Revealing cellular and molecular organization in Toxoplasma's invasion machinery after ionophore stimulation

Host cell invasion by Toxoplasma gondii and other important species within the phylum Apicomplexa, is an active process involving the coordinated action of a remarkable machine at their anterior end known as the apical complex (AC), for which the phylum is named. To date, fundamental details of the AC and capturing how the various apical organelles interact during invasion have been relatively elusive due, in part, to the fact that the entire complex is approximately a single diffraction-limited volume in size (in the visible). The conoid, a dynamic structure within the AC of Toxoplasma gondii and related coccidia, comprises a barrel of uniquely arranged tubulin-based filaments that protrudes during invasion. Numerous proteins have been shown to localize to the conoid, but many of their functions and precise locations have yet to be determined. Using cryogenic electron tomography, a powerful tool to study cellular architecture in situ, we visualize the parasite's AC in a near-native state. By averaging subvolumes of interest, we generated a density map that allowed us to accurately assign tubulins in the conoid fibrils and reveal the organization of tubulin-associated proteins. Using single-molecule, super-resolution fluorescence microscopy, we have determined with high resolution the molecular organization of Myosin H (Myo-H), a motor protein that associates with the conoid and is indispensable for parasite invasion. We similarly imaged tubulin in the parasites to provide context at the spatial scale necessary to determine how the Myo-H distribution organizes with respect to the AC as a whole. By imaging conoid-protruded and -retracted parasites, we monitor Myo-H reorganization in preparation for host cell invasion, bringing greater detail to this critical biological process.