Characterization of TgICMAP1 Figure 8. Daughter intra-conoid MTs are assembled during daughter formation within the mother cell. eGFP-TgICMAP1 expression (green) and anti-IMC1 staining (red) in a dividing parasite show that eGFP-TgICMAP1 is present at the intra-conoid MTs of both the mother (green arrow) and the daughter parasites (green arrowhead) as well as the duplicated centriole/spindle pole assembly (white arrowheads). Scale bar = 2 mm. doi:10.1371/journal.pone.0007406.g008 identification of a new MT-binding protein in T. gondii, TgICMAP1, which is localized predominantly to the intra-conoid MTs 2 two highly stable MTs in the apical complex. When TgICMAP1 was ectopically expressed in mammalian cells, it significantly increased the resistance of MTs to nocodazole treatment, suggesting that TgICMAP1 might play a role in stabilizing the two intra-conoid MTs. We also found colocalization of TgICMAP1 with perinuclear MTs when TgICMAP1 was expressed at a high level in mammalian cells. It has been shown that perinculear MTs are more stable than other MTs, but it is unclear if their perinuclear localization is the cause or the consequence of their stability [34–36]. The colocalization of TgICMAP1 with this subset of MTs therefore could be due to the preference of TgICMAP1 in binding to these MTs (recognizing, for example, certain distinct biochemical or structural properties of these MTs) or an indirect result of the stabilization effect of TgICMAP1 on MTs. Right now we cannot distinguish between these two possibilities. TgICMAP1 binds directly to the lattice of bare MTs polymerized in vitro, yet in T. gondii it preferentially binds to the intra-conoid MTs but not other tubulin containing structures, such as the conoid and the cortical MTs. How is this specific binding regulated? We showed previously that the conoid fibers are formed of protofilaments arranged in a ‘‘comma’’ like structure much different from that of a canonical MT [7] (c.f. Figure 1). This drastic structural difference between the conoid fibers and the intra-conoid MTs could well be the reason that TgICMAP1 is not found in the conoid. This cannot explain however, why TgICMAP1 does not bind to the cortical MTs, which are tubes with 13 protofilaments, just like the intra-conoid MTs. We speculate that the ‘‘masking’’ of the cortical MTs by other MT binding proteins might play a role in excluding TgICMAP1 from the cortical MTs. A second possibility is that differences in the post-translational modifications of tubulin in the intra-conoid MTs and the cortical MTs may affect the affinity of TgICMAP1 binding. In mammalian cells, for example, a number of MT- binding proteins and molecular motors preferentially bind detyrosinated MTs [37–41]. The identification of other proteins that are specifically localized to the intra-conoid MTs or the cortical MTs is necessary to test these hypotheses. Protein secretion through the apical complex from the membrane-bound invasion organelles, rhoptries and micronemes, is essential for parasite invasion into the host cell [42–45]. MT based cytoskeleton mediates the majority of vesicle traffic in eukaryotic cells [46–49]. Based on electron microscopy evidence, it was proposed that the intra-conoid MTs might serve as tracks for the secretion of proteins from the apical complex during T. gondii invasion [3,18]. Indeed, the length and position of the intra-conoid MTs are very consistent with their potential structural roles in secretion and the subsequent recycling of the lipid and proteins on the membrane of the emptied vesicles. However, due to the lack of knowledge of their protein composition other than tubulin, little is known about how the intra-conoid MTs function and how they are constructed during daughter formation. Using TgICMAP1 as a marker for the intra-conoid MTs, we showed that the intra-conoid MTs are formed during daughter parasite construction within the mother parasite. In addition, we noted that the anti-TgICMAP1 antibody recognized a doublet by Western blot, which may suggest that TgICMAP1 is post-translationally modified. Protein modifica- tions, such as phosphorylation, have been shown to regulate the binding of MT-associated proteins to MT in vertebrates [50]. Therefore, it will be very interesting to elucidate whether TgICMAP1 is post-translationally modified, and understand whether such modification regulates TgICMAP1 function. TgICMAP1 is the first molecular tool that can be used to dissect the biogenesis of the intra-conoid MTs. Future functional studies of TgICMAP1, such as the creation of TgICMAP1 deficient parasites and dominant negative mutants of TgICMAP1 will allow us to specifically interfere with the function of the intra-conoid MTs to understand the role of these two short MTs in the life of T. gondii. Materials and Methods Mammalian cell culture and transfection HeLa cells were grown in DMEM (Cat#10569, Invitrogen, CA) containing 10% (v/v) Hyclone Bovine Calf Serum (BCS) (Cat#SH30087.03, Fisher Scientific, PA) and 1x antibiotic- antimycotic (Cat#15240, Invitrogen) at 37uC and 5% CO2. DNA (1 mg/well) was transfected into subconfluent HeLa cell monolayers grown in six well plates by FuGene6 transfection reagent (Cat #11815091001, Roche, IN) at a FuGene:DNA ratio of 3:1 per manufacturer’s instruction. Parasite culture and transfection T. gondii RH tachyzoites were used in all experiments. The parasites were maintained by continuous passage in human PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7406