Fig. 3. The association of host mitochondria with the parasitophorous vacuole membrane (PVM) is inhibited in ROP2-deficient parasites. (A-B) Mitotracker red staining of wild-type (WT), ROP2AS-7 and ROP2AS-20 parasites. The morphology of parasites in B', illustrating both distention and occasional vacuolization, was observed in only a portion of antisense-expressing parasites. In comparison to WT, mitotracker labeling indicates that the host mitochondria are not localized to the parasite vacuole containing ROP2-deficient parasites, in which only the intracellular mitochondria are detected (A-A', arrows). (C-D) Transmission electron micrographs illustrate abrogation of mitochondrial association with the PVM (arrows) in ROP2AS (D) compared to WT (C). (E) Linear density of parasitophorous membrane that are associated with mitochondria in WT, ROP2AS-7 and ROP2AS-7 (recovered) as determined by EM morphometric analysis similar to that described in Sinai et al. (Sinai et al., 1977). ROP2AS-7 (recovered) is parasites in which ROP2 expression is recovered with continued passage. Random EM sections of parasite vacuoles containing WT (n=25), ROP2AS-7 (n=26) and ROP2AS-7 (recovered) (n=31) were examined by the double square overlay test system to calculate the percentage of PVM that is associated with host mitochondria. The percentage of PVM associated with mitochondria is reduced by ten-fold in ROP2AS-7 in comparison to WT (P<0.0001), whereas ROP2AS-7 (recovered) showed an increase by four-fold (P<0.001). Values shown in E are mean±s.d. Bar, 1 µM.