Dynamics and localization of PI3P in P falciparum–infected red cells. (A-B) In live P falciparum–infected red cells (3D7 strain; Pf3D7), transgenic expression of a secretory form of the PI3P-binding protein EEA1 fused to mCherry (SS-EEA1^WT-mCherry; red) reveals secretory PI3P in a perinuclear region in early ring parasites (A).²⁵  Not shown here, but as previously shown, a single-point mutant of EEA1 that fails to bind PI3P is secreted to the parasitophorous vacuole (PV),²⁵  marked by the dotted circles in the middle panels, where the fluorescence image is merged with the bright field (B). Perinuclear localization is also seen in later schizont stages. The boxed regions in the left-hand panels are magnified in the right-hand panels. (C) Time-lapse images of the parasite’s extracellular merozoite stage (arrowhead) invading red cells to become an intracellular ring (arrow). (D) In live Pf3D7, the transgenic cytosolic form of the PI3P-binding protein EEA1 (cEEA1^WT–green fluorescent protein) is seen associated with punctate vesicles and organelles of late trophozoites/schizonts (as was previously reported³⁰ ) and distinct from perinuclear foci seen for SS-EEA1^WT-mCherry (shown in panels A-B). The boxed region in the left-hand panel is magnified in the right-hand panel. Parasite nucleus stained with Hoechst 33242 (blue). Scale bar, 5 µm. Live cells were imaged using indicator-free RPMI1640 (Gibco) by DeltaVision Deconvolution microscopy²⁵  with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope on a temperature-controlled stage at 37°C and a Photometrics cooled custom CCD camera (CH350/LCCD) driven by DeltaVision Software from Applied Precision Inc. (Seattle, WA). (E) Cryo-IEM of PfNF54 (wild-type) parasites probed with antibodies to PI3P and secondary antibody 10-nm gold conjugate. Gold particles are detected in the apicoplast, food vacuole, and tubules (suggestive) of ER. Black arrows indicate PI3P in lumen of tubule; yellow arrows indicate cytoplasmic PI3P; double yellow arrows indicate PI3P vesicular clusters cytoplasmic to ER tubules. Experimental replicates, n = 3. Scale bar, 100 nm. Imaged in a Philips CM120 Electron Microscope (Eindhoven, The Netherlands) under 80 kV. BF, bright field; cEEA1, cytosolic form of the PI3P-binding protein EEA1; GFP, green fluorescent protein; P, parasite nucleus; R, red cell.