Characterization of TgICMAP1 Figure 5. TgICMAP1 binds MTs in vitro. Recombinant 6xHis-eGFP-TgICMAP1 (green) (A) or 6xHis-eGFP (B) (green) were incubated with in vitro polymerized MTs, spun onto coverslips, and stained with mouse-anti-tubulin B-5-1-2 (red). 6xHis GFP-TgICMAP1 protein bound along the MT lattice. No MT binding was observed in 6xHis-eGFP control samples. Scale bars = 10 mm. doi:10.1371/journal.pone.0007406.g005 recognized a closely spaced doublet band around 135 kDa, the predicted size of TgICMAP1 (Figure 6C). We do not know why TgICMAP1 ran as a doublet in reducing SDS-PAGE, but post- translational modification is a potential cause, and we are currently investigating this possibility. Because the conoid and the intra-conoid MTs are the only tubulin containing structures in the apical complex (Figure 1) [3], the highly restricted localization of TgICMAP1 in the apical complex strongly suggests that TgICMAP1 binds to the intra- conoid MTs. This was confirmed by electron microscopy, where the intra-conoid MTs were decorated with gold particles indicating the binding of anti-TgICMAP1 antibody (Figure 6D). Consistent with our previous experience in immunolabeling apical complex proteins, where antigen accessibility was often an issue [7], harsh extraction conditions were needed for the successful labeling of the intra-conoid MTs with the anti-TgICMAP1 antibody. For extracellular parasites we found that 10 mM deoxycholate extraction gave the best labeling of the intra-conoid MTs with the anti-TgICMAP1 antibody (Figure 6, E and F, arrows). This condition often resulted in the loss of the banana-like shape of the parasite, likely due to the release of the cortical MTs from other parasite membrane-cytoskeletal elements (Figure 6E). However in cases where the basal end of the parasite held together, we were surprised to find anti-tubulin staining in a ring-like structure at the basal end of the parasite (Figure 6F, arrowhead). This is the first time that a tubulin containing structure has been observed at the basal end of the parasite. Recently, another group has also made a similar observation independently (Personal communication, Louis Weiss, Albert Einstein College, NY). The first 474 amino acids of TgICMAP1 are important for intra-conoid MT association in the parasite To determine if the SMC containing N-terminal domain is important for MT binding, we made eGFP fusions of two TgICMAP1 truncation mutants and expressed them in T. gondii: eGFP- TgICMAP11–474, a N-terminal truncation that contains the SMC- like coiled-coil domain, and eGFP-TgICMAP1455–1231, a C-terminal truncation that excludes most of the SMC-like domain (c.f. Figure 3B). Expression of eGFP-TgICMAP11–474 recapitulated the intra-conoid MT localization of the full-length protein at the apical complex with, however, more prominent cytoplasmic pool localization (Figure 7A–B). Interestingly, eGFP-TgICMAP11–474 also displayed basal complex labeling (Figure 7B, inset). In contrast, deletion of the SMC-like domain in eGFP-TgICMAP1455–1231 resulted in the complete loss of apical complex localization, with the truncated protein predominantly localizing to the nucleus (Figure 7C). Together, these data show that the first 474 amino acids of TgICMAP1 are essential for the intra- conoid MT association in T. gondii. The role of the C-terminus is less clear, but may be important for regulating the efficiency of TgICMAP1 targeting to the apical complex or may affect the binding affinity of TgICMAP1 for MTs. TgICMAP1 is a marker for monitoring the intra-conoid MT construction process during daughter formation The apical complex is a highly intricate, motile cytoskeletal machine that is likely to be important in parasite invasion and replication [6]. Therefore, how the different parts of this machine are assembled together during daughter cell construction is of great interest. Due to the lack of EM evidence and appropriate markers, it is not known whether the two intra-conoid MTs are assembled into the apical complex when the daughters are still being constructed inside the mother or after the daughters have emerged from the mother and become independent entities. TgICMAP1, the first marker for the intra-conoid MTs, allowed us to address this question. Figure 8 shows the localization of eGFP-TgICMAP1 in a dividing parasite (green), where both the mother and two daughter cortical profiles were marked by an antibody recognizing IMC1 (red), a component of a protein meshwork underlying the parasite PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7406