JCS02673 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1A
• Supplemental Figure 1B -

  Fig. S1. Expression of PfERD2-GFP in transgenic parasites. (A) Immunoblot using wild-type (WT) and PfERD2-GFP (ERD2-GFP)-expressing parasites. A GFP fusion protein of approximately 53 kDa is recognized by the antibody in the transgenic but not in the WT parasite line. The smaller band is presumably a degradation product of the GFP fusion. (B) Fluorescence microscopy of fixed parasites expressing PfERD2-GFP (green) using anti-PfERD2 antibodies (red) and DAPI (blue) for DNA staining. PfERD2-GFP fluorescence accumulates in two discrete structures connected with a weaker perinuclear staining (a). PfERD2 specific antibodies stain two foci adjacent to the nucleus (b). (c) Merged image. Note that in contrast to PfERD2-specific antibodies PfERD2-GFP fluorescence additionally visualizes the perinuclear envelope. (d) Merge of fluorescent and bright-field images.

• Supplemental Figure 1 -

  Fig. S2. Live transgenic parasites expressing PfERD2-GFP were imaged by fluorescence microscopy. (a) At 8 hours post invasion a weak crescent-shaped fluorescence is observed in early ring stages. (b-c) At 16-24 hours post invasion two protrusions of intense PfERD2-GFP fluorescence are connected with weaker perinuclear staining. (d-e) By 32-40 hours post invasion ongoing schizogony is observed. Nuclear division is indicated by additional, multiple perinuclear distributions of PfERD2-GFP. (f) At 48 hours post invasion the late schizont stage is reached. Note that every forming merozoite displays one focused PfERD2-GFP signal.

• Movie 1 -

  Movie 1. Three-dimensional reconstruction of a P. falciparum-infected erythrocyte (8 hours post invasion) expressing PfGRASP-GFP. Confocal images of live cells were taken through the z-plane and reconstituted to create a three-dimensional image, which is rotated to show the spherical organization of the Golgi situated in close proximity to the nucleus. The nucleus is stained with DAPI. Low levels of background fluorescence are visible.

• Movie 2 -

  Movie 2. Three-dimensional reconstruction of a fixed P. falciparum-infected erythrocyte (8 hours post invasion) expressing PfGRASP-GFP. The spatial ER-Golgi relationship is investigated by visualizing the ER with antiPfBiP-specific antibodies (red). The ER forms an envelope around the nucleus. One protrusion of the ER that might represent an ER exit site is in close proximity but clearly separated from the Golgi.

• Movie 3 -

  Movie 3. Three-dimensional reconstruction of a fixed P. falciparum-infected erythrocyte (16 hours post invasion) expressing PfGRASP-GFP. Rab6 containing vesicles are visualized with antiPfRab6-specific antibodies (red) and are only partially associated with the Golgi.