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Figures
- Fig. 1.
A workflow for longitudinal intensity profiling of proteins during merozoite invasion. (A) Schematic of merozoite invasion showing stages of invasion, tight junction formation, micronemal and rhoptry release and surface protein shedding, with some of the key proteins involved. (B) To analyse the localisation of proteins during invasion with respect to the tight junction in an unbiased manner, invading merozoites labelled for RON4 and with another antibody of interest were chosen for imaging based on RON4 labelling and orientation and imaged by widefield fluorescence deconvolution microscopy. (i) Images were rotated so as to align invading left-to-right along the x-axis of the image and (ii) were then analysed using a max intensity plot profile along the long axis of the merozoite, centred to the brightest point in the RON4 labelling channel. (iii) Pixel intensity values were then summed to provide an average longitudinal distribution for each merozoite. (C) Heatmaps (left), normalised intensity plots (middle) and single-slice images (right) of four example merozoites (04, 11, 13 and 16) for the longitudinal intensity profiling of mouse RON4 (mRON4; green) versus rabbit RON4 (rRON4; red) antibody labelling, demonstrating the validity of this technique. See also Fig. S1A. (D) Confirmation of workflow accuracy. Three independent replicate images of a single invading merozoite (left) were analysed for correlation by plotting corresponding values from the normalised longitudinal intensity profiles against one another (right). The boxed regions in DIC images are shown magnified to the right. Arrows indicate the position of the RON4-labelled tight junction. White boxes denote approximate front and back position of merozoite as determined in the DIC channel. Scale bars: 1 μm.
- Fig. 2.
Longitudinal intensity profiling demonstrates antibody-specific variation in AMA1 detection at the tight junction. Heatmaps, normalised intensity plots and single-slice images of example merozoites for the longitudinal intensity profiling of (A) mRON4 (green) versus rAMA1 (red) or of (B) rRON4 (green) versus mAMA1 (red) antibody labelling. The boxed regions in DIC images are shown magnified to the right. Arrows indicate the position of the RON4-labelled tight junction. White boxes denote approximate front and back position of merozoite as determined in the DIC channel. See also Fig. S1B,C. Scale bars: 1 μm.
- Fig. 3.
Longitudinal intensity profiling localises actin and aldolase towards the rear of the invading merozoite tight junction. Heatmaps, normalised intensity plots and single-slice images of example merozoites for the longitudinal intensity profiling of mRON4 (green) versus (A) rabbit actin (rAct; red) or (B) rabbit aldolase (rAldo; red) antibody labelling. The boxed regions in DIC images are shown magnified to the right. Arrows indicate the position of the RON4-labelled tight junction. White boxes denote approximate front and back position of merozoite as determined in the DIC channel. See also Fig. S1D,E. Scale bars: 1 μm.
- Fig. 4.
MTRAP organisation and profile of domain-specific antibodies. (A) MTRAP consists of a signal sequence (SS, orange), two thrombospondin repeat domains (TSR, red) and a single transmembrane domain (TM, blue), with a short C-terminal cytoplasmic tail. In order to track the various cleavage products of MTRAP we generated a rabbit monoclonal antibody (3A3) against the unstructured mid domain (rM-mid) and rabbit antiserum (R1081) against the short C-terminal tail (rM-tail). These antibodies complement the previously published MTRAP TSR (R556) antiserum (rM-tsr). (B) Analysis of 3D7 schizont and mixed schizont and ring (schizont/ring)-stage parasites along with schizont/ring-stage parasites expressing a DDtm-tagged version of MTRAP (MTRAPDDtm), were used to test antibody recognition. Immunoblot analysis labelled with rM-tsr antiserum identified full-length wild-type (wt) MTRAP at ∼75 kDa, full-length MTRAPDDtm at ∼90 kDa, and the cleaved MTRAP TSR domain at ∼27 kDa in both late schizont/ring samples as expected. rM-mid antibodies also recognised the full-length products, demonstrating its ability to specifically label MTRAP. Additionally, rM-mid recognised at least one product at ∼44 kDa in 3D7 samples. A corresponding band running at ∼55 kDa in MTRAPDDtm parasites confirms that this is a cleavage product of MTRAP, which maintains its C-terminal tail. In some samples a second cleavage product running at ∼38 kDa was also seen (data not shown). rM-tail antiserum recognised the same full-length and cleavage products in 3D7 samples. Additionally, a faint band was consistently labelled running at ∼15 kDa in samples involving early ring stage parasites, but was absent in schizont stage parasites. We believe this corresponds to the MTRAP C-terminal tail stub following intramembrane cleavage. rM-tail antiserum was unable to label C-terminally-tagged versions of MTRAP.
- Fig. 5.
Longitudinal intensity profiling identifies unexpected localisation profiles for MTRAP during merozoite invasion. (A-C) Heatmaps, normalised intensity plots and single-slice images of example merozoites for the longitudinal intensity profiling of mRON4 (green) versus (A) rM-tail (red), (B) rM-mid (red) or (C) rM-tsr (red). Scale bars: 1 μm. The boxed regions in DIC images are shown magnified to the right. Arrows indicate the position of the RON4-labelled tight junction. White boxes denote approximate front and back position of merozoite as determined in the DIC channel. See also Fig. S2A–C. Scale bars: 1 μm.
- Fig. 6.
MTRAP is cleaved irrespective of invasion or egress from the schizont. (A) Immunoblot analysis of a timecourse of tightly synchronised schizonts and early ring samples grown in the presence or absence of inhibitors of egress (E-64) or invasion [heparin (hep)]. Labelling with rM-tail antibodies labels full-length MTRAP (∼75 kDa) and cleavage products (∼38/44 kDa), including the tail stub (∼15 kDa). (B) Immunoblot analysis of tightly synchronised late-schizont- and early-ring-stage parasites grown in the presence or absence of inhibitors of egress [antipain and leupeptin (A+L)] or E-64. Labelling with rM-tsr antisera identifies full-length MTRAP (∼75 kDa) and the cleaved TSR domain (∼25 kDa). rM-tail labelling identified products as in A. (C,D) Immunofluorescence analysis of very late-stage schizonts (C) treated with E-64 to prevent egress or (D) from untreated culture, co-labelled with mAMA1 (1F9) monoclonal antibodies (green) and rM-tail (left), rM-mid (middle) or rM-tsr (right) antibodies (red in respective images). Scale bars: 1 μm.
- Fig. 7.
MTRAP is cleaved within the micronemes. (A) Immunofluorescence analysis of Compound 1 (C1)-treated schizonts (versus DMSO control) co-labelled with mAMA1 (green) and rM-tsr (top), rM-mid (middle) or rM-tail (bottom) (red in respective images) antibodies. Scale bars: 1 μm. (B) Immunoblot analysis of tightly synchronised late-stage schizont and early ring parasites labelled for GFP (loading control), MSP1 (C1 treatment control), rM-TSR, rM-tail and rAMA1. (C) Schematic of pre- (left half) and post- (right half) invasion merozoites, showing the predominant distribution of each protein examined as part of this study. It should be noted that all proteins show a degree of variability in labelling distribution across merozoites.
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