Characterization of TgICMAP1 Figure 1. Schematic drawings of T. gondii (Adapted from [6]). Left and middle: a longitudinal section and a semitransparent projected view of the cytoskeleton of a dividing cell. Right: an enlarged view of the apical portion of the cytoskeleton. The cytoskeleton of T. gondii includes the apical and the basal complexes, and the cortical cytoskeleton, which contains the cortical MTs, and the Inner Membrane Complex (IMC), a set of flattened vesicles with an underlying filamentous protein network. The centrioles/spindle pole assembly is located close to the nucleus and is not shown in the drawings. The apical complex is formed of the conoid, the intra-conoid MTs and three ring-like structures. The conoid fibers are novel tubulin polymers, the structure of which is dramatically different from that of a canonical MT, such as the cortical MTs and the intra-conoid MTs. In the enlarged view of the apical portion of the cytoskeleton (right), EM cross-sections of a single conoid fiber and a canonical MT are shown. doi:10.1371/journal.pone.0007406.g001 one of the most prevalent parasites in warm-blooded animals and is the most common cause of congenital neurological defects in humans [9]. It also causes devastating opportunistic infections in immuno-compromised patients [10]. Many of its 5,000 relatives in phylum Apicomplexa are also important human or animal pathogens [11], including Plasmodium spp, which kills more than a million people every year. The damage caused by T. gondii, most commonly severe lytic cerebral and ocular lesions, absolutely depends on its ability to replicate [10,12–14]. In the absence of massive, uncontrolled expansion of the parasite population, the infections are benign [15–17]. The MT-based T. gondii cytoskel- eton is essential for parasite survival and proliferation, as it provides the framework for organelle replication and partition. In addition, it is likely to be important for parasite invasion. The intra-conoid MTs, for example, are closely associated with the secretory organelles, such as rhoptries and micronemes, and are speculated to provide structural support for protein secretion from these organelles during invasion [3,18]. As tubulins themselves are highly conserved between T. gondii and mamma- lian cells [19], novel MT binding proteins in T. gondii are prime drug targets. Here we report the identification and characterization of a novel T. gondii MT binding protein, intra-conoid microtubule associated protein1 (TgICMAP1, Accession number: TgME49_039300). TgICMAP1 is a large 135 kDa protein that contains a coiled-coil Structural Maintenance of Chromosomes (SMC)-like domain. Purified recombinant eGFP-TgICMAP1 protein binds to in vitro polymerized MTs, suggesting that its interaction with the MT is direct. Surprisingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid MTs. When over-expressed in mammalian cells, eGFP-TgICMAP1 coats and stabilizes MTs, suggesting that TgICMAP1 might play a role in stabilizing the intra-conoid MTs. The discovery of TgICMAP1 thus provides us the first molecular tool to dissect the function and the assembly pathway of the intra- conoid MTs. Results TgICMAP1 is a coiled-coil protein that binds to and stabilizes MTs when ectopically expressed in mammalian cells Despite the richness of novel tubulin containing structures in T. gondii, only a few MT binding proteins have been identified in T. gondii [6]. Previously, we identified ,170 proteins in the apical complex of T. gondii using comparative proteomics [6]. To quickly assess if any of the novel proteins could interact with MTs, eGFP fusions of a number of these proteins were constructed and expressed in mammalian cells. One of the proteins tested, designated TgICMAP1, showed binding along the lattice of MTs (Figure 2). TgICMAP1 is likely to have a high affinity for MTs, as heavy eGFP-TgICMAP1 coating correlated with much weaker anti-tubulin staining when compared with that of uncoated MT segments, suggesting that perhaps TgICMAP1 binding blocks the accessibility of tubulin antibody to the MTs (Figure 2, insets). To determine if TgICMAP1 homologues are present in other protozoan parasites species, the TgICMAP1 protein sequence was blasted against the available apicomplexan parasite genomic sequences (http://eupathdb.org/eupathdb/), including those of Plasmodium spp, Cryptosporidium spp, Theileria spp, and Neospora caninum. The search revealed a single homologue in one of the closest relatives of T. gondii, Neospora caninum, another parasite that belongs to the family of Sarcocystidae. Neospora ICMAP1 (NcICMAP1, Accession Number: Nc_LIV_ 070470) shares 82% identity and 89% similarity with TgICMAP1 (Figure S1). Approximately 50% of TgICMAP1 was predicted to form coiled-coil domains by coiled-coil domain prediction software (Figure 3A) [20]. When blasted against the NCBI non-redundant protein database, the sequence from amino acids 200 to 475 in TgICMAP1 was recognized to be homologous to the coiled-coil region in Structural Maintenance of Chromosomes (SMC) domains (EValue = 4e-09) (Figure 3B and S1) [21–26]. The PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7406