Characterization of TgICMAP1 Figure 6. TgICMAP1 localizes to the intra-conoid MTs in T. gondii. (A) In T. gondii, eGFP-TgICMAP1 (green) localized to the apical complex of the parasite (inset), in a region narrower than the width of the conoid marked by mCherryFP-TubA1 (red). eGFP-TgICMAP1 also labeled the centriole/ spindle pole area, which also incorporated mCherryFP-TubA1 (arrows). Inset is at 36 magnification. Scale bar = 2 mm. (B) Images of a parasite expressing mCherryFP-TubA1 (red) fixed and permeablized with methanol and labeled with anti-TgICMAP1 (green) show the prominent apical complex localization of endogenous TgICMAP1, which is similar to that of eGFP-TgICMAP1, but a specific localization in the centriole/spindle pole area could not be confirmed. The punctate labeling in the parasite body is likely to be the combined result of labeling for TgICMAP1 in the cytoplasmic pool and non-specific labeling, as some spots are seen outside the parasite. Inset is at 36 magnification. Scale bar = 2 mm. (C) Western blot of T. gondii total cell lysate showing that anti-TgICMAP1 antibody recognizes a ,135 kDa protein doublet (arrowheads) consistent with the predicted size of TgICMAP1. The blot was stripped and reprobed with mouse-anti-tubulin B-5-1-2 to use a-tubulin as a loading control. (D) Left: Two EM images of Ca2+ ionophore–treated, deoxycholate-extracted RH parasites, which were immunogold-labeled with anti-TgICMAP1 antibody and negatively stained with phosphotungstic acid. Scale bars = 0.5 mm. Middle: Higher magnification of the apical complex regions of the parasites shown in left panels, showing that the intra-conoid MTs (arrows and arrowheads) are decorated with gold particles, recapitulating the distribution of fluorescence in Figure 6 A and B. Scale bars = 0.2 mm. Right: schematic diagram of the T. gondii apical complex. (E and F) Parasites were extracted with deoxycholic acid, and subsequently fixed and stained with anti-tubulin B-5-1-2 (red) and anti-TgICMAP1 (green). The cortical cytoskeleton of the parasite in E was mostly destroyed with the cortical MTs splaying around the apical complex (white arrow). In (F) the cortical cytoskeleton of the parasite was more intact and a ring-like structure stained with anti-tubulin antibody can be observed at the basal end of the parasite (red arrowheads). Scale bars = 2 mm. doi:10.1371/journal.pone.0007406.g006 PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7406