PfRab6 but not PfRab7 or retromer rapidly redistributes to the ER upon brefeldin A treatment. (A) Distributions of DDmCherry-PfRab6, DD-mCherry-PfRab7 and PfVps35-YFP in live parasites after one hour in the presence of 5 mg/mL brefeldin A. Redistribution of DDmCherry-PfRab6 to the perinuclear ER is indicated with an arrow (top panel). FP, fluorescent protein. mCherry fluorescence is pseudocolored red, YFP fluorescence is pseudocolored green and Hoechst 33342 fluorescence is pseudocolored cyan. Scale bar, 2 mm. Golgi marker DD-mCherry-PfRab6 redistributed to the perinuclear ER. Association with the perinuclear ER was not observed for either Rab7 or Vps35. The contrasting responses of the Golgi apparatus and endosome to brefeldin A treatment reveal differences in the dynamics of vesicular traffic to and from these compartments.Krai P, Dalal S, Klemba M. Evidence for a Golgi-to-Endosome Protein Sorting Pathway in Plasmodium falciparum. PLoS One. 2014 9(2):e89771.

(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 mm.Krai P, Dalal S, Klemba M. Evidence for a Golgi-to-Endosome Protein Sorting Pathway in Plasmodium falciparum. PLoS One. 2014 9(2):e89771.

(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. Both PfVps29-YFP and PfVps35-YFP were expressed throughout the asexual blood stage. In live parasites expressing PfVps35-YFP, punctate structures were observed in trophozoites and schizonts amid a background of diffuse, presumably cytosolic fluorescence (A). As the parasites matured, the puncta became more numerous until in segmenting schizonts there appeared to be one retromer-labeled punctum for each daughter nucleus. Single puncta were present in egressed merozoites, which indicates that the retromer-labeled compartment is inherited. Surprisingly, the PfVps35-YFP-labeled puncta were no longer visible in early ring stage. The distribution of PfVps29-YFP across the asexual cycle was essentially identical to that of PfVps35-YFPKrai P, Dalal S, Klemba M. Evidence for a Golgi-to-Endosome Protein Sorting Pathway in Plasmodium falciparum. PLoS One. 2014 9(2):e89771.