PMC

Immediately following invasion, dense granule fusion causes local membrane extension and deposits export-associated regions as clusters of foci at the parasite periphery. Each focus contains multiple PTEX complexes as well as non-biochemically associated proteins, such as EXP1. Labelled regions tend to localize at the connexion point to PVM whorl-like extensions and at the tips of pseudopodia-like plasma membrane/PVM extensions. RESA export begins simultaneously, within minutes of invasion. In the hours following, the export-associated foci move to evenly surround the parasite, first as smaller clusters that often adopt a ‘ring-like’ structure and then as discrete foci, through which PEXEL-dependent protein export will ultimately travel.

Parasites were fixed at <15 min, 60–90 min and 20–21 h after invasion and labelled by IFA for EXP1 (red), EXP2 (green) and the nucleus (4′,6–diamidino-2-phenylindole (DAPI), blue). (a) Imaging of all samples by widefield deconvolution microscopy (scale bar, 1 μm), (b) and the 60–90 min sample by 3D-SIM (scale bar, 0.5 μm), suggested strong similarity between EXP1 and EXP2 localizations at the parasite periphery. (c) Using the 3D-SIM data set the percentage overlap (% colocalization), as calculated by the sum of pixel intensities, of EXP1 with EXP2 was calculated (n=17). EXP1 values were comparable with the positive control, HSP101HA <20 min (; n=32) and were substantially higher than the negative control, RAP1 60–90 min (; n=25). Also shown are data measuring percentage of GBP-DHFR-GFP (GDG) (; n=16) and RESA (; n=29) signal overlap with EXP2. Graph shows median and interquartile ranges. Statistics can be found in .

Widefield deconvolution microscopy of parasites fixed and labelled by IFA for EXP2 (green), the nucleus (4′,6–diamidino-2-phenylindole (DAPI), blue) and (a) PfEMP3 (red), a protein exported by PEXEL-dependent processes, or (b) PfEMP1, a protein likely to undergo PEXEL-independent export, showed some areas of possible association of both exported proteins and EXP2; however, results were inconclusive. Parasite ages are as shown. Scale bars, 1 μm. Parasites were labelled by IFA for EXP2 (green), RESA (red) and the nucleus (DAPI, blue) having been (c) fixed <12 min or 60–90 min after invasion and imaged by widefield deconvolution microscopy (scale bar, 1 μm) or (d) fixed <12 min after invasion and imaged by 3D-SIM (Grid=0.5 μm). Insets show individual PVM domains (scale bar, 0.1 μm) and gamma settings were altered for display purposes only. See also for a 3D rotation of the upper parasite. These results showed that RESA export is detectable in a small proportion of parasite in <12 min assays and in essentially all parasites at 60–90 min. Considerable association, although not direct overlap, was observed between RESA and EXP2 signals at the parasite periphery, particularly in the earlier time point.

(a) The GBP130-DHFR-GFP construct involves a 150 amino-acid leader sequence from the PEXEL containing protein GBP130, fused to mDHFR and GFP. (b) Imaging of live parasites by widefield microscopy showed that the GBP130-DHFR-GFP (GDG, green) reporter was exported in the absence of any drug (top) but was prevented from export when grown in the presence of 5 nM WR99210 (WR) drug (bottom). Scale bar, 1 μm. (c) 3D-SIM imaging of parasites grown in the absence of WR, fixed and labelled by IFA for GBP130-DHFR-GFP (GFP, green), EXP2 (red) and the nucleus (4′,6–diamidino-2-phenylindole (DAPI), blue) demonstrated that analysis of export under native conditions would be difficult using this system. Grid=0.5 μm. Gamma settings were altered on images for display purposes only. Parasites labelled similarly but grown in the presence of 5 nM WR (d) for 18–19 h or 20–21 h and imaged by widefield deconvolution microscopy (scale bar, 1 μm) or (e) for 18–19 h and imaged by 3D-SIM (Grid=0.5 μm; Gamma settings were altered on images for display purposes only) both showed accumulation of translocation intermediates adjacent to areas of EXP2 signal. See for a 3D rotation of the right hand parasite from e.

(a) Widefield deconvolution imaging of parasites fixed <12 min following erythrocyte invasion and labelled by IFA for EXP2 (green), the PV (RAP1, red) and the nucleus (4′,6–diamidino-2-phenylindole (DAPI), blue) showed EXP2 localized in puncta at the parasite periphery at this time point (scale bar, 1 μm). HSP101HA parasites were fixed <10 min, <20 min, <45 min and 15–16 h following erythrocyte invasion and labelled by IFA for EXP2 (green), HSP101HA (HA, red) and the nucleus (DAPI, blue). (b) Widefield deconvolution microscopy (scale bar, 1 μm) (a and b) and (c) 3D-SIM imaging (scale bar, 300 nm) both showed the presence of substantial regions of coincident fluorescence between EXP2 and HSP101HA (scale bar, 0.3 μm). (d) The percentage signal overlap (% colocalization), measured as the sum of pixel intensities, was quantified between HSP101HA with EXP2-labelled regions in 3D-SIM data sets across the time course period (<10 min, n=28; <20 min, n=32; <45 min, n=35; 15–16 h, n=36). Graph shows median with interquartile range. Statistics can be found in .

Parasites were fixed <12 min following merozoite invasion and labelled by IFA using standard markers for the PV (RAP1, red), parasite plasma membrane (MSP1-19, green) and nucleus (4′,6–diamidino-2-phenylindole (DAPI), blue). (a) Widefield deconvolution microscopy identified various morphological changes that had occurred within this period, including pseudopodia-like extensions (top) and whorl-like extensions (bottom). Scale bar, 2.5 μm. 3D-SIM imaging of similarly prepared and labelled parasites confirmed (b) pseudopodia-like and (c) whorl-like morphology changes, showing transitional stages from rounded merozoite-like to more flattened and extended amoeboid forms. Grid=0.5 μm. Gamma settings were altered on images for display purposes only. (d) Transmission electron microscopy (TEM) analysis of parasites <10 min post erythrocyte invasion identified IMC remnants present in cup-like (upper parasite and inset, arrows) and absent in more flattened parasites (lower parasite and inset). Representative section of a doubly infected erythrocyte is shown. Scale bar, 200 nm. (e) 3D modelling of serial sections by TEM confirmed pseudopodia-like (left) and whorl-like PVM (right) extensions. Light green=PVM, darker region=PM. Scale bar, 200 nm.

A time course of parasites fixed <12 min, 60–90 min, 11–12 h and 18–19 h after invasion was labelled by IFA for EXP2 (green), the PV (RAP1, red) and the nucleus (4′,6–diamidino-2-phenylindole (DAPI), blue). (a) 3D-SIM imaging confirmed that EXP2 localized as puncta to the parasite periphery throughout this period. Grid=0.5 μm. Gamma settings were altered on images for display purposes only. (b) EXP2-labelled regions (zooms) from the earlier two time points showed apparent clustering of bright foci (arrows). A representative example is shown. Scale bar, 0.15 μm. (c) In these earlier time points a recurring structural motif was also regularly identified in clusters (upper left). A representative example is presented, with the magnified cluster as orthogonal projection and 3D rendering (lower left) and serial slices (right) shown. Scale bar, 0.15 μm. (d) EXP2-labelled volumes (μm³) were calculated in 3D-SIM data sets (12 min, n=19; 60–90 min, n=27; 11–12 h, n=37; and 18–19 h, n=22) before (‘presegmentation’, blue bars) and after (‘segmented’, green bars) treatment with a region growing algorithm to isolate the previously observed foci within larger regions of fluorescence. Graph shows mean±s.e.m. Statistics can be found in .