TGME49_278870

Toxoplasma gondii Myosin F (MyoF) is a class XXII, alveolate-specific actin-based motor that organizes a dynamic cytosolic F-actin network to drive organelle inheritance and apical secretory-organelle positioning during parasite division and invasion (PMID:23695356, PMID:37732764, PMID:41995028). Biochemically, MyoF constructs lacking the C-terminal WD40 β-propeller tail form dimers that bind and translocate actin but are non-processive, whereas the WD40 domain oligomerizes multiple dimers into higher-order complexes that render the full-length motor processive on parasite actin filaments and bundles; the same WD40 domain binds microtubules and enables MyoF to slide actin filaments relative to microtubules (PMID:41995028). Consistent with this filament-translocating (rather than canonical cargo-transport) mode of action, MyoF adopts a dynamic filamentous cytosolic organization, and its loss remodels the interphase actin cytoskeleton and reduces actin dynamics (PMID:38416592). Functionally, MyoF-driven actomyosin activity elongates the apicoplast and promotes its association with the centrosomes, ensuring apicoplast inheritance and preventing accumulation of organelles in enlarged residual bodies (PMID:23695356, PMID:37732764). Through interaction with the rhoptry-associated armadillo-repeat protein TgARO, MyoF positions rhoptries apically, an actomyosin-dependent step required for host-cell invasion (PMID:23498954, PMID:26769898). MyoF folding and assembly depend on the dedicated myosin chaperone TgUNC, whose loss phenocopies MyoF depletion (PMID:28593938).

1. 2013 High

   Establishing that a class XXII myosin is required for organelle inheritance answered whether an actomyosin motor governs apicoplast segregation and centrosome positioning in a dividing parasite.

   Evidence Conditional knockdown and dominant-negative tail overexpression with live-cell imaging of apicoplast and centrosome phenotypes in T. gondii

   PMID:23695356

   Open questions at the time

   • Did not resolve whether MyoF acts as a cargo transporter or an actin-organizing motor
   • Molecular partners at the apicoplast/centrosome not identified
   • In vitro motor activity not measured
2. 2013 High

   Linking MyoF to the rhoptry scaffold TgARO answered how apical secretory organelles are positioned, showing rhoptry localization is an actomyosin-dependent process required for invasion.

   Evidence Co-immunoprecipitation of TgARO with MyoF, conditional TgARO depletion, actin/myosin inhibitor treatment, and rhoptry-positioning microscopy in T. gondii

   PMID:23498954

   Open questions at the time

   • Direct MyoF-binding interface on TgARO not mapped
   • Whether the interaction is direct or bridged not established
3. 2016 Medium

   The crystal structure of TgARO provided a structural rationale for the ARO–MyoF interaction by identifying a candidate myosin-binding surface resembling the UNC-45 myosin-binding domain.

   Evidence Crystal structure of TgARO with structure-guided ARM-repeat mutagenesis, interaction assays, and PfARO complementation in T. gondii

   PMID:26769898

   Open questions at the time

   • Predicted MyoF-binding patch not directly tested by mutagenesis of that patch
   • Structure of the MyoF domain that engages ARO not determined
4. 2017 Medium

   Identifying TgUNC as a dedicated myosin chaperone answered how MyoF and other parasite myosins achieve proper folding and assembly.

   Evidence Conditional TgUNC knockdown with phenotypic recapitulation of myosin loss and genetic epistasis in T. gondii

   PMID:28593938

   Open questions at the time

   • Direct chaperone binding to MyoF not biochemically demonstrated
   • Pathway placement rests on phenome comparison rather than direct interaction
5. 2023 High

   Live-cell imaging of apicoplast dynamics answered what MyoF actually does at the organelle, showing the actomyosin system drives apicoplast elongation and centrosome association rather than passive tethering.

   Evidence Conditional MyoF depletion, actin perturbation, and live-cell imaging of dynamic apicoplast morphologies in T. gondii

   PMID:37732764

   Open questions at the time

   • Molecular link between MyoF-driven actin and the apicoplast membrane not identified
   • How centrosome capture is achieved mechanistically unresolved
6. 2024 Medium

   Characterizing MyoF's cytosolic organization and effect on actin resolved its mode of action, reframing it as an actin-organizing protein acting via an unconventional mechanism rather than a canonical cargo motor.

   Evidence Live-cell imaging of MyoF-EmeraldFP with conditional loss-of-function and quantitative analysis of actin dynamics and cytoskeletal organization in T. gondii

   PMID:38416592

   Open questions at the time

   • The unconventional cargo-movement mechanism not defined at molecular level
   • Why actin organization during replication and motility is spared unexplained
7. 2026 High

   In vitro reconstitution answered how MyoF achieves processivity and organizes actin, showing WD40-mediated oligomerization builds processive higher-order motors that slide actin filaments relative to microtubules.

   Evidence Recombinant MyoF purification, in vitro motility and single-molecule assays, negative-stain EM, size-exclusion chromatography, microtubule-binding/sliding assays, and ΔWD40 domain-deletion mutagenesis

   PMID:41995028

   Open questions at the time

   • Stoichiometry and regulation of oligomer assembly in vivo not established
   • How TgARO and TgUNC interface with the oligomeric motor biochemically not tested

• How MyoF-generated actin dynamics are physically coupled to specific cargo membranes (apicoplast, rhoptries) and how oligomerization is regulated in the living parasite remain open.
• No identified membrane adaptor linking MyoF-driven actin to organelle surfaces
• Regulation of WD40 oligomerization in vivo unknown
• Direct biochemical demonstration of the MyoF–TgARO and MyoF–TgUNC interfaces lacking