Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii, apically attach to the ir host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we de monstrate that microneme discharge is regulated by parasite cytoplasmic fre e Ca2+ and that the micronemal contents, including the MIC2 adhesin, are re leased through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of ex ternal Ca2+, while chelation of intracellular Ca2+ prevented release. Mobil ization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are suffi cient to stimulate release. Following activation of secretion by the Ca2+ i onophore A23187, MIC2 initially occupied the apical surface of the parasite , but was then rapidly treadmilled to the posterior end and released into t he culture supernatant This capping and release of MIC2 by ionophore-stimul ated tachyzoites mimics the redistribution of MIC2 that occurs during attac hment and penetration of host cells, and both events are dependent on the a ctin-myosin cytoskeleton of the parasite. These studies indicate that micro neme release is a stimulus-coupled secretion system responsible for releasi ng adhesins involved in cell attachment.