Fig. 1
Conoid architecture and endogenous TgDCX localization. a Diagrams of the T. gondii cytoskeleton [modified from [6]], in which several tubulin containing structures (22 cortical microtubules, 2 intra-conoid microtubules, and 14 conoid fibers) are highlighted in red. EM images of a cross-section of each of those polymers [5] are also shown. Shown in brown are several rings mentioned later in the text. The apical polar ring is the anchoring structure for the 22 cortical microtubules. A complex structure (the preconoidal rings, unlabeled), rich in intricate detail, lying at the apical end of the conoid, is portrayed in this cartoon as two featureless rings. IMC: Inner Membrane Complex. A replicating parasite is also shown, with daughter parasites being built inside the mother. The cortical microtubules of the adult are omitted for clarity. On the right, a cartoon shows how the conoid responds to increasing [Ca2+] by extending and changing its shape. (b-d) Z-projections of SIM images of mCherryFP-TgDCX (red, “K-in mCh-TgDCX”) knock-in parasites [6] expressing mNeonGreenFP-β1-tubulin (green, mNe-TgTub). b Two interphase adult parasites. One adult is outlined with a dashed white border. The arrowhead indicates the apical complex of one parasite, shown 2x enlarged and contrast enhanced in the inset. Tubulin and TgDCX are co-localized in the conoid, appearing as an annulus with a ~ 0.2 μm central opening. c Two dividing parasites at an early stage of daughter formation, with two daughters in each adult. One of the developing daughter’s apical complex is indicated by the arrowhead, and enlarged 1.5x in the inset. d Parasites at a later stage of daughter formation. The daughter apical complexes (white arrowhead) are nearly mature, and daughter cortical microtubules have grown to ~ 1/3 of their length in the adult. e-g Electron microscope (EM) images of the conoid region of negatively stained whole-mount mCherryFP-TgDCX knock-in (e, “K-in mCh-TgDCX”), TgDCX knockout (f, “ΔTgDCX”) parasites (two images), and a complemented line generated by transfecting the TgDCX knockout parasite with a plasmid driving expression of TgDCX-EGFP (g, “Comp”). The conoids are shorter, distorted, and disordered in the TgDCX knockout parasites (f) compared to their parental strain in (e), but supplying TgDCX completely restores conoid structure (g)