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As expected, the counts of intracellular ATc-treated ��AROe/AROi-Ty were markedly reduced. The fact that extracellular ATc-treated ��AROe/AROi-Ty was not increased compared to AROi-Ty-expressing ��AROe/AROi-Ty or to TATi-1 suggested that AROi-Ty-depleted parasites are defective in host cell attachment. Indeed, when we performed the red/green assay with parasites pretreated with 1?��M cytochalasin D (CD), a drug which blocks parasite actin-based motility and invasion, we observed a significant reduction in attachment for AROi-Ty-depleted parasites (p value?selleck chemical host cell. Next, we addressed the question of whether AROi-Ty-depleted parasites were affected INSRR in their ability to egress from infected cells. For this purpose, ��AROe/AROi-Ty and TATi-1 were grown for 48 or 72?hr in presence or absence of ATc, and egress was induced by addition of the calcium ionophore A23187. ��AROe/AROi-Ty depleted in AROi-Ty showed no significant defect in egress, and the percentage of lysed vacuoles was almost 100%. Comparable counts were obtained for all strains treated?�� ATc, except for ��GAP45e/GAP45i-Ty, which we included as a positive control (Figures 2E and S1C; Fr��nal et?al., 2010). The same results were obtained when AROi-Ty was stimulated with 8% ethanol (Figure?S1D) or allowed to egress spontaneously (data not shown). Combined, these results clearly indicate that TgARO is not required for egress. Next, we assessed whether the observed phenotype in invasion arises from a defect in parasite BMS-754807 in vivo gliding motility. ��AROe/AROi-Ty and TATi-1 were grown for 48?hr?�� ATc, stimulated with ionomycin, and added to poly-L-Lysine-treated coverslips. The trails were visualized by staining for TgSAG-1 deposited in the tails of gliding?parasites and scored. Trails of AROi-Ty-depleted parasites were indistinguishable from trails formed by AROi-Ty-expressing?��AROe/AROi-Ty and by TATi-1 (Figure?2F). Taken together, these results establish that TgARO depletion severely and selectively affects T.?gondii host cell invasion. In contrast, other steps of the lytic cycle and most noteworthy egress from host cells are unaffected in ��AROe/AROi-Ty ATc-treated parasites. To understand the link between AROi-Ty depletion and the inability of ATc-treated ��AROe/AROi-Ty to invade host cells, we examined the nature of the rhoptry organelles and their content upon AROi-Ty knockdown by performing IFA analysis with ��-RON (uncharacterized anti-RON Abs) and ��-ROP markers (��-ROP1 and ��-ROP2 Abs). When AROi-Ty was expressed in ��AROe/AROi-Ty, ��-RON localized to the rhoptry neck and ��-ROP1 and ��-ROP2 to the rhoptry bulb indistinguishably from their localizations in TATi-1 strain (Figures 1A and 3A).
