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First published online 13 May 2008
doi: 10.1242/jcs.023150

Journal of Cell Science 121, 1937-1949 (2008)
Published by The Company of Biologists 2008
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A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum

Michelle D. Lazarus, Timothy G. Schneider and Theodore F. Taraschi*

Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA


Figure 1
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  		Fig. 1. Cytostomes are present during each intraerythrocytic stage. (A-C) Electron micrographs depicting the presence of cytostomes (arrowheads) in ring (A), trophozoite (B) and schizont (C) stage parasites. (D) Electron micrograph of a representative trophozoite stage PE in which three cytostomes (CYT) are visible (CYT 1, CYT 2, CYT 3) within a single thin section. (E) Trophozoite stage PE in which the cytostome is observed interacting with the FV. FV, food vacuole; P, parasite; PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane; RBCM, red blood cell membrane. Scale bar: 100 nm.

 

Figure 2
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  		Fig. 2. Hemoglobin-containing compartments are contiguous with the cytostome. Electron micrographs of serial thin sections (sections 1-9) from a representative trophozoite stage PE. Three cytostomes, CYT 1, CYT 2 and CYT 3, are apparent. CYT, cytostome; P, parasite; RBC, red blood cell; PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane. Scale bar: 100 nm.

 

Figure 3
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  		Fig. 3. Cytostomes possess an electron-dense collar. (A-D) Electron micrographs of representative trophozoite stage PE fixed with `mix-fix'. Arrowheads denote the electron-dense collars that encircle the cytostome neck. P, parasite; RBC, red blood cell. Scale bar: 100 nm.

 

Figure 4
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  		Fig. 4. Parasite actin distribution in the erythrocytic cycle. (A) Western blot of 15 µg of erythrocyte (E) or parasite (P) protein showing the anti-actin antibody is specific for parasite actin and non-reactive with erythrocyte actin. (B) Pfactin in the triton-soluble (gray) and triton-insoluble (black) fractions was quantified by densitometric analysis of western blots using a parasite specific anti-actin antibody in ring (n=4), trophozoite (n=6) and schizont (n=5) stage PE. (C) Triton-soluble (gray) and triton-insoluble (black) Pfactin fractions were separated from trophozoites following JAS (n=6) or CD (n=6) treatment, and quantified by densitometric analysis of western blots using a parasite specific anti-actin antibody. Data are expressed as a percentage of total Pfactin±s.e.m.

 

Figure 5
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  		Fig. 5. Pfactin distribution and localization in untreated, JAS- or CD-treated trophozoite stage PE. Representative confocal microscopy images showing Pfactin localization (green) in relation to the PVM/PPM (red) in untreated, JAS- and CD-treated trophozoite PE. Merge is a composite of the green and red images; merged+DIC is a composite of the DIC, green and red images. The electron-dense inclusion (hemozoin) in the DIC images indicates the location of the parasite FV. Bar, 2.0 µm.

 

Figure 6
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  		Fig. 6. Serial-section electron micrographs of JAS- and CD-treated PE. Representative serial thin-section electron micrographs depicting cytostome morphology in (A) JAS-treated and (B) CD-treated PE. Scale bars are in nm.

 

Figure 7
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  		Fig. 7. Single thin-section electron micrographs of untreated, JAS- or CD-treated trophozoite-stage PE. (A-C) Representative single thin-section electron micrographs of (A) untreated, (B) CD- or (C) JAS-treated PE. White arrowheads denote CS. CS, cytostomal section; CYT, cytostome; FV, food vacuole; N, nucleus; P, parasite; PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane; RBC, red blood cell; RBCM, red blood cell membrane. Scale bar: 100 nm

 

Figure 8
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  		Fig. 8. Electron micrographs showing cytostome collar morphology in JAS-treated PE. (A-D) Single thin-section electron micrographs of trophozoite PE fixed with `mix-fix' immediately following incubation with JAS. (A,B) The electron-dense ring is observed as two electron-dense collars (arrowheads). Note the radiating electron-dense pattern at the cytostome neck both in the horizontal view (C) and the top view (D). Scale bar: 100 nm.

 

Figure 9
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  		Fig. 9. The effects of JAS and CD on cytostome neck diameter. Dot plot displaying cytostome neck diameters analyzed in untreated (U; n=103), JAS- (n=47), and CD- (n=41) treated PE. Data are displayed in the natural log scale with the dots representing the means±s.e.m.

 

Figure 10
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  		Fig. 10. Immunoelectron microscopy depicting the localization of Pfactin in JAS-treated PE. (A-D) Representative electron micrographs depicting localization of Pfactin in JAS-treated trophozoite PE. (A-C) Pfactin associated with the neck and body of the cytostome (black arrowheads). (D) Pfactin is specifically labeled with the anti-actin antibody in the PVM/PPM (black arrowheads) and the parasite cytosol (white arrowheads). In addition, some Pfactin is present in the erythrocyte cytosol (pointed black arrowheads). Scale bar: 100 nm.

 

Figure 11
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  		Fig. 11. The effect of JAS on P. falciparum intraerythrocytic development. (A,B) Pf lactate dehydrogenase (PfLDH) activity was measured in the ring (00-04 hours post-invasion; n=3), late ring (12-16 hours post-invasion; n=3), early trophozoite (20-24 hours post-invasion; n=3) and late trophozoite (28-32 hours post-invasion; n=3) stages. (B) PfLDH activity was measured following a 3-hour incubation with (white bars) and without (black bars; n=3) JAS. Data are normalized to number of parasites and are expressed as a percentage of PfLDH activity in untreated PE; error bars denote ±standard error. Statistical significance (asterisks) was determined using Student's t-test, where P<0.05 when comparing untreated and JAS-treated PE within each intraerythrocytic stage. n, number of separate parasite cultures analyzed.

 

Figure 12
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  		Fig. 12. Effects of JAS and CD on transport of hemoglobin to the FV. Representative electron micrographs depicting the effects of actin-perturbing agents on accumulation of undigested hemoglobin within the FV. Arrowheads denote cytostomal sections (CS). CD, cytochalasin D; FV, food vacuole; JAS, jasplakinolide; P, parasite. Scale bar: 500 nm.

 

Figure 13
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  		Fig. 13. A new model for hemoglobin transport to the FV. (Steps 1-3) Cytostome formation. (1) The PVM invaginates after which (2) the PPM invaginates. (3) A double-membrane electron-dense collar forms around the cytostome. (Steps 4-6) Cytostome maturation. (4) The cytostome continues to fill with red blood cell cytosol and hemoglobin, and (5) elongates to appose the FV. (Steps 6-8) Hemoglobin deposition and degradation in the FV. (6) Fusion occurs between the matured cytostome and the FV (white arrowheads), while, simultaneously, the cytostome pinches off from the PVM and PPM (black arrowheads), resulting in the release (7) of a single-membrane-bound hemoglobin-filled vesicle into the FV lumen. During Step 6 (white arrowheads) content mixing occurs between the FV lumen and the PVS. (8) The membrane of this vesicle is degraded by FV-resident lipases, while the hemoglobin is degraded by FV-resident proteases. The resulting heme is polymerized into hemozoin (Hz). P, parasite; PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane; PVS, parasitophorous vacuolar space; RBC, red blood cell; RBCM, red blood cell membrane.

 

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© The Company of Biologists Ltd 2008