(A) The major structural reference points of the T. gondii cytoskeleton referred to throughout this paper are illustrated in the cartoon drawing of an interphase adult parasite. The cytoskeleton of T. gondii includes the apical and the basal complexes, the spindle pole, the centriole, and the cortical cytoskeleton, which includes all of the cytoskeletal elements aligning the parasite body (i.e., cortical microtubules, the IMC, and the filamentous network underlying the IMC), except for the apical and basal complexes.
(B) In interphase parasites, TgMORN1 (green, EGFP-TgMORN1) forms a cap at the extreme basal end of the parasite (green arrow), filling the gap at the basal end of the IMC (blue, labeled by anti-IMC1 monoclonal antibody plus Alexa350-anti-mouse IgG). TgMORN1 also weakly concentrates in the apical complex (dotted square indicated by the white arrow. Stronger contrast enhancement of TgMORN1 labeling is applied to this region to highlight the apical complex labeling of TgMORN1). TgMORN1 is also localized to the spindle pole (green arrowhead), which is juxtaposed to the centriole (red, mCherryFP-TgCentrin1, red arrowhead) during interphase.
(C) In interphase parasites, the basal labeling of TgMORN1 (green arrows) is clearly separated from the cortical microtubules (red, mCherryFP-TgTubA1, small gray arrows). The inset shows a magnified view of the centriole/spindle pole assembly, showing red TgTubA1 in the centriole and green TgMORN1 in the spindle pole. At this point in the cell cycle, tubulin labeling of the spindle pole is weak, but will become stronger in early cell division (cf. ). White arrow, conoid labeling by mCherryFP-TgTubA1.
(D) TgMORN1 and TgCentrin2 occupy different subcompartments within the basal complex, with the TgCentrin2 compartment (red arrow at the basal) located posterior to the TgMORN1 basal labeling (green arrow). These parasites are at the very end of interphase, as indicated by the recent duplication of the centriole in the parasite on the left, but not in the parasite on the right. Green, mCherryFP-TgMORN1 (pseudo-color, for consistency in the color scheme); red, EGFP-TgCentrin1 (pseudo-color); blue, anti-IMC1 antibody detected by Alexa350-anti-mouse IgG. Red arrowheads, TgCentrin2 labeling of the apical polar ring (cf. [A], []). Red arrows near the apical portion of the parasite apical, TgCentrin2 peripheral annuli (cf. [A], []).
Insets are at 2× magnification. The insets in (B) and (D) do not include the anti-IMC1 labeling in order to emphasize the differences in localization betweenTgMORN1/ TgCentrin1 (B) and TgMORN1/TgCentrin2 (D) labeling.
(E) An EM image of an EGFP-TgMORN1 transgenic parasite extracted with 0.5% TritonX-100, immunogold labeled with anti-GFP antibody and negatively stained with phosphotungstic acid. Numerous gold particles are located in the basal complex (bottom inset, green arrows). There is also a small concentration of gold particles in the apical polar ring (top inset, green arrowhead). The cluster of gold particles aligned together (black arrow) at the bottom half of the parasite body is likely to be the labeling of an EGFP-TgMORN1 fiber sometimes seen to form in extracellular parasites [].
(F) An immuno-EM image of an EGFP-TgCentrin2 transgenic parasite, extracted, labeled, and stained as described in (E). As previously reported [], there are clear concentrations of EGFP-TgCentrin2 labeling in the apical polar ring (top inset, red arrowheads) and several peripheral annuli (red arrows). Consistent with the light microscopy data, the basal complex labeling of EGFP-TgCentrin2 is considerably lighter than that of EGFP-TgMORN1 (bottom inset, red arrows). A concentration of gold particles within a ∼250 nm patch at the extreme basal end of the parasite is often seen, which likely corresponds to the concentration of TgCentrin2 basal labeling at the light microscopy level (cf. [D]).
Both TgMORN1 and TgCentrin2 display certain levels of localization along the parasite body, which is likely to be from the proteins in the cytoplasmic pool. Scale bars = 500 nm; Insets are at 1.5× magnification.

Images show a parasitophorous vacuole containing parasites whose centrioles have duplicated, but the spindle pole still appears as one single spot. No TgMORN1 labeling is seen around the centrosomal area other than in the spindle pole itself. In the three parasites at the bottom of the figure, the duplicated centrioles are still near the basal end of the nucleus, whereas in the other five, the centrioles have started or completed their return migration to the apical end of the nucleus.
Green, EGFP-TgMORN1; red, mCherryFP-TgCentrin1; blue, anti-IMC1 antibody detected by Alexa350-anti-mouse IgG.
Inset: 2× magnification of the region indicated by the dotted frame. The inset does not include the anti-IMC1 labeling in order to emphasize the difference in localization between TgMORN1 and TgCentrin1 in the centriole/spindle pole assembly.
All images are maximum intensity projections of deconvolved 3D stacks.

(A) Ring like structures containing TgMORN1 (green, EGFP-TgMORN1, green arrows) are constructed around the duplicated centrioles (red, mCherryFP-TgCentrin1, red arrows) after the duplicated centrioles return to the apical end of the nucleus.
(B) TgCentrin2 (pseudo-colored red, EGFP-TgCentrin2) is undetectable in these TgMORN1 rings (pseudo-colored green, mCherryFP-TgMORN1). Arrows, centriole labeling by TgCentrin2; arrowheads, the mother and the future daughter's apical polar ring labeling by TgCentrin2. Note that a weak concentration of TgCentrin2 fluorescence can be seen at the upper-right portion of the inset in the TgCentrin2 panel. The identity of this concentration is not clear at the present.
Blue, anti-IMC1 antibody detected by Alexa350-anti-mouse IgG. Lack of IMC1 antibody labeling in nascent daughters at this stage might be caused by poor epitope accessibility (cf. and ).
Insets: 2× magnification of regions indicated by the dotted frames.
All images are maximum intensity projections of deconvolved 3D stacks.

(A) A parasitophorous vacuole with parasites at similar stage as those in expressing mCherryFP-TgMORN1 (pseudo-colored green) and EGFP-TgIMC4 (pseudo-colored red). Faint planar concentrations of IMC (red arrows) are surrounded by the TgMORN1 rings (green arrows).
(B) Eight parasites in one parasitophorous vacuole expressing EGFP-TgMORN1 (green) and mCherryFP-TubA1 (red). The EGFP-TgMORN1 rings (green arrows close to the dotted frames in the TgMORN1 and the merged panels) are formed around the centrioles and possibly nascent conoids highlighted by mCherryFP-TgTubA1 (red arrows at the bottom of the TgTubA1 and the merged panels). Both TgMORN1 and TgTubA1 also highlight the spindle poles, indicated by yellow arrows in the merged panel and the corresponding green and red arrows in the TgMORN1 and TgTubA1 panels. Green arrowheads, mother basal complex; red arrowheads, mother conoid.
Insets: 2× magnification of regions indicated by the dotted frames.
All images are maximum intensity projections of deconvolved 3D stacks.

Images are selected time points from a time-lapse experiment tracking the cell division of four parasites expressing EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) (cf. ). See text for detailed description of the time sequence. Top and bottom panels are 2× magnification of regions indicated by the dotted frames. Yellow arrows (t = 0–40 min), the initiation sites of the basal complex around the centrioles; green arrows (t = 50–110 min), daughter basal ring complex; green arrowheads (t = 60–70 min), TgMORN1 labeling of the daughter apical complex; red arrowheads (t = 70 min), the centriole labeling that is just separated from the conoid labeling of mCherryFP-TgTubA1.
All images are maximum intensity projections of deconvolved 3D stacks.

Time-lapse experiment tracking the exchange of EGFP-TgMORN1 in the basal complexes of growing daughters using Fluorescence Recovery After Photo-bleaching (FRAP). The fluorescence of EGFP-TgMORN1 in the basal complexes of two daughters was partially bleached (arrowheads, insets) at time 0, and the recovery was well underway at ∼90 s.
Insets: 2× magnification of regions indicated by the arrowheads.
The merged image of EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) is the maximum intensity projection of a deconvolved 3D stack. All the gray scale images are non-deconvolved single optical planes.

A parasitophorous vacuole containing eight dividing parasites expressing EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) was treated with 2.5 μM oryzalin at time 0. No daughter cortical microtubules can be detected in these parasites, but the initiation (cf. , 33–48 min) and the construction (cf. , 60–120 min) of the basal complex are not affected. As previously reported [,], the spindle pole (arrowheads) fails to replicate in the presence of 2.5 μM oryzalin.
Insets: 2.5× magnification of regions indicated by the dotted frames.
All images are maximum intensity projections of deconvolved 3D stacks.

(A) TgCentrin2 is clearly localized to a ring structure (indicated by dotted frames) in daughter parasites with a partially constricted basal complex.
(B) TgCentrin2 basal labeling (indicated by dotted frames) in the daughter is pronounced during cytokinesis, when the daughters start to take over mother's plasma membrane.
(C) A TgCentrin2 ring (indicated by dotted frames) is also present in parasites that have just finished cytokinesis, but whose basal complexes are not yet fully closed.
Green, mCherryFP-TgMORN1 (pseudo-color); red, EGFP-TgCentrin2 (pseudo-color); blue, anti-IMC1 antibody detected by Alexa350-anti-mouse IgG.
Similar exposure time (0.8 s/plane for and [A and B], and 0.6 s/plane for [C]) and the same brightness, and contrast setting were applied to the TgCentrin2 panels in and this figure to enable comparison of the relative level of Tgcentrin2 in the daughter basal complexes at different stages.
Insets: 2× magnification of regions indicated by the dotted frames. The insets in the rightmost panels merge only TgCentrin2 and TgMORN1 images, and different brightness/contrast settings from the single channel images are applied to these panels to emphasize the difference in localization between these two proteins.
All images are maximum intensity projections of deconvolved 3D stacks.

A collection of the enlarged insets from (left) B; ; A–C and D (right) for side-by-side comparison of morphological and molecular composition differences among basal complexes at different stages of construction and maturation. For ease of comparison, all images are oriented such that the posterior end of the parasite is closer to the bottom. Scale bar = 0.5 μm

A parasite expressing EGFP-TgCentrin2 that has started its cytokinesis. EGFP-TgCentrin2 has already been recruited to the basal complex of the daughters (one of which is indicated by the arrows) at this point. Upon A23187 treatment, the EGFP-TgCentrin2 labeling clearly constricts from ∼0.9 μm (time 0) to ∼0.5 μm (time 0:09:13) in less than 10 min. The morphology of the basal complex in control parasites at a similar stage does not change when treated with 0.1% DMSO alone (unpublished data).
Arrowheads, TgCentrin2 basal complex labeling in the mother cell.
Insets: 2× magnification of the daughter basal complex indicated by the arrows.
All images except for the DIC image are maximum intensity projections of deconvolved 3D stacks.

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 color scheme is such that TgMORN1 containing structures are labeled green (e.g., TgMORN1 basal compartments in the mother and daughter, spindle pole), and all TgCentrin2 containing structures are labeled red (e.g., TgCentrin2 basal compartments in the mother and daughter, centrioles, peripheral TgCentrin2 annuli, and the apical polar ring). Cortical microtubules are labeled gray. The conoid and the plasma membrane are labeled white. The daughter and mother IMCs are labeled with different shades of blue. Cortical microtubules are always present in both the mother and in daughters from early stage of daughter development, but are shown only for the daughter parasites during cytokinesis and for the interphase adult parasite.