PMC

Fluorescence images (projections of a deconvolved 3D stack) of two different vacuoles (A–C) (D–F), each containing four parasites expressing both EGFP–TgDLC and mRFP–TgCentrin2. Blue brackets in (A–C) mark the position of the apical cap of dynein. Note that the ring of TgCentrin2 spots is always positioned at the lower border of this cap. The arrowheads in (D–F) indicate the faint basal ring of dynein that lies adjacent to the basal spot of centrin2.

(A) Combined DIC and epifluorescence image of the starting material, a suspension of extracellular YFP-α-tubulin (green) transgenic parasites.
(B) Epifluorescence image of detergent extracted, sonicated parasites. The fluorescent material, a small fraction of the total mass, is composed of small MT fragments (cyan arrows) and conoids (red arrows). The inset shows an intact “ghost,” a detergent extracted parasite, at 2× higher magnification. The total cytoskeleton prep was fractionated by differential centrifugation into the conoid-depleted (C) and conoid-enriched (D) fractions.

(A) EM image of a thin section through a pellet of the centrifuged conoid-enriched fraction.
(B) To give a rough visual estimate of the enrichment, material that is recognizably fragments of the conoid + polar rings has been colored red (C).
(C) EM analysis of the conoid-depleted fraction; MT are marked with cyan lines, which are much wider than the image of the MT at this magnification.
(D) Enlarged view of the region within the yellow box, showing small vesicles and MT (cyan arrows).

Combined DIC and fluorescence images are shown for the beginning (t = 0) and end (t = 250 min) of the sequence, during which a vacuole of four parasites progresses from the onset of mitosis through completion of budding out of the eight daughters. See text for a detailed description of the sequence of events.

(A–B) Two EM images of Triton-extracted cytoskeletons from a transgenic parasite line expressing T. gondii EGFP–centrin-2, immunogold-labeled with anti-GFP antibody, and negatively stained with phosphotungstic acid. Specific staining is seen at the preconoidal rings (black arrows), and in five ~200nm annular patches (white arrows). After deoxycholate extraction (C), the annular patches disappear almost completely but the preconoidal ring staining remains. The large dark blob in the center of the parasites is primarily attributable to accumulation of the phosphotungstic acid in thicker regions, but may also include some cytoplasmic and centriolar concentrations of centrin-2.

(Upper) The major structural reference points are labeled in a cell containing two growing daughters. The maternal IMC is shown in brown. The unlabeled daughter IMC/scaffold is shown in pink.
(Middle and Lower) The cell cycle is traversed starting with an interphase adult cell at the top left, and proceeding clockwise through early, mid, and late stages of daughter formation. The remnants of the mother are left behind as a “residual body” when the daughters bud out (lower left). The variable color scheme for the organelles is intended to emphasize colocalization (e.g., green Centrin2 + blue DLC = cyan; red MORN1 + blue DLC = magenta; multiple color overlaps are shown in white). Subpellicular MTs are always present in mothers and in daughters from very early on, but are shown only for the interphase adult cell.

(A) LM localization of a T. gondii protein (TgIMC4) with weak similarity to articulin family members, and weaker similarity to TgIMC1. Fluorescence LM images of living transgenic parasites expressing TgIMC4 fused to the C-terminus of EGFP. Fluorescence is observed on both the mother and daughter IMC. FRAP reveals significant differences in the turnover of TgIMC1 and TgIMC4 (unpublished data).
(B and C) Fluorescence LM images of living transgenic parasites expressing GFP-CAM1 (B) and GFP-CAM2 (C). Bright fluorescence is observed in the conoid region of both mother and daughters (arrows).
(D and E) EM images of T. gondii cytoskeletons from the same two lines of transgenic parasites as in (B) and (C [immunogold-labeled with anti-GFP antibody and negatively stained with phosphotungstic acid]). Specific staining occurs over the conoid itself, and lightly over the subpellicular MTs. In (D), diagonal lines of gold particles are visible, tracing the conoid fibers.

(A) Combined DIC and epifluorescence image of human fibroblasts infected with transgenic T. gondii expressing GFP-tubulin (green). Parasitophorous vacuoles containing 1, 2, 4, or 8 parasites are seen.
(B) Drawings of T. gondii (modified from [] and []). Left: a longitudinal section of a dividing cell. Lobes of the dividing nucleus bordered by ER, Golgi (yellow), and developing rhoptry (mauve) are surrounded by the developing daughters' scaffolds (red). Maternal and daughter conoids are shown in green, secretory organelles (rhoptries) in purple. T. gondii has three membranes: a plasma membrane (black) and two additional layers (IMC, red) formed from a patchwork of flattened vesicles. Right: semitransparent view showing subpellicular MT (green).
(C) Enlarged view of the apical complex cytoskeleton, showing the conoid (green), preconoidal, and polar rings (brown), and two intraconoid MT (green). The conoid is formed of 14 fibers of tubulin (not MT), 430 nm long, arranged in a left-handed spiral []. Cytoskeletal elements, including the subpellicular MT (green) and a 2-dimensional lattice of intermediate filament-like proteins (not shown), are closely associated with the cytoplasmic face of the IMC.

(A) Fluorescence LM image of a vacuole containing eight living transgenic parasites expressing EGFP-DLC. Bright fluorescence is observed in the conoid region of both mother and daughters and in an apical cap. See bracket in (B), in centrioles, and in spindle poles; arrows are identification of the centrioles, and spindle poles is based on colocalization with centrin and tubulin [,].
(B) Time-lapse imaging of live EGFP-TgDLC transgenic parasites. Elapsed time after addition of the Ca²⁺ ionophore A23187 is shown at the bottom of each frame. The bright spot of EGFP at the apical end of the parasite (arrow) elongates as the conoid protrudes through the polar ring in response to the elevated intracellular [Ca²⁺] (). After conoid protrusion, the fluorescence is seen to be concentrated in two bands of unequal width. The entire apical end of the cell narrows and elongates at the same time.
(C) Two EM images of Ca²⁺ ionophore–treated, deoxycholate-extracted EGFP–TgDLC transgenic parasites, immunogold-labeled with anti-GFP antibody and negatively stained with phosphotungstic acid. Numerous gold particles are located on the apical and basal ends of the conoid fibers as well as on the polar ring at the sites of attachment of the subpellicular MT, but are sparse along the length of the fibers and MTs, recapitulating the distribution of fluorescence in (B).

(A–C) Single optical section of living transgenic parasites expressing YFP-α-tubulin (green) and mRFP-TgMORN1 (red). Bright red fluorescence is observed at the spindle poles (yellow arrows), near the ends of the growing IMC in developing daughters (pink arrowheads), and in a conical patch at the basal end of the mature parasite. Weaker fluorescence is observed in the region of the conoids of both mother and daughters (blue arrows). Note in (B), showing YFP-tubulin only, that two small green dots are seen in each daughter. The more apical dot is the centriole, the more basal dot (yellow arrow) is the spindle pole [,].
(C) TgMORN1 accumulates at the basal dot (spindle pole, yellow arrow) but not the centriole.
(D–F) Double transgenic parasites expressing IMC1-YFP (green) and mRFP-TgMORN1 (red).
(D) Projection of a deconvolved 3D stack of images. MORN1 is clearly seen to form a ring around the basal end of the developing daughter scaffold.
(E) Projection of a deconvolved 3D stack of images of an extracellular parasite, showing a cytoplasmic fiber of TgMORN1. No daughters are present. Same scale as (D).
(F) Parasite culture treated with oryzalin for 24 h before imaging. A vacuole containing four grossly distorted parasites. IMC1 accumulates as large shells and smaller blobs. f′ & f′′: higher magnification views (rotated 90°) of IMC1 blobs from (F) revealing a core of TgMORN1.

(A–C) Fluorescence LM images of a vacuole containing eight living transgenic parasites expressing mRFP-centrin-2 and YFP-α-tubulin. Bright fluorescence is observed in the extreme apical region (yellow arrows), centrioles (blue arrows), five spots around the circumference near the apical end (green arrows; only two or three are visible in one focal plane with intact parasites), and a single small patch at the basal end (magenta arrows). The left inset in (C) shows an enlarged view of the apical region. The pair of smaller insets show two projected views of a deconvolved 3D stack of images, looking from the side (left) and from the apical end (right) of a single parasite. White arrowheads point to the apical spot; five other spots are organized in a ring around the circumference of the parasite.
(D) Projection of a 3D stack of images of a vacuole containing four parasites from a transgenic parasite line expressing T. gondii EGFP-centrin-2 (green), after 24 h of oryzalin treatment. DNA (blue) is visualized with the cell-permeant dye Hoechst 33342. A host cell nucleus abutting the parasitophorous vacuole (upper right corner of the image) has been digitally attenuated for clarity. Clusters of EGFP-TgCentrin2 dots are dispersed throughout the cells. A minority of the dots occur as closely spaced pairs, presumably daughter centrioles (red arrowheads).