and dense granule discharge, establishing MIC2 secretion as the earliest step in the invasion sequence.","method":"Immunofluorescence, immunoelectron microscopy, quantitative immunoassay during live invasion","journal":"European journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (IF, IEM, quantitative immunoassay), replicated in subsequent studies","pmids":["9208224"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"Microneme secretion of MIC2 is regulated by parasite intracellular free Ca2+: Ca2+ ionophores trigger apical release and subsequent treadmilling/capping of MIC2 to the posterior end, while chelation of intracellular Ca2+ prevents release. This redistribution depends on the actin-myosin cytoskeleton.","method":"Ca2+ ionophore stimulation, intracellular Ca2+ chelation (BAPTA), thapsigargin/NH4Cl treatment, immunofluorescence, cytoskeletal inhibitor assays","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple pharmacological perturbations with orthogonal methods, replicated across multiple papers","pmids":["10027960"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"The full-length, cell-surface form of MIC2 (but not the proteolytically shed soluble form) binds specifically to host cells, indicating that proteolytic processing inactivates the adhesive function of MIC2.","method":"Host cell binding assay comparing full-length vs. proteolytically modified secreted MIC2","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay with two forms of the protein, single lab","pmids":["11207555"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"A staurosporine-sensitive kinase activity regulates microneme discharge (including MIC2 release) and is required for parasite invasion of host cells.","method":"Staurosporine treatment, microneme secretion assay, invasion assay","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological inhibition with invasion readout, single lab","pmids":["11207555"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"finding":"MIC2 is proteolytically processed at multiple sites by two parasite-derived proteases: MPP1 removes the C-terminal domain upon release into the culture supernatant, while MPP2 (a serine/cysteine protease-sensitive activity) trims the N-terminus prior to release.","method":"Biochemical protease inhibitor panel, protein secretion assays, N- and C-terminal domain mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assays with inhibitor profiling, multiple processing sites mapped","pmids":["10799515"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"finding":"Two conserved cytoplasmic tail motifs (SYHYY and EIEYE) of MIC2 are both necessary and sufficient for targeting proteins to micronemes; an artificial tail containing only these two motifs in preserved spacing redirects the surface protein SAG1 to micronemes.","method":"Site-directed mutagenesis, chimeric protein (SAG1 fusion) expression, immunofluorescence and immunoelectron microscopy","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis plus gain-of-function chimera, orthogonal localization methods","pmids":["10982850"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"TgM2AP physically associates with TgMIC2, forming a stable complex within 15 min of synthesis. M2AP remains associated with MIC2 in the micronemes, on the parasite surface during invasion, and in the culture medium after shedding. M2AP propeptide is removed in the Golgi, and M2AP is further processed on the parasite surface after microneme discharge.","method":"Co-immunoprecipitation, pulse-chase labeling, immunofluorescence, immunoelectron microscopy","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, pulse-chase, and orthogonal localization; replicated in subsequent work","pmids":["11532123"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"MPP1 cleaves MIC2 within its membrane-spanning domain (intramembrane proteolysis); the cleavage site mapped to a conserved motif (IAGG) in the transmembrane domain of TgMIC2 and TgMIC6, and MPP1 is constitutively active at the parasite surface. Fusion of the SAG1 ectodomain with MIC2/MIC6 transmembrane domains showed the ectodomain is not required for cleavage recognition.","method":"Site-specific mutagenesis, chimeric SAG1-MIC2/MIC6 transmembrane domain fusions, protein secretion and surface expression assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of cleavage site combined with chimeric constructs, mechanistic mapping","pmids":["11927542"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"C-terminal proteolytic processing of MIC2 (removal of the C-terminal domain upstream of the transmembrane domain) is essential for invasion: mutations preventing cleavage caused stable surface expression of MIC2 and increased host cell adhesion but severely impaired invasion, demonstrating that shedding is required for cell entry.","method":"Site-directed mutagenesis of extracellular domain, invasion and adhesion assays, surface expression quantification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with defined phenotypic readout, clear structure-function relationship","pmids":["12471033"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Disruption of M2AP (MIC2-associated protein) leads to >80% impairment in host cell entry because MIC2 accumulates in the ER/Golgi and cannot be rapidly secreted upon host cell attachment, demonstrating that the MIC2-M2AP complex is required for efficient invasion.","method":"Gene knockout, immunofluorescence, invasion assay, secretion assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with quantified invasion phenotype and mechanistic follow-up on MIC2 trafficking","pmids":["12727875"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"MIC2 and M2AP form a stable hexameric complex of ~450 kDa consisting of three αβ dimers, as determined by native blue gels and gel filtration of parasite lysates. The cytoplasmic C-domain of MIC2 is not required for MIC2-M2AP complex formation or microneme trafficking.","method":"Native blue gel electrophoresis, gel filtration, transgenic expression of C-domain deletion mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution with size analysis, mutagenesis in parasite","pmids":["14670959"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"The integrin-like A-domain of MIC2 binds specifically to heparin (a sulfated proteoglycan of the extracellular matrix) as a high-molecular-weight multimeric species; this binding does not depend on the MIDAS divalent cation-binding motif.","method":"Recombinant A-domain fusion protein binding assays, heparin binding assay, size analysis of binding species","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro binding assay with recombinant protein and MIDAS mutagenesis, single lab","pmids":["15003840"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"Eimeria tenella MIC1 (the ortholog of TgMIC2) can substitute for TgMIC2 to enable the first TgMIC2 knockout clone; in TgMIC2 KO parasites, M2AP is misprocessed and mistrafficked to the parasitophorous vacuole rather than the micronemes, demonstrating mutual dependence for correct trafficking.","method":"Trans-genera complementation, gene knockout, immunofluorescence, Western blot","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout enabled by trans-genera complementation, orthogonal methods for trafficking analysis","pmids":["15236644"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"Aldolase bridges MIC2 to filamentous actin via the MIC2 cytoplasmic tail, coupling the adhesin to the actomyosin motor. The penultimate tryptophan and flanking acidic residues in the MIC2 cytoplasmic tail are required for aldolase binding.","method":"In vitro binding assay (aldolase-MIC2 tail interaction), homology modeling, mutagenesis","journal":"Cell host & microbe (referenced in Starnes 2006); original aldolase-MIC2 interaction established in prior work cited by Starnes GL et al.","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding assay with mutagenesis, single lab at this stage","pmids":["16923803"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"cGMP-dependent protein kinase (PKG) acts downstream of calcium-dependent events to regulate MIC2 secretion, gliding motility, host cell attachment, and invasion; PKG inhibition blocks MIC2 adhesin secretion even when calcium is elevated by ionophores, and PKG-refractory transgenic strains show reduced sensitivity to this inhibition.","method":"Pharmacological inhibition (compound 1/trisubstituted pyrrole), transgenic PKG mutant strains, immunofluorescence, invasion/motility assays","journal":"International journal for parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — transgenic validation with PKG mutant strains plus pharmacological data, multiple assays","pmids":["15003497"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Eimeria tenella","strain":""}]},{"year":2005,"finding":"TgROM5, a rhomboid intramembrane serine protease localized predominantly to the posterior of the parasite, colocalizes with MIC2 at the posterior cleavage site during invasion and is the only T. gondii rhomboid able to cleave MIC adhesins in a cell-based assay, identifying it as the likely MPP1 protease responsible for MIC2 shedding.","method":"Cloning and expression of five TgROMs, GFP localization, cell-based cleavage assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-based cleavage assay plus colocalization, but later challenged by ROM4 studies","pmids":["15753289"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2005,"finding":"Human ICAM-1 immunoprecipitates the mature surface form of MIC2, indicating a direct interaction between parasite MIC2 and host ICAM-1 that contributes to transepithelial migration; soluble ICAM-1 and ICAM-1 antibodies inhibit parasite transmigration across cellular barriers.","method":"Immunoprecipitation of parasite surface MIC2 with soluble human ICAM-1, transmigration inhibition assay","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional inhibition, single lab","pmids":["15760456"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"finding":"Conditional suppression of MIC2 causes mistrafficking of M2AP, markedly defective host-cell attachment and invasion, loss of helical gliding motility, and inability to establish lethal infection in mice, establishing the MIC2-M2AP complex as a major virulence determinant.","method":"Conditional (anhydrotetracycline-regulatable) MIC2 expression system, endogenous mic2 knockout, invasion/motility/attachment assays, murine infection model","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with multiple orthogonal phenotypic readouts including in vivo virulence","pmids":["16933991"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"finding":"Two separate acidic residue clusters in the MIC2 cytoplasmic tail are essential for parasite survival: the C-terminal cluster is required for direct aldolase binding, while an upstream acidic cluster is essential but does not mediate aldolase binding, implying a second critical cytoskeletal interaction.","method":"Alanine point mutagenesis, in vitro aldolase binding assay, transgenic complementation, parasite survival assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with in vitro binding assay and in vivo complementation","pmids":["16923803"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"finding":"The M2AP propeptide is required for the MIC2-M2AP complex to exit an early endosomal compartment; deletion of the propeptide traps the complex in the early secretory pathway, while propeptide cleavage-resistant mutants reach micronemes but cannot rapidly mobilize to the parasite surface. Both defects impair invasion and reduce virulence.","method":"Propeptide deletion and cleavage-site mutagenesis, immunofluorescence/immunoelectron microscopy, invasion assay, in vivo virulence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis of propeptide with orthogonal localization and functional readouts","pmids":["16914527"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"Aldolase is essential for efficient host cell invasion through its ability to bridge MIC2 cytoplasmic tail to actin filaments; mutations in TgALD1 that selectively abolish MIC2 tail binding (without affecting glycolytic activity) decrease invasion efficiency without affecting motility, while mutations affecting glycolysis reduce motility.","method":"Structure-guided mutagenesis of TgALD1, conditional knockout complementation, in vitro MIC2 tail binding assay, motility and invasion assays","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure-guided mutagenesis, in vitro binding, and genetic complementation with delineated functions","pmids":["19380114"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgROM4 is the primary rhomboid protease responsible for shedding of surface adhesins including MIC2; conditional knockout of TgROM4 decreases MIC2 release into supernatant, increases surface MIC2 accumulation, disrupts normal gliding (causing twirling), and severely impairs invasion despite enhanced host cell binding.","method":"Conditional knockout, surface adhesin shedding assay, supernatant MIC2 quantification, gliding motility and invasion assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with multiple orthogonal mechanistic readouts","pmids":["20421941"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgSUB1 (a GPI-anchored microneme subtilisin protease) is responsible for proteolytic trimming of MIC2 (and MIC4, M2AP) on the parasite surface after secretion; loss of TgSUB1 abolishes this trimming, decreases cell attachment and gliding efficiency, reduces invasion rates, and lowers virulence in mice.","method":"Gene deletion, MIC2 processing assay, attachment and invasion assays, in vivo virulence","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean gene deletion with multiple orthogonal phenotypic and biochemical readouts","pmids":["20678172"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"ROM4 (not ROM5) is the primary rhomboid protease for MIC2 cleavage in T. gondii tachyzoites; ROM4 single knockout blocks MIC2 shedding and causes surface accumulation, nonproductive attachment, altered gliding, impaired moving junction formation, and reduced invasion. Triple knockout (ROM1/ROM4/ROM5) parasites are viable, demonstrating ROM-independent mechanisms for adhesin removal during actual cell entry.","method":"Single, double, and triple rhomboid knockouts, MIC2 shedding assay, motility and invasion assays, moving junction analysis","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic single/double/triple KO approach with multiple mechanistic readouts; reconciles prior ROM5 data","pmids":["25336455"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Crystal structures of N-terminal proMIC2 and MIC2 fragments reveal a closed conformation of the von Willebrand A (VWA) domain, its association with the TSR1 domain, a proline-rich pigtail loop in TSR1 overlapping the VWA domain, α-C-linked mannose on Trp-276, and a mobile α5-helix with a disordered region replacing the α6-helix in a putative activation mechanism. M2AP binds as a 1:1 monomer to TSR6 in a highly elongated complex.","method":"X-ray crystallography of MIC2-M2AP ectodomain and sub-fragments, small-angle X-ray scattering","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determination with multiple constructs and functional domain mapping","pmids":["24639528"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"MIC2 contributes to initial adhesion of T. gondii tachyzoites to vascular endothelium under shear stress but is not required for adhesion strengthening, as shown by comparison of wild-type and MIC2-deficient parasites in a microfluidic system.","method":"Microfluidics, time-lapse fluorescence microscopy, MIC2-deficient parasite comparison under shear stress","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MIC2 knockout with defined adhesion phenotype, single lab, specialized assay","pmids":["24692639"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"The NMR solution structure of M2AP globular domain reveals a modified galectin fold similar to MIC1 C-terminal domain; hydrophobic residues on one face of this galectin fold mediate binding to the sixth TSR domain of MIC2 (TSR6), as validated by NMR and immunoprecipitation.","method":"NMR structure determination, immunoprecipitation, domain mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional binding validation by IP, single lab","pmids":["25411252"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"ROM4 is the primary contributor to MPP1 activity (intramembrane cleavage of MIC2 and MIC6), while ROM5 is dispensable for MIC2 shedding; ROM4 knockout parasites engage predominantly in twirling motility and show enhanced attachment but impaired invasion, whereas ROM5 knockout does not alter MIC2 shedding or invasion.","method":"Cre-recombinase and CRISPR-Cas9 knockouts of ROM4 and ROM5, MIC2/MIC6 cleavage assay, motility and invasion assays","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent gene disruption methods, multiple assays, confirms ROM4 primacy","pmids":["25846828"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Glideosome-associated connector (GAC), an armadillo/PH domain protein, directly binds filamentous actin and specifically associates with TgMIC2, connecting the MIC2 adhesin to the actomyosin system to mediate gliding motility, invasion, and egress. Apical localization of TgGAC depends on TgAKMT (apical lysine methyltransferase). GAC's PH domain also binds phosphatidic acid, a lipid linked to microneme exocytosis.","method":"Co-immunoprecipitation (TgGAC-TgMIC2), F-actin binding assay, localization by IFA, conditional knockdown of TgGAC, TgAKMT perturbation","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro binding, conditional knockdown with defined phenotypes, multiple orthogonal methods","pmids":["27978434"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2017,"finding":"MIC2 null mutant parasites are deficient in surface attachment but retain near-normal gliding speed and host cell invasion once initiated; deletion of mic2 causes delayed host cell egress in vitro, and causes only mild attenuation of virulence in vivo (slight delay, not prevention of lethal disease).","method":"Gene deletion (null mutant), 3D motility analysis, attachment under shear stress, invasion assay, in vivo murine infection","journal":"Wellcome open research","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean null mutant with careful quantification, multiple assays; provides nuanced refinement of MIC2 role","pmids":["28630943"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"MIC2 TSR domains are modified by O-fucosylation (GlcFuc disaccharide added by POFUT2/NST2) and C-mannosylation; loss of POFUT2 or NST2 markedly reduces cellular MIC2 levels, causes partial mislocalization from micronemes, and impairs host cell attachment and invasion comparable to the mic2 knockout, demonstrating that O-fucosylation is required for efficient MIC2 processing and function.","method":"Mass spectrometry glycan mapping, gene knockout (Δpofut2, Δnst2), immunofluorescence, invasion and attachment assays, anti-GlcFuc antibody detection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — MS glycan characterization combined with genetic knockouts and functional phenotyping","pmids":["30538131"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"TgPOFUT2 O-glycosylates MIC2 TSR domains (≥3 O-linked sites with >95% occupancy confirmed by MS/MS), but loss of TgPOFUT2 has only a modest impact on MIC2 levels and parasite invasion in this study, suggesting the O-glycan may be dispensable in T. gondii tachyzoites (in contrast to findings in another study).","method":"Mass spectrometry (MS/MS), enzymatic digestion, TgPOFUT2 gene knockout, plaque and invasion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous MS characterization of glycosylation sites, but functional conclusion contradicts PMID 30538131; single lab, conflicting evidence lowers confidence","pmids":["30514763"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"C-mannosylation of MIC2 TSR domains is catalyzed by apicomplexan DPY19 homologs; T. gondii and P. falciparum DPY19 enzymes modify WXXWXXC motifs in TRAP/MIC2 TSR domains when expressed in mammalian cells deficient in C-mannosylation, and can transfer mannose to WXXWXXC but not WXXW peptides in vitro.","method":"Expression of apicomplexan DPY19 in C-mannosylation-deficient mammalian cells, in vitro C-mannosyltransferase assay with peptide substrates","journal":"Glycobiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay and cell-based complementation, clear substrate specificity mapping","pmids":["29432542"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"Deletion of dpy19 (C-mannosyltransferase) reduces MIC2 protein levels and impairs parasite adhesion, motility, host cell invasion, and egress; attenuation of virulence in Δdpy19 parasites cannot be fully explained by reduced MIC2 alone, suggesting C-mannosylation stabilizes additional invasion-related proteins.","method":"Gene knockout (Δdpy19), immunofluorescence, motility and invasion assays, in vivo virulence, Western blot for MIC2 levels","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with multiple assays and in vivo validation","pmids":["31862733"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"During intracellular replication, micronemes (tracked by MIC2 labeling) are recycled from mother to daughter parasites via a highly dynamic F-actin network; this recycling is F-actin dependent, while de novo microneme synthesis is F-actin independent. The residual body acts as a storage and sorting station for micronemal vesicles.","method":"Dual labeling, super-resolution microscopy, F-actin perturbation, live imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — live super-resolution imaging plus genetic/pharmacological perturbation of F-actin","pmids":["31519913"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"3D traction force mapping shows that a moving T. gondii parasite generates periodic, inward-directed forces at a fixed circular attachment zone on the extracellular matrix; parasites lacking TgMIC2 fail to generate these inward-directed forces and lack the constriction in the plasma membrane at this zone, and display less straight trajectories, demonstrating that TgMIC2 mediates the annular attachment zone that constrains directional motility.","method":"3D traction force microscopy, TgMIC2-null parasites, live imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — novel biophysical method (3D traction force mapping) with genetic null mutant validation","pmids":["36519527"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"Crystal structures of M2AP alone and in complex with MIC2 TSR6 reveal that the non-layered side of TSR6, centered on hotspot residue His-620, binds M2AP; TSR6 residue Y602 shifts from a layered to a flipped-out state upon M2AP binding, stabilizing the complex. This interaction is required for MIC2-M2AP complex maturation and transport to the parasite surface.","method":"X-ray crystallography of M2AP alone and M2AP-TSR6 complex, mutagenesis (H620A and Y602 mutants), functional validation","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus mutagenesis, builds on prior NMR study","pmids":["37652989"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgMIC2 is a transmembrane adhesin stored in micronemes and released apically upon Ca2+/PKG-dependent stimulation; it treadmills posteriorly via the actomyosin motor (connected through its cytoplasmic tail to aldolase/F-actin and GAC), mediates a circumferential attachment zone that directs gliding motility, binds host cell surface receptors (including heparin and ICAM-1) through its von Willebrand A and TSR domains, and is shed from the parasite surface by the rhomboid intramembrane protease ROM4 to complete cell entry. MIC2 forms a heterohexameric complex with M2AP (bound at TSR6) that is required for correct microneme trafficking of both proteins and for efficient invasion; the complex is stabilized by O-fucosylation and C-mannosylation of TSR domains, and multiple proteases (SUB1 for surface trimming, ROM4 for intramembrane shedding) sequentially process MIC2 to regulate adhesion and cell entry."},"narrative":{"mechanistic_narrative":"TgMIC2 is a transmembrane microneme adhesin that acts as the earliest effector of the Toxoplasma gondii invasion sequence, coupling host-cell recognition to the parasite's substrate-dependent gliding motility [PMID:9208224, PMID:16933991]. It is stored in micronemes and discharged apically at the tight attachment zone upon a Ca2+/PKG-dependent signal, after which the adhesin treadmills posteriorly in an actomyosin-dependent manner and is capped at the posterior pole [PMID:10027960, PMID:15003497]. Two conserved cytoplasmic-tail motifs (SYHYY and EIEYE) are necessary and sufficient for microneme targeting [PMID:10982850], and the same tail engages the cytoskeletal machinery: a C-terminal acidic cluster and penultimate tryptophan mediate direct aldolase binding that bridges MIC2 to F-actin, while an additional upstream acidic cluster and the glideosome-associated connector GAC provide further links to the actomyosin motor [PMID:16923803, PMID:19380114, PMID:27978434]. On its extracellular face, MIC2 recognizes host ligands through its integrin-like von Willebrand A domain (which binds sulfated proteoglycan/heparin) and its thrombospondin type-1 repeats, and its mature surface form directly engages host ICAM-1 to support transepithelial migration [PMID:15003840, PMID:15760456, PMID:24639528]. MIC2 forms a stable ~450 kDa heterohexameric complex of three αβ dimers with M2AP, which binds the TSR6 domain; this interaction is mutually required for correct microneme trafficking of both partners and for efficient invasion [PMID:11532123, PMID:12727875, PMID:14670959, PMID:24639528, PMID:25411252, PMID:37652989]. The TSR domains are further stabilized by O-fucosylation and C-mannosylation, modifications required for efficient MIC2 processing, trafficking, and function [PMID:30538131, PMID:29432542, PMID:31862733]. Cell entry requires sequential proteolysis: TgSUB1 trims the surface ectodomain, and the rhomboid intramembrane protease ROM4 sheds MIC2 from the membrane, because shedding rather than adhesion per se is the step that permits productive invasion [PMID:12471033, PMID:20421941, PMID:20678172, PMID:25336455]. Functionally, MIC2 mediates a circumferential attachment zone that generates periodic inward-directed traction forces to constrain directional gliding, and its loss impairs surface attachment and delays egress while leaving core gliding and invasion machinery partly operational [PMID:24692639, PMID:28630943, PMID:36519527].","teleology":[{"year":1997,"claim":"Establishing when MIC2 acts in invasion showed that its micronemal release is the first molecular event of host-cell entry, placing MIC2 upstream of rhoptry and dense-granule discharge.","evidence":"Immunofluorescence, immunoelectron microscopy and quantitative immunoassay during live invasion","pmids":["9208224"],"confidence":"High","gaps":["Did not identify the trigger for release","Did not define the host receptor bound at the attachment zone"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"The signal controlling MIC2 discharge was unknown; pharmacological manipulation showed intracellular Ca2+ triggers apical release and actomyosin-dependent posterior capping, linking secretion to motility.","evidence":"Ca2+ ionophore and BAPTA chelation with immunofluorescence and cytoskeletal inhibitor assays","pmids":["10027960"],"confidence":"High","gaps":["Downstream kinase effectors not yet defined","Molecular link between MIC2 and the actomyosin motor not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"It was unclear whether proteolysis affected adhesion; binding assays showed only the full-length surface form of MIC2 binds host cells, establishing that shedding inactivates the adhesin, and that a staurosporine-sensitive kinase governs discharge.","evidence":"Host-cell binding assays comparing full-length vs shed MIC2, and staurosporine inhibition of secretion/invasion","pmids":["11207555"],"confidence":"Medium","gaps":["Kinase identity not established","Single-lab binding comparison"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"The targeting signal and processing events of MIC2 were undefined; mutagenesis identified two cytoplasmic-tail motifs sufficient for micronemal delivery and mapped distinct N- and C-terminal cleavages by parasite proteases MPP1/MPP2.","evidence":"Site-directed mutagenesis, SAG1 chimera targeting, and protease inhibitor domain-mapping assays","pmids":["10982850","10799515"],"confidence":"High","gaps":["Protease identities (MPP1/MPP2) not yet molecularly resolved","Trafficking machinery reading the tail motifs unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"claim":"The composition of the MIC2 secretory unit was unknown; co-IP and pulse-chase showed M2AP associates with MIC2 rapidly after synthesis and remains bound through the micronemes, surface and shed forms.","evidence":"Reciprocal co-immunoprecipitation, pulse-chase labeling and orthogonal localization","pmids":["11532123"],"confidence":"High","gaps":["Functional consequence of the association not yet tested","Stoichiometry of the complex not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"Whether intramembrane cleavage and C-terminal shedding matter for entry was open; mapping of an intramembrane IAGG cleavage site and mutants blocking C-terminal cleavage showed shedding is required for invasion despite increased adhesion.","evidence":"Cleavage-site mutagenesis, SAG1-TMD chimeras, and invasion/adhesion assays","pmids":["11927542","12471033"],"confidence":"High","gaps":["Rhomboid responsible for intramembrane cleavage not yet identified","Mechanistic reason adhesion must be released not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"The functional role of the MIC2-M2AP association was untested; knockout and biochemical sizing showed the two proteins are mutually dependent for correct trafficking and form a ~450 kDa hexamer of three αβ dimers required for efficient invasion.","evidence":"M2AP gene knockout with trafficking/invasion assays, and native gel/gel filtration sizing","pmids":["12727875","14670959"],"confidence":"High","gaps":["Structural basis of the interaction not resolved","Cytoplasmic C-domain shown dispensable for complex, its function unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"claim":"How MIC2 recognizes host surfaces and couples to the motor was unclear; the VWA A-domain was shown to bind heparin MIDAS-independently, aldolase was shown to bridge the tail to F-actin, and PKG was placed downstream of Ca2+ controlling secretion.","evidence":"Recombinant A-domain heparin-binding assays, aldolase-tail binding with mutagenesis, and PKG pharmacology with transgenic PKG-refractory strains","pmids":["15003840","16923803","15003497"],"confidence":"High","gaps":["In vivo relevance of heparin binding not established","Nature of a second tail-cytoskeleton link not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Eimeria tenella","strain":""}]},{"year":2005,"claim":"The identity of the shedding protease and a host receptor were open; TgROM5 was proposed as the posterior MIC-cleaving rhomboid, and host ICAM-1 was shown to bind mature surface MIC2 and support transmigration.","evidence":"Rhomboid cloning/cleavage assays with GFP localization, and ICAM-1 immunoprecipitation with transmigration inhibition","pmids":["15753289","15760456"],"confidence":"High","gaps":["ROM5 assignment later revised in favor of ROM4","ICAM-1 co-IP from single lab without structural mapping"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"claim":"The in vivo importance and tail requirements of MIC2 were untested; conditional MIC2 suppression established the complex as a virulence determinant, tail mutagenesis defined two essential acidic clusters, and M2AP propeptide mutants revealed a trafficking checkpoint.","evidence":"Conditional MIC2 knockout with in vivo virulence, tail alanine-scanning with aldolase binding, and M2AP propeptide deletion/cleavage mutants","pmids":["16933991","16923803","16914527"],"confidence":"High","gaps":["Identity of the second tail-binding cytoskeletal factor unknown","Compartment gating the propeptide-dependent exit not fully defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Whether aldolase acts as a moonlighting motor bridge versus a glycolytic enzyme was ambiguous; structure-guided TgALD1 mutants separated MIC2-tail binding (needed for invasion) from glycolysis (needed for motility).","evidence":"Structure-guided TgALD1 mutagenesis with conditional-knockout complementation, in vitro tail binding, and motility/invasion assays","pmids":["19380114"],"confidence":"High","gaps":["How force is transmitted through aldolase to actin not directly measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"The proteases executing MIC2 processing were reassessed; conditional knockout identified ROM4 as the primary intramembrane sheddase and gene deletion showed TgSUB1 performs surface trimming, both required for efficient attachment and invasion.","evidence":"Conditional ROM4 knockout and TgSUB1 gene deletion with shedding, motility, invasion and virulence assays","pmids":["20421941","20678172"],"confidence":"High","gaps":["Whether ROM4 is essential during actual cell entry not resolved","Order of SUB1 vs ROM4 action not fully sequenced"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The rhomboid assignment and complex architecture were clarified; systematic ROM knockouts confirmed ROM4 (not ROM5) as the main sheddase and revealed ROM-independent entry mechanisms, while crystallography defined the elongated MIC2-M2AP ectodomain, a closed VWA domain, C-mannosylation, and 1:1 M2AP binding to TSR6.","evidence":"Single/double/triple rhomboid knockouts with motility/invasion/moving-junction analysis, and X-ray crystallography with SAXS; microfluidic shear-adhesion assay","pmids":["25336455","24639528","24692639"],"confidence":"High","gaps":["Nature of ROM-independent adhesin removal during entry unknown","Activation mechanism of the VWA domain inferred but not captured in an open state"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"The relative rhomboid contributions were confirmed and refined; independent knockouts established ROM4 as the primary MPP1 activity while ROM5 was dispensable for MIC2 shedding and invasion.","evidence":"Cre-recombinase and CRISPR-Cas9 knockouts of ROM4 and ROM5 with cleavage, motility and invasion assays","pmids":["25846828"],"confidence":"High","gaps":["Residual shedding pathway during entry still unaccounted for"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The structural basis of M2AP recognition was defined; the NMR structure of M2AP revealed a galectin fold whose hydrophobic face binds MIC2 TSR6.","evidence":"NMR structure determination with immunoprecipitation and domain mapping","pmids":["25411252"],"confidence":"High","gaps":["Atomic detail of the TSR6 interface awaited co-crystal structure"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"A second cytoskeletal connector was sought; GAC was shown to directly bind F-actin and MIC2, and to bind phosphatidic acid via its PH domain, providing the additional motor link implied by the earlier upstream acidic-cluster requirement.","evidence":"Reciprocal Co-IP, in vitro F-actin binding, conditional GAC knockdown and TgAKMT perturbation","pmids":["27978434"],"confidence":"High","gaps":["Whether GAC and aldolase act on the same or distinct MIC2 tail motifs not fully resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2017,"claim":"The strict essentiality of MIC2 was re-examined; a clean null mutant showed MIC2 is deficient in attachment but retains near-normal gliding speed and invasion once initiated, delays egress, and is only mildly attenuated in vivo, refining MIC2 to an attachment-focusing rather than obligatory motility factor.","evidence":"mic2 null mutant with 3D motility, shear attachment, invasion and murine infection assays","pmids":["28630943"],"confidence":"High","gaps":["Molecular basis of functional redundancy with other adhesins not defined","Reconciliation with earlier conditional-knockout severe phenotype not fully explained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"The role of TSR glycosylation was tested; MS mapping plus knockouts showed O-fucosylation (POFUT2/NST2) and C-mannosylation (DPY19) modify MIC2 TSRs, with O-fucosylation loss reducing MIC2 levels and invasion, though a second study found only modest impact of POFUT2 loss.","evidence":"MS/MS glycan mapping, Δpofut2/Δnst2 knockouts, DPY19 in vitro C-mannosyltransferase assays, and functional phenotyping","pmids":["30538131","30514763","29432542"],"confidence":"High","gaps":["Conflicting POFUT2 functional phenotypes between labs","Whether glycans act on folding, stability or trafficking specifically not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"The cellular impact of C-mannosylation and micronemal dynamics were addressed; dpy19 deletion reduced MIC2 and impaired adhesion/invasion/egress beyond MIC2 alone, and imaging showed MIC2-marked micronemes are recycled from mother to daughter via an F-actin network.","evidence":"Δdpy19 knockout with functional/virulence assays, and super-resolution live imaging with F-actin perturbation","pmids":["31862733","31519913"],"confidence":"High","gaps":["Additional C-mannosylated invasion proteins not enumerated","Machinery driving F-actin-dependent microneme recycling not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"The biophysical role of MIC2 in directing motility was quantified; 3D traction force mapping showed MIC2 is required to form an annular attachment zone generating periodic inward forces that constrain directional gliding.","evidence":"3D traction force microscopy with TgMIC2-null parasites and live imaging","pmids":["36519527"],"confidence":"High","gaps":["How MIC2 organizes the circular zone molecularly not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"The atomic basis of MIC2-M2AP assembly was resolved; co-crystal structures showed TSR6 hotspot His-620 and a Y602 conformational flip stabilize M2AP binding, an interaction required for complex maturation and surface transport.","evidence":"X-ray crystallography of M2AP alone and M2AP-TSR6 with H620A/Y602 mutagenesis and functional validation","pmids":["37652989"],"confidence":"High","gaps":["Structure of the full hexamer not determined","Link between TSR6 binding and downstream trafficking machinery not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The ROM-independent mechanism that removes MIC2 during actual cell entry, and the identity of the second (non-aldolase) cytoskeletal factor binding the upstream tail cluster, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["Adhesin disengagement pathway during entry in the absence of rhomboids unknown","Full molecular inventory of the MIC2 tail-cytoskeleton interface incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MIC2-M2AP heterohexamer (~450 kDa, three αβ dimers; M2AP bound at TSR6)"],"partners":["M2AP (MICRONEME PROTEIN, TSR6-BINDING PARTNER)","ALDOLASE (TGALD1; MIC2 CYTOPLASMIC-TAIL-TO-F-ACTIN BRIDGE)","GAC / GLIDEOSOME-ASSOCIATED CONNECTOR (F-ACTIN AND MIC2 BINDING)","HOST ICAM-1 (HOST RECEPTOR FOR MATURE SURFACE MIC2)","HEPARIN / SULFATED PROTEOGLYCAN (VWA A-DOMAIN LIGAND)","ROM4 (RHOMBOID INTRAMEMBRANE SHEDDASE)","TGSUB1 (SUBTILISIN SURFACE-TRIMMING PROTEASE)","POFUT2/NST2 (O-FUCOSYLATION OF TSR DOMAINS)","DPY19 (C-MANNOSYLTRANSFERASE OF TSR DOMAINS)","PKG (CGMP-DEPENDENT KINASE REGULATING SECRETION)"],"other_free_text":["MIC2 cytoplasmic tail contains SYHYY and EIEYE motifs necessary and sufficient for micronemal targeting.","Two acidic clusters in the tail are essential; the C-terminal cluster (with penultimate Trp) binds aldolase, an upstream cluster mediates a distinct cytoskeletal link.","TSR domains carry O-fucosylation (GlcFuc) and C-mannosylation required for efficient processing/function.","Only the full-length surface form is adhesive; proteolytic shedding inactivates adhesion and is required for productive cell entry.","MIC2 mediates a circular attachment zone generating periodic inward-directed traction forces that direct gliding."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"9208224","id":"PMC_9208224","title":"Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts.","date":"1997","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9208224","citation_count":565,"is_preprint":false},{"pmid":"10027960","id":"PMC_10027960","title":"Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii.","date":"1999","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/10027960","citation_count":296,"is_preprint":false},{"pmid":"11207555","id":"PMC_11207555","title":"Secretion of micronemal proteins is associated with toxoplasma invasion of 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host cell binding at the tight attachment zone between the parasite and host cell, prior to rhoptry and dense granule discharge, establishing MIC2 secretion as the earliest step in the invasion sequence.\",\n      \"method\": \"Immunofluorescence, immunoelectron microscopy, quantitative immunoassay during live invasion\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (IF, IEM, quantitative immunoassay), replicated in subsequent studies\",\n      \"pmids\": [\"9208224\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Microneme secretion of MIC2 is regulated by parasite intracellular free Ca2+: Ca2+ ionophores trigger apical release and subsequent treadmilling/capping of MIC2 to the posterior end, while chelation of intracellular Ca2+ prevents release. This redistribution depends on the actin-myosin cytoskeleton.\",\n      \"method\": \"Ca2+ ionophore stimulation, intracellular Ca2+ chelation (BAPTA), thapsigargin/NH4Cl treatment, immunofluorescence, cytoskeletal inhibitor assays\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple pharmacological perturbations with orthogonal methods, replicated across multiple papers\",\n      \"pmids\": [\"10027960\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The full-length, cell-surface form of MIC2 (but not the proteolytically shed soluble form) binds specifically to host cells, indicating that proteolytic processing inactivates the adhesive function of MIC2.\",\n      \"method\": \"Host cell binding assay comparing full-length vs. proteolytically modified secreted MIC2\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay with two forms of the protein, single lab\",\n      \"pmids\": [\"11207555\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"A staurosporine-sensitive kinase activity regulates microneme discharge (including MIC2 release) and is required for parasite invasion of host cells.\",\n      \"method\": \"Staurosporine treatment, microneme secretion assay, invasion assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological inhibition with invasion readout, single lab\",\n      \"pmids\": [\"11207555\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MIC2 is proteolytically processed at multiple sites by two parasite-derived proteases: MPP1 removes the C-terminal domain upon release into the culture supernatant, while MPP2 (a serine/cysteine protease-sensitive activity) trims the N-terminus prior to release.\",\n      \"method\": \"Biochemical protease inhibitor panel, protein secretion assays, N- and C-terminal domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assays with inhibitor profiling, multiple processing sites mapped\",\n      \"pmids\": [\"10799515\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Two conserved cytoplasmic tail motifs (SYHYY and EIEYE) of MIC2 are both necessary and sufficient for targeting proteins to micronemes; an artificial tail containing only these two motifs in preserved spacing redirects the surface protein SAG1 to micronemes.\",\n      \"method\": \"Site-directed mutagenesis, chimeric protein (SAG1 fusion) expression, immunofluorescence and immunoelectron microscopy\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis plus gain-of-function chimera, orthogonal localization methods\",\n      \"pmids\": [\"10982850\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TgM2AP physically associates with TgMIC2, forming a stable complex within 15 min of synthesis. M2AP remains associated with MIC2 in the micronemes, on the parasite surface during invasion, and in the culture medium after shedding. M2AP propeptide is removed in the Golgi, and M2AP is further processed on the parasite surface after microneme discharge.\",\n      \"method\": \"Co-immunoprecipitation, pulse-chase labeling, immunofluorescence, immunoelectron microscopy\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, pulse-chase, and orthogonal localization; replicated in subsequent work\",\n      \"pmids\": [\"11532123\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MPP1 cleaves MIC2 within its membrane-spanning domain (intramembrane proteolysis); the cleavage site mapped to a conserved motif (IAGG) in the transmembrane domain of TgMIC2 and TgMIC6, and MPP1 is constitutively active at the parasite surface. Fusion of the SAG1 ectodomain with MIC2/MIC6 transmembrane domains showed the ectodomain is not required for cleavage recognition.\",\n      \"method\": \"Site-specific mutagenesis, chimeric SAG1-MIC2/MIC6 transmembrane domain fusions, protein secretion and surface expression assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of cleavage site combined with chimeric constructs, mechanistic mapping\",\n      \"pmids\": [\"11927542\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"C-terminal proteolytic processing of MIC2 (removal of the C-terminal domain upstream of the transmembrane domain) is essential for invasion: mutations preventing cleavage caused stable surface expression of MIC2 and increased host cell adhesion but severely impaired invasion, demonstrating that shedding is required for cell entry.\",\n      \"method\": \"Site-directed mutagenesis of extracellular domain, invasion and adhesion assays, surface expression quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with defined phenotypic readout, clear structure-function relationship\",\n      \"pmids\": [\"12471033\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Disruption of M2AP (MIC2-associated protein) leads to >80% impairment in host cell entry because MIC2 accumulates in the ER/Golgi and cannot be rapidly secreted upon host cell attachment, demonstrating that the MIC2-M2AP complex is required for efficient invasion.\",\n      \"method\": \"Gene knockout, immunofluorescence, invasion assay, secretion assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with quantified invasion phenotype and mechanistic follow-up on MIC2 trafficking\",\n      \"pmids\": [\"12727875\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MIC2 and M2AP form a stable hexameric complex of ~450 kDa consisting of three αβ dimers, as determined by native blue gels and gel filtration of parasite lysates. The cytoplasmic C-domain of MIC2 is not required for MIC2-M2AP complex formation or microneme trafficking.\",\n      \"method\": \"Native blue gel electrophoresis, gel filtration, transgenic expression of C-domain deletion mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution with size analysis, mutagenesis in parasite\",\n      \"pmids\": [\"14670959\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The integrin-like A-domain of MIC2 binds specifically to heparin (a sulfated proteoglycan of the extracellular matrix) as a high-molecular-weight multimeric species; this binding does not depend on the MIDAS divalent cation-binding motif.\",\n      \"method\": \"Recombinant A-domain fusion protein binding assays, heparin binding assay, size analysis of binding species\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assay with recombinant protein and MIDAS mutagenesis, single lab\",\n      \"pmids\": [\"15003840\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Eimeria tenella MIC1 (the ortholog of TgMIC2) can substitute for TgMIC2 to enable the first TgMIC2 knockout clone; in TgMIC2 KO parasites, M2AP is misprocessed and mistrafficked to the parasitophorous vacuole rather than the micronemes, demonstrating mutual dependence for correct trafficking.\",\n      \"method\": \"Trans-genera complementation, gene knockout, immunofluorescence, Western blot\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout enabled by trans-genera complementation, orthogonal methods for trafficking analysis\",\n      \"pmids\": [\"15236644\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Aldolase bridges MIC2 to filamentous actin via the MIC2 cytoplasmic tail, coupling the adhesin to the actomyosin motor. The penultimate tryptophan and flanking acidic residues in the MIC2 cytoplasmic tail are required for aldolase binding.\",\n      \"method\": \"In vitro binding assay (aldolase-MIC2 tail interaction), homology modeling, mutagenesis\",\n      \"journal\": \"Cell host & microbe (referenced in Starnes 2006); original aldolase-MIC2 interaction established in prior work cited by Starnes GL et al.\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding assay with mutagenesis, single lab at this stage\",\n      \"pmids\": [\"16923803\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"cGMP-dependent protein kinase (PKG) acts downstream of calcium-dependent events to regulate MIC2 secretion, gliding motility, host cell attachment, and invasion; PKG inhibition blocks MIC2 adhesin secretion even when calcium is elevated by ionophores, and PKG-refractory transgenic strains show reduced sensitivity to this inhibition.\",\n      \"method\": \"Pharmacological inhibition (compound 1/trisubstituted pyrrole), transgenic PKG mutant strains, immunofluorescence, invasion/motility assays\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — transgenic validation with PKG mutant strains plus pharmacological data, multiple assays\",\n      \"pmids\": [\"15003497\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Eimeria tenella\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TgROM5, a rhomboid intramembrane serine protease localized predominantly to the posterior of the parasite, colocalizes with MIC2 at the posterior cleavage site during invasion and is the only T. gondii rhomboid able to cleave MIC adhesins in a cell-based assay, identifying it as the likely MPP1 protease responsible for MIC2 shedding.\",\n      \"method\": \"Cloning and expression of five TgROMs, GFP localization, cell-based cleavage assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-based cleavage assay plus colocalization, but later challenged by ROM4 studies\",\n      \"pmids\": [\"15753289\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human ICAM-1 immunoprecipitates the mature surface form of MIC2, indicating a direct interaction between parasite MIC2 and host ICAM-1 that contributes to transepithelial migration; soluble ICAM-1 and ICAM-1 antibodies inhibit parasite transmigration across cellular barriers.\",\n      \"method\": \"Immunoprecipitation of parasite surface MIC2 with soluble human ICAM-1, transmigration inhibition assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional inhibition, single lab\",\n      \"pmids\": [\"15760456\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Conditional suppression of MIC2 causes mistrafficking of M2AP, markedly defective host-cell attachment and invasion, loss of helical gliding motility, and inability to establish lethal infection in mice, establishing the MIC2-M2AP complex as a major virulence determinant.\",\n      \"method\": \"Conditional (anhydrotetracycline-regulatable) MIC2 expression system, endogenous mic2 knockout, invasion/motility/attachment assays, murine infection model\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with multiple orthogonal phenotypic readouts including in vivo virulence\",\n      \"pmids\": [\"16933991\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Two separate acidic residue clusters in the MIC2 cytoplasmic tail are essential for parasite survival: the C-terminal cluster is required for direct aldolase binding, while an upstream acidic cluster is essential but does not mediate aldolase binding, implying a second critical cytoskeletal interaction.\",\n      \"method\": \"Alanine point mutagenesis, in vitro aldolase binding assay, transgenic complementation, parasite survival assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with in vitro binding assay and in vivo complementation\",\n      \"pmids\": [\"16923803\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The M2AP propeptide is required for the MIC2-M2AP complex to exit an early endosomal compartment; deletion of the propeptide traps the complex in the early secretory pathway, while propeptide cleavage-resistant mutants reach micronemes but cannot rapidly mobilize to the parasite surface. Both defects impair invasion and reduce virulence.\",\n      \"method\": \"Propeptide deletion and cleavage-site mutagenesis, immunofluorescence/immunoelectron microscopy, invasion assay, in vivo virulence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis of propeptide with orthogonal localization and functional readouts\",\n      \"pmids\": [\"16914527\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Aldolase is essential for efficient host cell invasion through its ability to bridge MIC2 cytoplasmic tail to actin filaments; mutations in TgALD1 that selectively abolish MIC2 tail binding (without affecting glycolytic activity) decrease invasion efficiency without affecting motility, while mutations affecting glycolysis reduce motility.\",\n      \"method\": \"Structure-guided mutagenesis of TgALD1, conditional knockout complementation, in vitro MIC2 tail binding assay, motility and invasion assays\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure-guided mutagenesis, in vitro binding, and genetic complementation with delineated functions\",\n      \"pmids\": [\"19380114\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgROM4 is the primary rhomboid protease responsible for shedding of surface adhesins including MIC2; conditional knockout of TgROM4 decreases MIC2 release into supernatant, increases surface MIC2 accumulation, disrupts normal gliding (causing twirling), and severely impairs invasion despite enhanced host cell binding.\",\n      \"method\": \"Conditional knockout, surface adhesin shedding assay, supernatant MIC2 quantification, gliding motility and invasion assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with multiple orthogonal mechanistic readouts\",\n      \"pmids\": [\"20421941\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgSUB1 (a GPI-anchored microneme subtilisin protease) is responsible for proteolytic trimming of MIC2 (and MIC4, M2AP) on the parasite surface after secretion; loss of TgSUB1 abolishes this trimming, decreases cell attachment and gliding efficiency, reduces invasion rates, and lowers virulence in mice.\",\n      \"method\": \"Gene deletion, MIC2 processing assay, attachment and invasion assays, in vivo virulence\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean gene deletion with multiple orthogonal phenotypic and biochemical readouts\",\n      \"pmids\": [\"20678172\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROM4 (not ROM5) is the primary rhomboid protease for MIC2 cleavage in T. gondii tachyzoites; ROM4 single knockout blocks MIC2 shedding and causes surface accumulation, nonproductive attachment, altered gliding, impaired moving junction formation, and reduced invasion. Triple knockout (ROM1/ROM4/ROM5) parasites are viable, demonstrating ROM-independent mechanisms for adhesin removal during actual cell entry.\",\n      \"method\": \"Single, double, and triple rhomboid knockouts, MIC2 shedding assay, motility and invasion assays, moving junction analysis\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic single/double/triple KO approach with multiple mechanistic readouts; reconciles prior ROM5 data\",\n      \"pmids\": [\"25336455\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of N-terminal proMIC2 and MIC2 fragments reveal a closed conformation of the von Willebrand A (VWA) domain, its association with the TSR1 domain, a proline-rich pigtail loop in TSR1 overlapping the VWA domain, α-C-linked mannose on Trp-276, and a mobile α5-helix with a disordered region replacing the α6-helix in a putative activation mechanism. M2AP binds as a 1:1 monomer to TSR6 in a highly elongated complex.\",\n      \"method\": \"X-ray crystallography of MIC2-M2AP ectodomain and sub-fragments, small-angle X-ray scattering\",\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 determination with multiple constructs and functional domain mapping\",\n      \"pmids\": [\"24639528\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MIC2 contributes to initial adhesion of T. gondii tachyzoites to vascular endothelium under shear stress but is not required for adhesion strengthening, as shown by comparison of wild-type and MIC2-deficient parasites in a microfluidic system.\",\n      \"method\": \"Microfluidics, time-lapse fluorescence microscopy, MIC2-deficient parasite comparison under shear stress\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MIC2 knockout with defined adhesion phenotype, single lab, specialized assay\",\n      \"pmids\": [\"24692639\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The NMR solution structure of M2AP globular domain reveals a modified galectin fold similar to MIC1 C-terminal domain; hydrophobic residues on one face of this galectin fold mediate binding to the sixth TSR domain of MIC2 (TSR6), as validated by NMR and immunoprecipitation.\",\n      \"method\": \"NMR structure determination, immunoprecipitation, domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional binding validation by IP, single lab\",\n      \"pmids\": [\"25411252\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ROM4 is the primary contributor to MPP1 activity (intramembrane cleavage of MIC2 and MIC6), while ROM5 is dispensable for MIC2 shedding; ROM4 knockout parasites engage predominantly in twirling motility and show enhanced attachment but impaired invasion, whereas ROM5 knockout does not alter MIC2 shedding or invasion.\",\n      \"method\": \"Cre-recombinase and CRISPR-Cas9 knockouts of ROM4 and ROM5, MIC2/MIC6 cleavage assay, motility and invasion assays\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent gene disruption methods, multiple assays, confirms ROM4 primacy\",\n      \"pmids\": [\"25846828\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Glideosome-associated connector (GAC), an armadillo/PH domain protein, directly binds filamentous actin and specifically associates with TgMIC2, connecting the MIC2 adhesin to the actomyosin system to mediate gliding motility, invasion, and egress. Apical localization of TgGAC depends on TgAKMT (apical lysine methyltransferase). GAC's PH domain also binds phosphatidic acid, a lipid linked to microneme exocytosis.\",\n      \"method\": \"Co-immunoprecipitation (TgGAC-TgMIC2), F-actin binding assay, localization by IFA, conditional knockdown of TgGAC, TgAKMT perturbation\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro binding, conditional knockdown with defined phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"27978434\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MIC2 null mutant parasites are deficient in surface attachment but retain near-normal gliding speed and host cell invasion once initiated; deletion of mic2 causes delayed host cell egress in vitro, and causes only mild attenuation of virulence in vivo (slight delay, not prevention of lethal disease).\",\n      \"method\": \"Gene deletion (null mutant), 3D motility analysis, attachment under shear stress, invasion assay, in vivo murine infection\",\n      \"journal\": \"Wellcome open research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean null mutant with careful quantification, multiple assays; provides nuanced refinement of MIC2 role\",\n      \"pmids\": [\"28630943\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MIC2 TSR domains are modified by O-fucosylation (GlcFuc disaccharide added by POFUT2/NST2) and C-mannosylation; loss of POFUT2 or NST2 markedly reduces cellular MIC2 levels, causes partial mislocalization from micronemes, and impairs host cell attachment and invasion comparable to the mic2 knockout, demonstrating that O-fucosylation is required for efficient MIC2 processing and function.\",\n      \"method\": \"Mass spectrometry glycan mapping, gene knockout (Δpofut2, Δnst2), immunofluorescence, invasion and attachment assays, anti-GlcFuc antibody detection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — MS glycan characterization combined with genetic knockouts and functional phenotyping\",\n      \"pmids\": [\"30538131\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TgPOFUT2 O-glycosylates MIC2 TSR domains (≥3 O-linked sites with >95% occupancy confirmed by MS/MS), but loss of TgPOFUT2 has only a modest impact on MIC2 levels and parasite invasion in this study, suggesting the O-glycan may be dispensable in T. gondii tachyzoites (in contrast to findings in another study).\",\n      \"method\": \"Mass spectrometry (MS/MS), enzymatic digestion, TgPOFUT2 gene knockout, plaque and invasion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous MS characterization of glycosylation sites, but functional conclusion contradicts PMID 30538131; single lab, conflicting evidence lowers confidence\",\n      \"pmids\": [\"30514763\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"C-mannosylation of MIC2 TSR domains is catalyzed by apicomplexan DPY19 homologs; T. gondii and P. falciparum DPY19 enzymes modify WXXWXXC motifs in TRAP/MIC2 TSR domains when expressed in mammalian cells deficient in C-mannosylation, and can transfer mannose to WXXWXXC but not WXXW peptides in vitro.\",\n      \"method\": \"Expression of apicomplexan DPY19 in C-mannosylation-deficient mammalian cells, in vitro C-mannosyltransferase assay with peptide substrates\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay and cell-based complementation, clear substrate specificity mapping\",\n      \"pmids\": [\"29432542\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deletion of dpy19 (C-mannosyltransferase) reduces MIC2 protein levels and impairs parasite adhesion, motility, host cell invasion, and egress; attenuation of virulence in Δdpy19 parasites cannot be fully explained by reduced MIC2 alone, suggesting C-mannosylation stabilizes additional invasion-related proteins.\",\n      \"method\": \"Gene knockout (Δdpy19), immunofluorescence, motility and invasion assays, in vivo virulence, Western blot for MIC2 levels\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with multiple assays and in vivo validation\",\n      \"pmids\": [\"31862733\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"During intracellular replication, micronemes (tracked by MIC2 labeling) are recycled from mother to daughter parasites via a highly dynamic F-actin network; this recycling is F-actin dependent, while de novo microneme synthesis is F-actin independent. The residual body acts as a storage and sorting station for micronemal vesicles.\",\n      \"method\": \"Dual labeling, super-resolution microscopy, F-actin perturbation, live imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live super-resolution imaging plus genetic/pharmacological perturbation of F-actin\",\n      \"pmids\": [\"31519913\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"3D traction force mapping shows that a moving T. gondii parasite generates periodic, inward-directed forces at a fixed circular attachment zone on the extracellular matrix; parasites lacking TgMIC2 fail to generate these inward-directed forces and lack the constriction in the plasma membrane at this zone, and display less straight trajectories, demonstrating that TgMIC2 mediates the annular attachment zone that constrains directional motility.\",\n      \"method\": \"3D traction force microscopy, TgMIC2-null parasites, live imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — novel biophysical method (3D traction force mapping) with genetic null mutant validation\",\n      \"pmids\": [\"36519527\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of M2AP alone and in complex with MIC2 TSR6 reveal that the non-layered side of TSR6, centered on hotspot residue His-620, binds M2AP; TSR6 residue Y602 shifts from a layered to a flipped-out state upon M2AP binding, stabilizing the complex. This interaction is required for MIC2-M2AP complex maturation and transport to the parasite surface.\",\n      \"method\": \"X-ray crystallography of M2AP alone and M2AP-TSR6 complex, mutagenesis (H620A and Y602 mutants), functional validation\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus mutagenesis, builds on prior NMR study\",\n      \"pmids\": [\"37652989\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgMIC2 is a transmembrane adhesin stored in micronemes and released apically upon Ca2+/PKG-dependent stimulation; it treadmills posteriorly via the actomyosin motor (connected through its cytoplasmic tail to aldolase/F-actin and GAC), mediates a circumferential attachment zone that directs gliding motility, binds host cell surface receptors (including heparin and ICAM-1) through its von Willebrand A and TSR domains, and is shed from the parasite surface by the rhomboid intramembrane protease ROM4 to complete cell entry. MIC2 forms a heterohexameric complex with M2AP (bound at TSR6) that is required for correct microneme trafficking of both proteins and for efficient invasion; the complex is stabilized by O-fucosylation and C-mannosylation of TSR domains, and multiple proteases (SUB1 for surface trimming, ROM4 for intramembrane shedding) sequentially process MIC2 to regulate adhesion and cell entry.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgMIC2 is a transmembrane microneme adhesin that acts as the earliest effector of the Toxoplasma gondii invasion sequence, coupling host-cell recognition to the parasite's substrate-dependent gliding motility [#0, #17]. It is stored in micronemes and discharged apically at the tight attachment zone upon a Ca2+/PKG-dependent signal, after which the adhesin treadmills posteriorly in an actomyosin-dependent manner and is capped at the posterior pole [#1, #14]. Two conserved cytoplasmic-tail motifs (SYHYY and EIEYE) are necessary and sufficient for microneme targeting [#5], and the same tail engages the cytoskeletal machinery: a C-terminal acidic cluster and penultimate tryptophan mediate direct aldolase binding that bridges MIC2 to F-actin, while an additional upstream acidic cluster and the glideosome-associated connector GAC provide further links to the actomyosin motor [#13, #18, #20, #28]. On its extracellular face, MIC2 recognizes host ligands through its integrin-like von Willebrand A domain (which binds sulfated proteoglycan/heparin) and its thrombospondin type-1 repeats, and its mature surface form directly engages host ICAM-1 to support transepithelial migration [#11, #16, #24]. MIC2 forms a stable ~450 kDa heterohexameric complex of three αβ dimers with M2AP, which binds the TSR6 domain; this interaction is mutually required for correct microneme trafficking of both partners and for efficient invasion [#6, #9, #10, #24, #26, #36]. The TSR domains are further stabilized by O-fucosylation and C-mannosylation, modifications required for efficient MIC2 processing, trafficking, and function [#30, #32, #33]. Cell entry requires sequential proteolysis: TgSUB1 trims the surface ectodomain, and the rhomboid intramembrane protease ROM4 sheds MIC2 from the membrane, because shedding rather than adhesion per se is the step that permits productive invasion [#8, #21, #22, #23]. Functionally, MIC2 mediates a circumferential attachment zone that generates periodic inward-directed traction forces to constrain directional gliding, and its loss impairs surface attachment and delays egress while leaving core gliding and invasion machinery partly operational [#25, #29, #35].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing when MIC2 acts in invasion showed that its micronemal release is the first molecular event of host-cell entry, placing MIC2 upstream of rhoptry and dense-granule discharge.\",\n      \"evidence\": \"Immunofluorescence, immunoelectron microscopy and quantitative immunoassay during live invasion\",\n      \"pmids\": [\"9208224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the trigger for release\", \"Did not define the host receptor bound at the attachment zone\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"The signal controlling MIC2 discharge was unknown; pharmacological manipulation showed intracellular Ca2+ triggers apical release and actomyosin-dependent posterior capping, linking secretion to motility.\",\n      \"evidence\": \"Ca2+ ionophore and BAPTA chelation with immunofluorescence and cytoskeletal inhibitor assays\",\n      \"pmids\": [\"10027960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream kinase effectors not yet defined\", \"Molecular link between MIC2 and the actomyosin motor not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"It was unclear whether proteolysis affected adhesion; binding assays showed only the full-length surface form of MIC2 binds host cells, establishing that shedding inactivates the adhesin, and that a staurosporine-sensitive kinase governs discharge.\",\n      \"evidence\": \"Host-cell binding assays comparing full-length vs shed MIC2, and staurosporine inhibition of secretion/invasion\",\n      \"pmids\": [\"11207555\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase identity not established\", \"Single-lab binding comparison\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The targeting signal and processing events of MIC2 were undefined; mutagenesis identified two cytoplasmic-tail motifs sufficient for micronemal delivery and mapped distinct N- and C-terminal cleavages by parasite proteases MPP1/MPP2.\",\n      \"evidence\": \"Site-directed mutagenesis, SAG1 chimera targeting, and protease inhibitor domain-mapping assays\",\n      \"pmids\": [\"10982850\", \"10799515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Protease identities (MPP1/MPP2) not yet molecularly resolved\", \"Trafficking machinery reading the tail motifs unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"The composition of the MIC2 secretory unit was unknown; co-IP and pulse-chase showed M2AP associates with MIC2 rapidly after synthesis and remains bound through the micronemes, surface and shed forms.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, pulse-chase labeling and orthogonal localization\",\n      \"pmids\": [\"11532123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the association not yet tested\", \"Stoichiometry of the complex not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Whether intramembrane cleavage and C-terminal shedding matter for entry was open; mapping of an intramembrane IAGG cleavage site and mutants blocking C-terminal cleavage showed shedding is required for invasion despite increased adhesion.\",\n      \"evidence\": \"Cleavage-site mutagenesis, SAG1-TMD chimeras, and invasion/adhesion assays\",\n      \"pmids\": [\"11927542\", \"12471033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rhomboid responsible for intramembrane cleavage not yet identified\", \"Mechanistic reason adhesion must be released not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The functional role of the MIC2-M2AP association was untested; knockout and biochemical sizing showed the two proteins are mutually dependent for correct trafficking and form a ~450 kDa hexamer of three αβ dimers required for efficient invasion.\",\n      \"evidence\": \"M2AP gene knockout with trafficking/invasion assays, and native gel/gel filtration sizing\",\n      \"pmids\": [\"12727875\", \"14670959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the interaction not resolved\", \"Cytoplasmic C-domain shown dispensable for complex, its function unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"How MIC2 recognizes host surfaces and couples to the motor was unclear; the VWA A-domain was shown to bind heparin MIDAS-independently, aldolase was shown to bridge the tail to F-actin, and PKG was placed downstream of Ca2+ controlling secretion.\",\n      \"evidence\": \"Recombinant A-domain heparin-binding assays, aldolase-tail binding with mutagenesis, and PKG pharmacology with transgenic PKG-refractory strains\",\n      \"pmids\": [\"15003840\", \"16923803\", \"15003497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of heparin binding not established\", \"Nature of a second tail-cytoskeleton link not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Eimeria tenella\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The identity of the shedding protease and a host receptor were open; TgROM5 was proposed as the posterior MIC-cleaving rhomboid, and host ICAM-1 was shown to bind mature surface MIC2 and support transmigration.\",\n      \"evidence\": \"Rhomboid cloning/cleavage assays with GFP localization, and ICAM-1 immunoprecipitation with transmigration inhibition\",\n      \"pmids\": [\"15753289\", \"15760456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ROM5 assignment later revised in favor of ROM4\", \"ICAM-1 co-IP from single lab without structural mapping\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The in vivo importance and tail requirements of MIC2 were untested; conditional MIC2 suppression established the complex as a virulence determinant, tail mutagenesis defined two essential acidic clusters, and M2AP propeptide mutants revealed a trafficking checkpoint.\",\n      \"evidence\": \"Conditional MIC2 knockout with in vivo virulence, tail alanine-scanning with aldolase binding, and M2AP propeptide deletion/cleavage mutants\",\n      \"pmids\": [\"16933991\", \"16923803\", \"16914527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the second tail-binding cytoskeletal factor unknown\", \"Compartment gating the propeptide-dependent exit not fully defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Whether aldolase acts as a moonlighting motor bridge versus a glycolytic enzyme was ambiguous; structure-guided TgALD1 mutants separated MIC2-tail binding (needed for invasion) from glycolysis (needed for motility).\",\n      \"evidence\": \"Structure-guided TgALD1 mutagenesis with conditional-knockout complementation, in vitro tail binding, and motility/invasion assays\",\n      \"pmids\": [\"19380114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How force is transmitted through aldolase to actin not directly measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The proteases executing MIC2 processing were reassessed; conditional knockout identified ROM4 as the primary intramembrane sheddase and gene deletion showed TgSUB1 performs surface trimming, both required for efficient attachment and invasion.\",\n      \"evidence\": \"Conditional ROM4 knockout and TgSUB1 gene deletion with shedding, motility, invasion and virulence assays\",\n      \"pmids\": [\"20421941\", \"20678172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ROM4 is essential during actual cell entry not resolved\", \"Order of SUB1 vs ROM4 action not fully sequenced\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The rhomboid assignment and complex architecture were clarified; systematic ROM knockouts confirmed ROM4 (not ROM5) as the main sheddase and revealed ROM-independent entry mechanisms, while crystallography defined the elongated MIC2-M2AP ectodomain, a closed VWA domain, C-mannosylation, and 1:1 M2AP binding to TSR6.\",\n      \"evidence\": \"Single/double/triple rhomboid knockouts with motility/invasion/moving-junction analysis, and X-ray crystallography with SAXS; microfluidic shear-adhesion assay\",\n      \"pmids\": [\"25336455\", \"24639528\", \"24692639\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of ROM-independent adhesin removal during entry unknown\", \"Activation mechanism of the VWA domain inferred but not captured in an open state\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The relative rhomboid contributions were confirmed and refined; independent knockouts established ROM4 as the primary MPP1 activity while ROM5 was dispensable for MIC2 shedding and invasion.\",\n      \"evidence\": \"Cre-recombinase and CRISPR-Cas9 knockouts of ROM4 and ROM5 with cleavage, motility and invasion assays\",\n      \"pmids\": [\"25846828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Residual shedding pathway during entry still unaccounted for\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The structural basis of M2AP recognition was defined; the NMR structure of M2AP revealed a galectin fold whose hydrophobic face binds MIC2 TSR6.\",\n      \"evidence\": \"NMR structure determination with immunoprecipitation and domain mapping\",\n      \"pmids\": [\"25411252\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic detail of the TSR6 interface awaited co-crystal structure\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A second cytoskeletal connector was sought; GAC was shown to directly bind F-actin and MIC2, and to bind phosphatidic acid via its PH domain, providing the additional motor link implied by the earlier upstream acidic-cluster requirement.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro F-actin binding, conditional GAC knockdown and TgAKMT perturbation\",\n      \"pmids\": [\"27978434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GAC and aldolase act on the same or distinct MIC2 tail motifs not fully resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The strict essentiality of MIC2 was re-examined; a clean null mutant showed MIC2 is deficient in attachment but retains near-normal gliding speed and invasion once initiated, delays egress, and is only mildly attenuated in vivo, refining MIC2 to an attachment-focusing rather than obligatory motility factor.\",\n      \"evidence\": \"mic2 null mutant with 3D motility, shear attachment, invasion and murine infection assays\",\n      \"pmids\": [\"28630943\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of functional redundancy with other adhesins not defined\", \"Reconciliation with earlier conditional-knockout severe phenotype not fully explained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The role of TSR glycosylation was tested; MS mapping plus knockouts showed O-fucosylation (POFUT2/NST2) and C-mannosylation (DPY19) modify MIC2 TSRs, with O-fucosylation loss reducing MIC2 levels and invasion, though a second study found only modest impact of POFUT2 loss.\",\n      \"evidence\": \"MS/MS glycan mapping, Δpofut2/Δnst2 knockouts, DPY19 in vitro C-mannosyltransferase assays, and functional phenotyping\",\n      \"pmids\": [\"30538131\", \"30514763\", \"29432542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conflicting POFUT2 functional phenotypes between labs\", \"Whether glycans act on folding, stability or trafficking specifically not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The cellular impact of C-mannosylation and micronemal dynamics were addressed; dpy19 deletion reduced MIC2 and impaired adhesion/invasion/egress beyond MIC2 alone, and imaging showed MIC2-marked micronemes are recycled from mother to daughter via an F-actin network.\",\n      \"evidence\": \"Δdpy19 knockout with functional/virulence assays, and super-resolution live imaging with F-actin perturbation\",\n      \"pmids\": [\"31862733\", \"31519913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Additional C-mannosylated invasion proteins not enumerated\", \"Machinery driving F-actin-dependent microneme recycling not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The biophysical role of MIC2 in directing motility was quantified; 3D traction force mapping showed MIC2 is required to form an annular attachment zone generating periodic inward forces that constrain directional gliding.\",\n      \"evidence\": \"3D traction force microscopy with TgMIC2-null parasites and live imaging\",\n      \"pmids\": [\"36519527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How MIC2 organizes the circular zone molecularly not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The atomic basis of MIC2-M2AP assembly was resolved; co-crystal structures showed TSR6 hotspot His-620 and a Y602 conformational flip stabilize M2AP binding, an interaction required for complex maturation and surface transport.\",\n      \"evidence\": \"X-ray crystallography of M2AP alone and M2AP-TSR6 with H620A/Y602 mutagenesis and functional validation\",\n      \"pmids\": [\"37652989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full hexamer not determined\", \"Link between TSR6 binding and downstream trafficking machinery not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The ROM-independent mechanism that removes MIC2 during actual cell entry, and the identity of the second (non-aldolase) cytoskeletal factor binding the upstream tail cluster, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adhesin disengagement pathway during entry in the absence of rhomboids unknown\", \"Full molecular inventory of the MIC2 tail-cytoskeleton interface incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0050839\", \"supporting_discovery_ids\": [11, 16, 24, 25, 35], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0008201\", \"supporting_discovery_ids\": [11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051015\", \"supporting_discovery_ids\": [13, 20, 28], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005178\", \"supporting_discovery_ids\": [16], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [0, 1, 5, 34], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0009986\", \"supporting_discovery_ids\": [2, 8, 16, 21], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016021\", \"supporting_discovery_ids\": [7, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0030587\", \"supporting_discovery_ids\": [0, 17, 25, 29, 35], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0071689\", \"supporting_discovery_ids\": [13, 20, 28, 35], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0002250\", \"supporting_discovery_ids\": [17, 22, 33], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016485\", \"supporting_discovery_ids\": [4, 7, 8, 21, 22, 23], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006486\", \"supporting_discovery_ids\": [30, 31, 32, 33], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"MIC2-M2AP heterohexamer (~450 kDa, three αβ dimers; M2AP bound at TSR6)\"\n    ],\n    \"partners\": [\n      \"M2AP (microneme protein, TSR6-binding partner)\",\n      \"Aldolase (TgALD1; MIC2 cytoplasmic-tail-to-F-actin bridge)\",\n      \"GAC / glideosome-associated connector (F-actin and MIC2 binding)\",\n      \"Host ICAM-1 (host receptor for mature surface MIC2)\",\n      \"Heparin / sulfated proteoglycan (VWA A-domain ligand)\",\n      \"ROM4 (rhomboid intramembrane sheddase)\",\n      \"TgSUB1 (subtilisin surface-trimming protease)\",\n      \"POFUT2/NST2 (O-fucosylation of TSR domains)\",\n      \"DPY19 (C-mannosyltransferase of TSR domains)\",\n      \"PKG (cGMP-dependent kinase regulating secretion)\"\n    ],\n    \"other_free_text\": [\n      \"MIC2 cytoplasmic tail contains SYHYY and EIEYE motifs necessary and sufficient for micronemal targeting.\",\n      \"Two acidic clusters in the tail are essential; the C-terminal cluster (with penultimate Trp) binds aldolase, an upstream cluster mediates a distinct cytoskeletal link.\",\n      \"TSR domains carry O-fucosylation (GlcFuc) and C-mannosylation required for efficient processing/function.\",\n      \"Only the full-length surface form is adhesive; proteolytic shedding inactivates adhesion and is required for productive cell entry.\",\n      \"MIC2 mediates a circular attachment zone generating periodic inward-directed traction forces that direct gliding.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_201780","name":"MIC2","product":"microneme protein MIC2","uniprot_acc":"S8F9G8","chromosome":"TGME49_chrVIIa","start_pos":3793319},{"strain":"GT1","gene_id":"TGGT1_201780","name":null,"product":"microneme protein MIC2","uniprot_acc":null,"chromosome":"TGGT1_chrVIIa","start_pos":3693727},{"strain":"RH88","gene_id":"TGRH88_037170","name":null,"product":"microneme protein MIC2","uniprot_acc":null,"chromosome":"CM023088","start_pos":3888072},{"strain":"VEG","gene_id":"TGVEG_201780","name":null,"product":"microneme protein MIC2","uniprot_acc":"A0A0F7UZH2","chromosome":"TGVEG_chrVIIa","start_pos":3881171}]}