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. 2017 Feb 1;28(3):411–428. doi: 10.1091/mbc.E16-08-0587

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© 2017 Nagayasu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — SIM images of mCherryFP-TgDCX (red) and NeonGreenFP-β-tubulin (green) in mCherryFP-TgDCX knock-in parasites. (A) A Z-projection of a 3D stack showing two parasites at the initiation of daughter formation. A 0.5-μm-thick subregion of the stack containing one nascent daughter (dashed cyan border) has been cut out and shown enlarged to the right. The nascent daughters appear as a central bright spot of tubulin with five surrounding lobes, a “five-petaled flower,” which will develop into the daughter apical cytoskeleton. A trace of TgDCX, marking the beginning of daughter conoid formation, is seen just beneath the spot of tubulin. (B) Two parasites with developing daughters at a slightly later stage (roughly 5–10 min) of daughter formation. Microtubules of the spindle are now visible (white arrows). Right, enlargements of the regions bordered by the dashed cyan and magenta borders. The cyan-bordered images are from a 1.6-μm-thick subregion that includes the five-petaled flowers superimposed on the underlying spindle and spindle poles. One daughter is viewed along the future apical basal axis (gray circle), and the other is viewed from the side (gray oval). A thinner subregion (0.125 μm, a single optical section) including only the five-petaled flower of one daughter is shown in the magenta-bordered images. The flower is now larger, and the amount of mCherryFP-TgDCX is increased compared with A. Note that the red channel of the images in the enlargements of A and B have been strongly contrast enhanced to make the small spot of mCherryFP-TgDCX visible. Its true intensity at this stage of daughter formation is ∼1% of the adult mCherryFP-TgDCX spot, that is, not visible in this display without contrast enhancement. (C) Two parasites with much larger daughters, roughly 1 h after B (estimated based on Hu et al., 2002a). The cortical microtubules in the daughters have grown to about one-half of their adult length, and the daughter conoids contain almost the full adult content of TgDCX. (D) Cartoon of a parasite showing the relationship of the developing daughters (green blobs) to the nucleus (purple) and mitotic spindle (cyan rods) during the closed-nuclear division of Toxoplasma. The nucleus appears in B as a dark region from which the diffuse cytoplasmic fluorescence is excluded.