Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its h ost cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin m onomers could play a key role in parasite actin dynamics. We isolated a 27- kDa tachyzoite protein on the basis of its ability to bind muscle G-actin a nd demonstrated that it interacts with parasite G-actin. Cloning and sequen ce analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofi lin not only bound to G-actin and inhibited actin polymerization as an acti n-sequestering protein but also slowed down F-actin disassembly through a f ilament end capping activity. In addition, when green fluorescent protein-t agged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamic s of actin stress fibers was drastically impaired, whereas green fluorescen t protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cy toplasm, including the rear end of the parasite, whereas in intracellular t achyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.