ATPase domain in typical SMC proteins is absent in TgICMAP1, but the presence of the coiled-coil region in the SMC domain internal to TgICMAP1 may suggest anti-parallel dimerization of this molecule through intermolecular association [21]. When expressed at a high level, eGFP-TgICMAP1 colocalizes with perinuclear MTs (Figure 2). To determine if there is any correlation between TgICMAP1 binding and tubulin post-transla- tional modifications, immunofluorescence experiments with anti- acetylated tubulin antibodies were performed. Analysis showed that MTs that were heavily coated with eGFP-TgICMAP1 were also acetylated (Figure 4A). Since acetylation is often a marker for stable MTs [27–29], we wanted to directly test if the binding of eGFP- TgICMAP1 indeed increases MT stability. Upon treatment with 2 mM nocodazole for four hours, 62% of HeLa cells transfected with eGFP-TgICMAP1 still contained high levels of MTs, significantly higher than that of untransfected cells on the same slide, which was 28% (p,0.01) (Figure 4, B and C). High eGFP-TgICMAP1 expression level accentuated this effect, as 86% of cells expressing high level of eGFP-TgICMAP1 (defined by fluorescence levels three times greater than that of untransfected cells) contained significant amount of MTs (p,0.001) (Figure 4, B and C). TgICMAP1 binds MTs in vitro The fact that a T. gondii protein can bind to MTs in a ‘‘foreign’’ environment, the cytosol of mammalian cells, suggests that TgICMAP1 likely binds to the MTs directly rather than through other MT binding proteins. To determine if the interaction between TgICMAP1 and MTs is indeed direct, recombinant Characterization of TgICMAP1 6xHis-eGFP-TgICMAP1 was expressed and purified from E. coli, and incubated with in vitro polymerized MTs stabilized with the GTP analog GMPcPP. The 6xHis-eGFP-TgICMAP1 and MT suspension was then spun onto coverslips and MTs were visualized by staining with anti-tubulin antibody (Figure 5A). TgICMAP1 showed binding along the MT lattice. No MT binding was observed in the negative control sample, where 6xHis-eGFP [30] was used in place of 6xHis-eGFP-TgICMAP1 (Figure 5B). TgICMAP1 preferentially localizes to the intra-conoid MTs in T. gondii Despite its ability to bind MT directly, TgICMAP1 only binds to a subset of tubulin containing structures in T. gondii. Colocalization studies showed that eGFP-TgICMAP1 predomi- nately localized to a thin focal point at the apical end of the parasite (Figure 6A), much narrower in width when compared to that of the conoid, marked by co-expression of mCherryFP- TubA1 in the same cell. eGFP-TgICMAP1 also localized to the centriole/spindle pole area (arrows) (also marked by the expression of mCherryFP-TubA1 [8]), although the intensity of this labeling varied depending on the expression level of eGFP-TgICMAP1. To investigate the localization of endogenous TgICMAP1, an anti- TgICMAP1 antibody was produced (Figure 6B–F). Immunoflu- orescence assays using the anti-TgICMAP1 antibody confirmed that endogenous TgICMAP1 had similar apical localization as eGFP-TgICMAP1 in the apical complex, but a specific localiza- tion in the centriole/spindle pole area could not be confirmed (Figure 6B). By Western blot, the anti-TgICMAP1 antibody Figure 4. Ectopically expressed eGFP-TgICMAP1 stabilizes MTs in mammalian cells. (A) eGFP-TgICMAP1 transfected cells (green) labeled with rat anti-tubulin YL1/2 antibody (red) and mouse anti-acetylated tubulin antibody (cyan). eGFP-TgICMAP1 coated MTs display higher acetylation level compared with adjacent ‘‘uncoated’’ MTs (insets). Insets are at 26magnification. Scale bar = 10 mm. (B) Graph showing the mean percentages of untransfected (blue bar, ‘‘untransfected’’); eGFP-TgICMAP1 transfected (green bar, ‘‘transfected’’); low (yellow bar) and high (dark green bar) eGFP- TgICMAP1 expressing HeLa cells containing high levels of MTs after nocadazole treatment. The error bars indicate standard error of the mean (SEM). ‘‘*’’ : p,0.01, ‘‘**’’: p,0.001. (C) Representative images of untransfected and transfected cells expressing low and high levels of eGFP-TgICMAP1 after nocodazole treatment. Scale bar = 10 mm. doi:10.1371/journal.pone.0007406.g004 PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7406