(A) Wide-field epifluorescence images of live parasites expressing PfVps35-YFP. Parasites are shown at ring (R), trophozoite (T), schizont (Sz), segmenter (Sg), and extracellular merozoite (M) stages. Hoechst 33342 fluorescence (DNA) is pseudocolored red. (B) Images of live parasites expressing PfVps29-YFP. (C) Images of live parasites co-expressing PfVps35-YFP and PfVps29-mCherry in trophozoite (T) and schizont (Sz) stages. mCherry (mC) fluorescence is pseudocolored red. (D) Co-localization of PfVps35-YFP or PfVps35-HA and organellar markers in fixed parasites (except for MitoTracker, which was imaged live). PMV, plasmepsin V; ACP, acyl carrier protein; MT, MitoTracker Red CM-H2Xros; RAP1, rhoptry associated protein 1; AMA1, apical membrane antigen 1. The AMA1 panel shows free merozoites; all others are intraerythrocytic. Organelles labeled by the markers are indicated in parenthesis. Marker-derived fluorescence is pseudocolored red. (E) PfVps35-YFP is adjacent to developing rhoptries in a 2N parasite. Hoechst 33342 fluorescence (DNA) is pseudocolored cyan. In all panels, YFP fluorescence is pseudocolored green. Scale bars, 2 µm.