Fig. 7: The intraconoidal microtubules and functional segregation of secretory organelles within the conoid complex revealed by cryo-FIB milling and cryo-ET.

a–c’ Tomographic slices (a–c: original; a’–c’: pseudo-colored) show: (a, a’) five regularly spaced vesicles (light blue), of which four are tracking along one of the microtubules of the ICMT (light green) and are connected by inter-vesicular linkers (pink); (b, b’) a rhoptry (rose) interacting with the plasma membrane (PM) via the most-apical vesicle (light blue) and the “rosette” docking complex (yellow); and (c, c’) a spiraling scaffold (dark rose) associated with the rhoptry membrane in the rhoptry-neck region. d, e Slices of the subtomogram averaged 8-nm repeats of the ICMTs viewed in cross-sectional (d) and longitudinal (e) orientations. f Occasionally, more than two microtubules were observed in the ICMT complex. Shown here is an example of three microtubules (white arrowheads). g Thirteen-fold rotationally averaged ICMT from 53 subtomograms of detergent-extracted Toxoplasma cells. The arrows indicate the clockwise skew of the protofilaments when the ICMT are viewed from apical to basal, indicating that the minus ends of the ICMTs are oriented apically in the parasite. h, h’ The basal ends of the ICMTs showed flared ends and different lengths of protofilaments, which is usually associated with dynamic plus-ends of MTs. i A tomographic slice of a protruded conoid shows the organization of micronemes. Insert: the subtomogram average of 25 microneme apical tips shows a flattened, electron-dense cap. j, k Tomographic slices provide a side (j) and a top cross-sectional view (k) of two secretory organelles, a microneme (M) and a rhoptry-associated vesicle (V), that are docked side-by-side to the plasma membrane (the vesicle through the rosette (Ro)), but with distinct docking sites (44 nm apart). Note that both organelles are tethered (pink and blue arrows) to the same plasma membrane-anchored ridge (yellow arrows). The white line in (j) indicates the location of the slice in (k). Scale bars: 100 nm (in I); 50 nm (in a–c, f, h, j, k); 20 nm (in i insert), 10 nm (in d, e, g).