LM and EM Localization of T. gondii Dynein

<div><p>(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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020013#ppat-0020013-b007" target="_blank">7</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020013#ppat-0020013-b009" target="_blank">9</a>].</p><p>(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⁺] (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020013#ppat-0020013-sv001" target="_blank">Video S1</a>). 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.</p><p>(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).</p>