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Blood, Vol. 105, Issue 10, 4078-4087, May 15, 2005
Trafficking of the major virulence factor to the surface of transfected P falciparum–infected erythrocytes
Blood Knuepfer et al.
105: 4078
Supplemental materials for: Knuepfer et al, Vol 105, Issue 10, 4078-4087
Files in this Data Supplement:
- Document S1. Supplemental information on constructs for transfection (PDF, 141 KB)
- Figure S1. Localization of 3D7-NTS-DBL1α-GFP in live transgenic parasites (JPG, 19 KB)
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The top row of panels shows the intrinsic GFP fluorescence, the second row represents DAPI staining of the nuclei, the third row displays a phase contrast image, and the bottom row of panels are overlays of the three previous images. Shown are ring, trophozoite and schizont stage parasites. This figure shows that the DBLα domain of PfEMP1 fused to GFP is retained in the cytoplasm of the parasite.
- Figure S2. Localization of GFP fluorescence in 3D7-K60CIDR2β10 transfectants (JPG, 22 KB)
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Transgenic parasites expressing the N-terminal 60 amino acids of KAHRP fused to the 10 amino acids of the C-terminus of the CIDR2β domain of PfEMP1 (PVLKPEEEAT) fused to GFP show export of the GFP fusion protein into the erythrocyte cytosol. The 60 amino acids of KAHRP contain the PEXEL motif required for export across the PVM. The intrinsic GFP fluorescence, nuclei stained with DAPI, phase-contrast, and merged images of the ring, trophozoite, and schizont stages of the parasites are shown. The chimeric protein is exported to the erythrocyte cytosol but does not associate with Maurer clefts, nor does it form a rim fluorescence, suggesting that the protein is retained within the cytoplasm.
- Figure S3. Control for IFA experiments on live cells infected with parental 3D7 parasites (JPG, 18 KB)
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(A) αKAHRP-His and αGFP antibodies show no reaction in live parental 3D7 parasites. The first 2 images display live cells incubated with αGFP or αKAHRP-His antibody; the third image shows DAPI-stained nuclei, and the fourth image is a phase-contrast image. (B) Fixed cells with 3D7 parasites were labelled with αKAHRP 3´ repeats (first image) or αKAHRP-His antibodies (second image). In both cases the antibodies label endogenous KAHRP protein. The third image shows an overlay of both antibody reactions, and the fourth image is a phase-contrast image. This experiment proves that the αGFP and αKhis antibodies do not react with the surface of live intact P falciparum-infected erythrocytes. When the parasites are fixed they become reactive to the internally located KAHRP and αKhis antibodies by virtue of endogenous KAHRP protein.
- Figure S4. The KAHRP-His domain cannot be detected on D10-K119TmATS parasites (JPG, 50 KB)
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(A) Live D10-K119TmATS parasites were incubated with αGFP (top row, middle panel) and anti–KAHRP-His (second row, middle panel) antibodies. The third panel in each row displays phase-contrast images. No surface expression of the KAHRP-His domain could be detected, in contrast to 3D7-K119TmATS (Figure 5). (B) Control experiment on a fixed parental D10 cell line and a D10-K119TmATS-GFP cell line, showing that the αKAHRP-His antibody can detect the K119TmATS GFP fusion protein. In the absence of endogenous KAHRP in the D10 parasites, no antibody reactivity was shown. The middle panels in both rows represent DAPI-stained nuclei, and the panels on the right are phase-contrast images. (C) Control experiment showing labelling of fixed D10-K119TmATS and 3D7-K119TmATS labelling with anti–KAHRP-His (first image in both panels) and αGFP antibodies (middle panel in both rows) showing complete colocalization in the D10 transfectants but only partial colocalization in the 3D7 transfectants, due to reactivity with endogenous KAHRP on top of reactivity with the GFP fusion. The third panel in each row represents an overlay of the 2 antibodies, and the fourth image in each row is a phase-contrast image.
- Video S1. Three-dimensional reconstruction image of a P falciparum-infected erythrocyte expressing the K119TmATS-GFP chimera (AVI, 910 KB)
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Confocal images were taken through the z-plane and reconstituted to create a 3-dimensional image, which is rotated to show the Maurer cleft structures. The erythrocyte is infected with two parasites. This movie shows the K119TmATS-GFP chimera and its transient localization to Maurer clefts, which are distributed within the infected erythrocyte's cytoplasm.
- Video S2. Dynamics of the K119TmATS-GFP chimera in transfected D10 parasites (AVI, 2.23 MB)
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The organization of the K119TmATS-GFP chimera was examined by repeated imaging of an erythrocyte infected with a D10 transfectant before and after 2 bleach pulses. The positions of the bleach pulses are indicated by the green arrows. Recovery of fluorescence into the bleach region occurs on a time scale of several seconds. The overexposed region in the top right corner of the cell corresponds to the more intense fluorescence arising from the parasite. In some of the images of the infected erythrocyte, bright "particles" that move position between successive images are visible. Note that the image series was obtained using a nonlinear time scale. The relative time at which each image was obtained relative to the first image is indicated on the image (in seconds). The individual frames have been adjusted with respect to brightness and contrast to take into account bleaching during image acquisition.
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