Figure 4. Immunofluorescence microscopy of PfCRT-GFP over-expressing parasites treated with either Brefeldin A or Dynasore.

PfCRT was over-expressed as a GFP-fusion protein using an ATet-inducible expression system and treated with either BFA (5 µg/mL) for 3 h or Dynasore (40 µM/mL) for 2 h. As a control a second parasite population was treated with an equivalent volume of carrier alone (ethanol and DMSO, respectively). Following the BFA treatment, immunofluorescence microscopy was performed on fixed cells with mouse anti-GFP, Alexa-594 goat anti-mouse IgG and DAPI. Representative parasites in trophozoite and schizont stage are shown for control and treated parasites. (A) The control parasites show the restricted FV localisation of PfCRT-GFP (false-coloured in green). (B) BFA treated parasites show an accumulation of fluorescence around the DAPI-stained nuclei, consistent with ER localisation in addition to the FV localisation (Figure 1). (C) Treatment of parasites with Dynasore resulted in an accumulation of fluorescence around the DAPI-stained nuclei in addition to the FV membrane localisation, similar to the observed effect of BFA treatment (B).