Ca2+-dependence of conoid extrusion in Toxoplasma gondii tachyzoites

The role of Ca2+ in conoid extrusion was investigated in isolated Toxoplasma gondii tachyzoites by treatment with Ca2+-ionophores, Ca2+-chelating agents and an inhibitor of the Ca2+-ATPase at the endoplasmic reticulum. The results were evaluated by light phase-contrast microscopy and electron microscopy. Ionomycin (0.5-1 mu M) caused an immediate and sustained extrusion of the conoid in up to 80% of the tachyzoites, depending on the concentrations of ionophore and Ca2+ in the medium. However, over 50% of the tachyzoites extruded the conoid when treated with ionomycin in Ca2+-free saline complemented with EGTA. The effect of ionomycin was reversible and could be induced a second time in about half of the responsive population. Similar results were obtained with A23187. Conoid extrusion induced by ionomycin in Ca2+-free medium was almost completely abolished when the tachyzoites were previously loaded with a permeable compound known to chelate intracellular Ca2+ (BAPTA/AM; 25 mu M). On the other hand, exposure of tachyzoites to the Ca2+-ATPase inhibitor thapsigargin (0.5-1 mu M) produced significant extrusion of the conoid. Tachyzoites loaded with BAPTA/AM as well as those treated with ionomycin, i.e. with conoids paralyzed in opposite positions, had a diminished capacity to invade cultured epithelial cells. A substantial reduction in the response to stimulation by ionomycin was found also in parasites treated with cytochalasin-D, a drug that depolymerizes actin-filaments. The results suggest that Ca2+-release from internal stores may act as a key signal to activate a mechanism of conoid extrusion probably mediated, at least in part, by actin-filaments.