Fig. 8. Ligand-mediated relocalization of S9(S+T)-CAD-GFP to the apicoplast occurs at low temperature. (A) Effect of low temperature on apicoplast targeting. T. gondii stably transfected with S9(S+T)-CAD-GFP were grown for 3 days without ligand, and transferred to a water bath equilibrated at 15°C. After 15 minutes, pre-chilled medium containing ligand was added and the cells incubated for the indicated times. Samples were prepared for IFA and probed with anti-GFP and quantum red streptavidin. A merge of GFP, quantum red streptavidin and DAPI is shown. (B) Effect of low temperature on secretion. T. gondii stably transfected with S9(S)-CAD-GFP were grown overnight in the absence of ligand and transferred to a water bath equilibrated at 15°C. After 15 minutes, ligand was added as above and the cultures were incubated for 3.5 hours before mild fixation. Cells were viewed for GFP fluorescence and transmitted light. Arrows indicate parasitophorous vacuole. Cartoons are overlaid on the images to facilitate identification of the parasites and the parasitophorous vacuolar membrane. Bar, 5 µm.