Figure 5
Live fluorescent imaging of the PTEX components EXP2 and PTEX88.
(a) Recombination strategy for the endogenous tagging of EXP2. Double crossover integration into the wild-type locus yields recombinant parasites with their endogenous locus tagged by mCherry-3xMyc. (b) Intravital competition assay of WT and exp2-mCherry parasites. Parasite multiplication rates for WT and exp2-mCherry parasites were 10.0 and 11.2, respectively (non-significant). (c) Western blot analysis of exp2-mCherry parasites. The predicted size for tagged EXP2 is 62 kDa and was identified correctly using an anti-mCherry antibody. (d) Purified exp2-mCherry × GFPPV-infected erythrocytes were lysed with hypotonic buffer (input) and spun at 100 000 × g. The supernatant (hypotonic lysate sup) along with proteins released from the pellet after Triton X-100 treatment (TX-100 sup) and the remaining insoluble pellet were analyzed by SDS-PAGE and Western blotting using anti-mCherry (EXP2-mCh) and anti-GFP (GFPPV) antibodies. (e) Micrographs of live exp2-mCherry-infected erythrocytes. Shown are representative images for three asexual developmental stages including the fluorescent EXP2-mCherry signal (top), a merge of EXP2-mCherry and cytoplasmic GFP (middle) and differential interference contrast images (DIC, bottom). Inset, free merozoite. (f) Co-localization of EXP2-mCherry (centre left) with the parasitophorous vacuole (GFPPV, centre). The line in the merge (centre right) indicates profiling of the fluorescent signal (right). (g) Co-localization of HSP101-mCherry (centre left) with PTEX88-GFP (centre). The line in the merge (centre right) indicates profiling of the fluorescent signal (right). Scale bars, 5 μm.
