BPS2026 - Revealing the nanoscale organization of myosin H in the apical complex of Toxoplasma gondii through 3D single-molecule localization microscopy of gel-expanded samples

Approximately 1 billion people globally are infected by the parasite Toxoplasma gondii . While most infections are asymptomatic, serious disease can occur in the immunocompromised and in fetuses. Despite this significance to human health, there are many gaps in our understanding of its basic biology. The parasite uses an apical complex (AC) comprised of several organelles and structures to invade virtually any animal cell. However, fundamental details of AC organization are only just coming to light, with direct observation hindered by the fact that the AC is approximately a single diffraction-limited volume in size (in the visible). The motor protein myosin H (MyoH) is indispensable for parasite motility and invasion and associates with the conoid, a barrel of uniquely arranged tubulin filaments in the AC. Precisely localizing MyoH is difficult, however, due to the AC’s small spatial extent. To circumvent this, we utilized 3D single-molecule localization microscopy with the double-helix point spread function to determine the protein’s position with 12-nanometer lateral and 22-nanometer axial precision. Utilizing two parasite strains with a C- and N-terminal ALFA tag fused to MyoH, we resolved the truncated-cone arrangement of MyoH in the AC and measured the N terminus to be ∼13 nm radially further away from the C-terminus, thus determining the orientation of the motor protein. We additionally imaged MyoH and tubulin in gel-expanded samples. These two-color reconstructions, improved in labeling efficiency and resolution, determined the organization of MyoH relative to the conoid. Thus, by combining the resolution afforded by 3D single-molecule super-resolution microscopy with the labeling enhancement from gel-based expansion, we determine precisely the organization of this indispensable motor protein, bringing nanoscale detail to a biological process with vast public health implications.