Intracellular Δ<i>Tg</i>CDPK3 parasites are defective in calcium-stimulated microneme secretion.

<p>A) Live fluorescent time lapse microscopy of wild-type and Δ<i>Tg</i>CDPK3 parasites, transiently expressing an ectopic DsRed protein which is targeted to the PVM, and treated with cytD to disrupt motility. Wild-type parasites permeabilize the PVM coincident with the timing of normal egress following addition of A23187, as seen by diffusion of DsRed from the PVM through the host cell. Δ<i>Tg</i>CDPK3 parasites cannot permeabilize the PVM, and DsRed remains within the PVM. Calcium ionophore is added at 30 sec time point. B) Following stimulation with A23187, wild-type parasites display microneme proteins <i>Tg</i>SUB1 and <i>Tg</i>MIC11 at the apical tip, indicative of microneme secretion, as seen by co-staining with the surface marker <i>Tg</i>SAG1. <i>Tg</i>GRA1 diffuses from the PVM through the host cell coincident with microneme secretion by wild-type parasites, confirming DsRed permeabilization results. Ionophore-treatment of Δ<i>Tg</i>CDPK3 parasites stimulates no such microneme protein secretion, and <i>Tg</i>GRA1 remains within the PVM. C) Stimulating calcium signaling in extracellular Δ<i>Tg</i>CDPK3 parasites by treatment with either 1% EtOH or A23187 shows no defect in secretion of a range of microneme proteins in supernatant samples, as compared to wild-type parasites. “*” denotes weak banding of unprocessed <i>Tg</i>MIC2 in Δ<i>Tg</i>CDPK3 supernatant samples, indicating a small level of inadvertent parasite lysis, but not sufficient to explain levels of microneme proteins seen in Δ<i>Tg</i>CDPK3 supernatant.