Localization of Apicomplexa Motor Myosin A with Axial Nanometric Precision Using Graphene Energy Transfer.

Apicomplexan parasites exhibit gliding motility, a fast locomotion mode, yet the precise localization of the migration motor myosin A (MyoA) remains unresolved because of limitations in light microscopy axial resolution, leading to two competing models: one placing MyoA in the cytoplasm and the linear model positioning it between the plasma membrane and the inner membrane complex (IMC), a doubled membrane underneath. To distinguish between these models, we applied graphene energy transfer (GET), a fluorescence-based axial ruler with nanometer precision, and determined the vertical position of MyoA relative to IMC1, a structural IMC marker. GET measured IMC dimensions with accuracy comparable to electron microscopy while preserving molecular specificity. The results were independently validated using live stimulated emission depletion (STED) imaging of vertically oriented parasites in agarose cylinders and expansion microscopy STED. Our data localize a major fraction of MyoA within the membrane-IMC space, supporting a linear motor model and establishing GET for nanoscale protein mapping.