Fig. 6. MIC8 is essential for the formation of the moving junction. (A) Formation of evacuoles is abolished in parasites depleted of MIC8. MIC8KOi, grown in the presence or absence of ATc, was incubated with host cells in the presence of CD. Formation of evacuoles (green) and an MJ (red) was analysed with monoclonal antibodies against Rop2-5 and polyclonal antibodies against RON4. No evacuoles were observed when MIC8KOi was grown in the presence of ATc. Blue staining indicates DAPI (nuclei). (B) Extracellular RON4 was detected by immunofluorescence analysis on non-permeabilised parasites expressing cytosolic GFP as a viability marker. MIC8KOi parasites were allowed to attach to host cells in the presence or absence of CD for 20 minutes. Red indicates an MJ. (C) Quantification of extracellular RON4, the presence of which indicates the formation of an MJ. Approximately 200 parasites were analysed in each assay (error bars indicate s.d. obtained from five independent assays). The units on the y-axis are percent of parasites with RON4 signal. (D) Depletion of MIC8 does not affect RON4 expression levels. Immunoblots from MIC8KOi and RH parasites, grown for 48 hours in the presence (+) or absence (–) of ATc, show efficient downregulation of MIC8 expression in the presence of ATc. No differences in RON4 expression levels could be detected. Scale bars: 15 µm (A); 20 µm (B).