Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

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The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii. Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites. INTRODUCTIONToxoplasma gondii is a ubiquitous protozoan parasite, chronically infecting 10 -90% of human populations worldwide. Sexual differentiation occurs only in the cat, but asexual T. gondii parasites can invade, proliferate, and encyst in virtually any nucleated cell (Frenkel, 1973;Bonhomme et al., 1992;Smith, 1995;Dubey, 1998;Dubey et al., 1998). Primary infection during pregnancy poses a risk of abortion or severe birth defects. Reactivation of dormant parasite tissue cysts (bradyzoites) gives rise to rapidly replicating tachyzoites, which may be fatal in immunocompromised individuals. Pathogenesis in both congenital infection and immunosuppressed patients is directly attributable to parasite proliferation (Frenkel, 1973;Dubey, 1998). Understanding the replication process of this parasite is therefore essential for the development of improved treatment but little is known about cell cycle control in these parasites.Like other members of the phylum Apicomplexa, T. gondii is an obligate intracellular parasite. Haploid tachyzoites invade into host cells, establishing a parasitophorous vacuole whose membrane is derived from the host plasma membrane (Joiner et al., 1994;Suss-Toby et al., 1996;Mordue et al., 1999; Figure 1A). Two parasites are typically produced in each mitotic cell cycle (ϳ7-10 h), and replication proceeds synchronously, resulting in geometric expansion of clonal progeny until the host cell is lysed, ϳ48 h postinfection.In contrast to replication by binary fission (as observed in most animal, plant, and bacterial cells), parasite replication proceeds via assembly of daughters within the mother (Figure 1B). Because asexual replication of T. gondii tachyzoites typically produces two parasites per mitotic cell cycle, this process is often termed "endodyogeny" (Sheffield and Melton, 1968). In c...

SummaryAssay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein±protein or protein± carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.

Membrane skeletons play an important role in the maintenance of cell shape and integrity in many cell types. In the protozoan parasite Toxoplasma gondii this function is performed by the subpellicular network, a resilient structure composed of tightly interwoven 10-nm filaments. We report here that this network is assembled at an early stage in the development of daughter parasites. The networks of immature and mature parasites differ dramatically with respect to their stability. Although in immature parasites the network is completely solubilized by detergent, the network in mature parasites is entirely detergent-resistant. Conversion of the detergent-labile to the detergent-resistant network occurs late in daughter cell development and appears to be coupled to proteolytic processing of the carboxyl terminus of TgIMC1, the major subunit of the network filaments. A single cysteine residue in the TgIMC1 carboxyl terminus was found to be essential for this processing event. The dramatic change in resistance to detergent extraction probably reflects an overall change in structural stability of the subpellicular network that accompanies maturation of daughter parasites and allows a switch from an assembly-competent but loose structure to one that is rigid and offers mechanical strength to the mature parasite.Membrane skeletons, composed of the plasma membrane and its organized underlying coat, are found in many cell types and are essential for mechanical strength and the maintenance of cell shape. Two widely divergent examples are the spectrinbased membrane skeletal system of the erythrocyte and the articulin-based system in the unicellular Euglena. In the erythrocyte, spectrin along with its associated proteins such as ankyrin form a meshwork underlying the plasma membrane (1). In the Euglena, two articulin proteins interact stoichiometrically to form a membrane skeletal system consisting of 40 interdigitating strips (2-4). Along with the articulins of the Euglena, a number of unique cytoskeletal systems have been described in protists, including the giardins (5, 6), assemblins (7), and tetrins (8) as well as the components of the subpellicular network, a novel cytoskeletal structure recently identified in the protozoan parasite Toxoplasma gondii (9).T. gondii is an obligate intracellular parasite that infects a wide range of nucleated cells. Human infection with the para-