A series of diagrams summarising in greater detail the main conclusions relating to post-invasion MSP1₁₉ endocytosis and its progression through the asexual cycle.

<p>In each panel a portion of the parasite is shown in blue, with the adjacent RBC in red, and with the parasitophorous vacuole space between the two. The fragments of MSP1 are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003085#pone-0003085-g007" target="_blank">Figure 7</a>.</p

The distribution of MSP1 within late trophozoites and schizonts.

<p>Panels A–C show the appearance of the single food vacuole, prepared for EM morphology. Panel A shows a late trophozoite containing a typically dilated food vacuole, containing widely spaced hemozoin and several profiles of internal membranous structures. In panel B an early schizont stage vacuole contains some hemozoin crystals and internal membrane profiles, while the vacuole wall shows signs of inward folding (arrowheads). C shows another schizont where a lipid body has formed adjacent to the food vacuole. Panels D–G show specific immunogold labeling of early to mid-stage schizonts labeled with MSP1₁₉-specific antibodies, in all cases detecting the protein at the vacuole wall. In panel D a limited area of labeling is present along one side of the food vacuole (small arrows), and in E a similar distribution is seen on a more folded vacuole wall. Panels F and G show labeling along obliquely sectioned folds of the wall (arrows), where the label also lies close to hemozoin crystals. Panels H and I show food vacuoles of late-stage schizonts containing closely packed large hemozoin crystals and are almost completely surrounded by MSP1₁₉ labeled vacuole wall membrane. In Panel J (and the inset of a portion at higher magnification) an early-/mid-stage schizont has been immunostained with antibody reacting with N-terminal regions of MSP1 but not MSP1₁₉. This antibody labels the newly synthesized (full-length) MSP1 on the parasite's plasma membrane but fails to label the food vacuole, indicating that the MSP1₁₉-specific labeling, seen in panels D–I, is specific for MSP1₁₉ carried in on the parasite surface at invasion rather than new MSP1 recently synthesized by the schizont. <i>Abbreviations</i>: FV–food vacuole; Hz–hemozoin; Int mem–internal membranous structures; RBC–red blood cell.</p

The distribution of MSP1₁₉ in ring-stage parasites detected by IEM.

<p>Panels D and E show MSP1₁₉-specific labeling of the surface of early rings, and in panel E, a clear endocytic vacuole is also labeled. Panels F–H show that MSP1₁₉ has been endocytosed into the small dense food vacuoles, and is located mainly at the inner vacuolar membrane surface; in Panel G the food vacuoles have a tubular appearance. In Panel I, a late ring is labeled, showing the incorporation of MSP1₁₉ into larger vacuoles containing small hemozoin crystals (Hz), with the label again associated with the vacuole membrane. <i>Abbreviations</i>: Fvtu- tubular food vacuoles; Hz- hemozoin.</p

Radiolabeling of MSP1₁₉, the final processed fragment of MSP1, showing that it remains attached to the parasite after merozoite invasion of erythrocytes, and that it persists within the parasite throughout its life cycle.

<p><i>P. falciparum</i> schizonts were radiolabeled with ³⁵S Met/Cys at 45 hours post-invasion for 1.5 hour and a proportion of these parasites were harvested (S, schizonts). The remaining parasites were allowed to undergo invasion, synchronized (2-hour window) and cultured for the next intraerythrocytic cycle. Parasites were harvested every 6 hours from 0 to 42 hours. Rabbit anti-MSP1-specific antibodies were used to immunoprecipitate labeled protein from NP40-extracts of these parasites. The resulting precipitates were analyzed by SDS-PAGE using a 5–12.5% gradient gel under reducing conditions and visualized by fluorography using X-ray film. The polyclonal anti-MSP1 serum recognized the full-length precursor as indicated by the top arrow in the labeled schizont extract, (S), and the 19-kDa fragment in newly formed ring stages (0 hours), indicated by the bottom arrow. The processed fragment of MSP7, MSP7₃₃ is also detected as part of the MSP1 complex in schizonts (middle arrow). The remaining lanes represent parasites harvested every 6 hours from 6 to 42 hours post-invasion. The intensity and the mobility of the MSP1₁₉ band remained constant until 42 hours when there was some decrease in intensity; this reduction in intensity could be due to rupture of some mature forms releasing MSP1₁₉ into the culture medium. The mobility of protein size markers is indicated at the right of the panel according to their size (kDa).</p

Immunofluorescence microscopy showing the localization of MSP1₁₉ and CRT in parasites that had invaded in the presence of mAb 1E1; showing that MSP1₁₉ moves to the food vacuole.

<p>Schizonts were allowed to release merozoites that invaded fresh erythrocytes in the presence of mAb 1E1, then ring-stage parasites were cultured and samples harvested every 3 hours over the asexual cycle. The mAb 1E1 antibody associates with MSP1₁₉ on the surface of the merozoite, and is detected within the ring-stage parasite; it was used to follow MSP1₁₉ in smears counterstained with rabbit anti-MSP1₁₉ (panel A) and rabbit anti-CRT (panel B), which is a marker of the food vacuole. Each row of four panels show an identical field from 1% formaldehyde-fixed thin smears of <i>P. falciparum</i> ring-stage parasites; columns A1 and B1 show mAb 1E1 associated with the parasite detected with Alexa Fluor 488 conjugated anti-mouse IgG (green); columns A2 rabbit anti-MSP1₁₉ and B2 rabbit anti-CRT, detected with Alexa Fluor 594 conjugated anti-rabbit IgG (red); columns A3 and B3 are composites of columns 1 and 2 with DAPI staining of the nucleus (blue) and any green and red fluorescence overlap is displayed in yellow. In columns A4 and B4, the parasitised erythrocyte is visualized by light microscopy, showing the location of the parasite within the infected erythrocyte. Only five time points are shown: 0, 6, 9, 12 and 18 hours post-invasion. The two anti-MSP1₁₉ antibodies, in panels A1 and A2, are seen to co-localize in panel A3, showing that 1E1 is still associated with MSP1₁₉ in the young parasite. CRT is clearly detectable from 6 to 9 hours onwards post-invasion (panel B2) and appears to be closely associated with MSP1₁₉ (panel B3). Pigment granules are clearly visible by light microscopy from about 18 hours (panels A4 and B4). Similar results were obtained in a separate experiment in which mAb 1E1 was used to detect MSP1₁₉ on formaldehyde fixed parasites at the same time points (data not shown) and at 24 hours (panel C). Panels C1–C4 show 1E1, (C1, green), rabbit anti-CRT (C2, red), composites of these antibodies with DAPI staining merged (C3) and light visualization (C4), as described above. CRT was clearly detectable (C2) and largely co-localized with MSP1₁₉ (C3). Both MSP1₁₉ and CRT were associated with the pigment detected by light microscopy in panel C4 and no longer around the surface of the parasite.</p

A diagram summarising the general fate of MSP1 and the C-terminal fragment MSP1₁₉ through the asexual cycle.

<p>In panel A, MSP1 on the surface of the invading merozoite undergoes cleavage to release the N-terminal portion (orange) and leave the C-terminal MSP1₁₉ (green) attached to the surface of the parasite: firstly as a newly internalized merozoite; and later as it transforms into a ring stage. MSP1₁₉ is then endocytosed from the ring surface into small food vacuoles as the parasite begins to feed on and metabolise hemoglobin. In B, all the MSP1₁₉ has been endocytosed and is associated with the membrane of the now single food vacuole, where it remains through the mid-schizont stage (C) and is then incorporated into the residual body at the end of schizogony (D).</p

IFA location of MSP1₁₉, CRT and new full length MSP1 during schizogony.

<p>Mature parasites at different stages of nuclear division were compared. Each row in panels A and B shows an identical field from 1% formaldehyde-fixed thin smears of late-stages parasites at the 1-, 2-, 4-, and 8-nuclei stage. Panels A1 and B1 show anti-MSP1₁₉(rabbit) and rabbit anti-CRT(rabbit) antibody labeling respectively (the secondary antibody was Alexa Fluor 488-conjugated anti-rabbit IgG antibody (green). Panels A2 and B2 show the same fields merged with the corresponding DAPI stained nucleus images (blue). Panels A3 and B3 show the same fields visualized by light microscopy - note the pigment is clearly visible. Rabbit-anti-MSP1₁₉ antibody only detects MSP1₁₉ associated with the food vacuole until the two-nuclei stage. Once new full length MSP1 is synthesized and transported to the parasite's plasma membrane the presence of MSP1₁₉ from the previous cycle is now obscured. In contrast, rabbit-anti-CRT antiobody is clearly detectable throughout parasite maturation and is associated with the food vacuole. Panel C, rows (a) and (b), shows that MSP1 and CRT are associated with the residual body that is released upon schizont rupture. Thin smears made at the time of schizont rupture were probed consecutively with mAb 1E1 and rabbit anti-CRT antibodies. These antibodies were detected using Alexa Fluor 488-conjugated anti-mouse IgG, and Alexa Fluor 594-conjugated anti-rabbit IgG, respectively (columns 1 and 2). Column 4 shows the bright field microscopy images of the same field and clearly indicate the presence of malarial pigment. Column 5 shows the absence of nuclear material in the residual bodies, as confirmed by the lack of DAPI staining. Column 3 shows the merged images from columns 1, 2 and 5 - both CRT and MSP1₁₉, or MSP1, which cannot be distinguished by the antibody, are associated with the released pigment in the residual body.</p