Plaque formation and invasion assays were performed as previously described27 ,28 (link). Microneme secretion was assayed by monitoring the release of MIC2 into the culture medium following stimulation with 3%FBS and 2% ethanol, 15 min at 37°C, as previously described20 (link). Samples were resolved by SDS-PAGE, blotted and probed with mouse-α-MIC2 (mAb 6D10), and mouse-α-GRA1 (mAb Tg17-43) and quantified by phosphoimager analysis. Egress and PVM permeabilization were monitored by video microscopy following stimulation with 8 μM calcium-ionophore A23187 (Calbiochem). When noted, parasites were pre-treated for 10 min with 2μM Cytochalasin D (Calbiochem) to block motility. The extent and rate of egress, as well as degree of vacuole permeability was quantified using Openlab v. 4.1 (Improvision) as described in the supplementary materials. Host cell lysis was assayed by staining monolayers with crystal violet, 3 days after infection at an MOI of 1.