PT - JOURNAL ARTICLE AU - Li-av Segev-Zarko AU - Peter D. Dahlberg AU - Stella Y. Sun AU - Daniël M. Pelt AU - James A. Sethian AU - Wah Chiu AU - John C. Boothroyd TI - Ionophore-stimulation promotes re-organization of the invasion machinery of <em>Toxoplasma gondii</em> AID - 10.1101/2022.01.12.476068 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.01.12.476068 4099 - http://biorxiv.org/content/early/2022/01/12/2022.01.12.476068.short 4100 - http://biorxiv.org/content/early/2022/01/12/2022.01.12.476068.full AB - Host cell invasion by intracellular, eukaryotic parasites, like the many important species within the phylum Api-complexa, is a remarkable and active process involving the coordinated action of many apical organelles and other structures. To date, capturing how these various structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to generate images of the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through addition of a calcium ionophore. Using AI-based image-processing we were able to annotate 48 tomograms to identify and extract densities of the relevant subcellular organelles and accurately analyze features in 3D. We describe an interaction between an anteriorly located apical vesicle and a rhoptry tip that occurs only in the ionophore-stimulated parasites and that is associated with dramatic changes in the vesicle’s shape in what appears to be a stalled fusion event. We also present information to support the presumption that this vesicle originates from the well-described vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. Lastly, we show that a previously described rosette is found associated with more than just the anterior-most apical vesicle, indicating that multiple such vesicles are primed to enable rhoptry secretion.Competing Interest StatementThe authors have declared no competing interest.