Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method

Tonkin, Christopher J.; Dooren, Giel G. van; Spurck, Timothy P.; Struck, Nicole S.; Good, Robert T.; Handman, Emanuela; Cowman, Alan F.; McFadden, Geoffrey I.

doi:10.1016/j.molbiopara.2004.05.009

Cited by 415 publications

(398 citation statements)

References 36 publications

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“…For confocal microscopy, infected red blood cells were labelled with 2.5 mM Bodypi-TR-C_5-ceramide for 30 min at 37°C as described 31 and imaged with a Fluoview 1000 confocal microscope (Olympus) equipped with a Â 100/1.4 oil immersion lens. Immunofluorescence assays for the MSP1 co-localization were carried out according to established procedures using fixation in 4% formaldehyde, 0.0075% glutaraldehyde in PBS 38 but with parasites coated on multiwell slides using concanavalin A 39 with the following modifications: before the primary antibody incubation the cells were blocked and permeablized with 3% BSA, 100 mM glycine, 0.2% Triton-X-100 in PBS for 1 h. Primary antibodies were applied in 3% BSA, 100 mM glycine in PBS overnight and secondary antibodies for 1 h in 3% BSA in PBS. Antibodies used were rabbit antimerozoite surface protein 1, MSP1, (1: 500), a kind gift of M. Blackman and mouse monoclonal anti-GFP (Roche; 1:500).…”

Section: Methodsmentioning

confidence: 99%

Identity of a Plasmodium lactate/H+ symporter structurally unrelated to human transporters

et al. 2015

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Maintenance of a high glycolytic flow rate is critical for the rapid growth and virulence of malarial parasites. The parasites release two moles of lactic acid per mole of glucose as the anaerobic end product. However, the molecular identity of the Plasmodium lactate transporter is unknown. Here we show that a member of the microbial formate-nitrite transporter family, PfFNT, acts as a lactate/proton symporter in Plasmodium falciparum. Besides L-lactate, PfFNT transports physiologically relevant D-lactate, as well as pyruvate, acetate and formate, and is inhibited by the antiplasmodial compounds phloretin, furosemide and cinnamate derivatives, but not by p-chloromercuribenzene sulfonate (pCMBS). Our data on PfFNT monocarboxylate transport are consistent with those obtained with living parasites. Moreover, PfFNT is the only transporter of the plasmodial glycolytic pathway for which structure information is available from crystals of homologous proteins, rendering it amenable to further evaluation as a novel antimalarial drug target.

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Section: Methodsmentioning

confidence: 99%

Identity of a Plasmodium lactate/H+ symporter structurally unrelated to human transporters

et al. 2015

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“…The prepared slides were then stored at −80°C with desiccants until use. The smears were thawed, fixed in 4% paraformaldehyde containing 0.0075% glutaraldehyde [25] (Nacalai Tesque) in PBS at RT for 15 min, rinsed with 50 mM glycine (Wako) in PBS. Sporozoites were permeabilized with 0.05% Triton X-100 in PBS for 5 min.…”

Section: Indirect Immunofluorescence Assay (Ifa)mentioning

confidence: 99%

Expression and localization of rhoptry neck protein 5 in merozoites and sporozoites of Plasmodium yoelii

Mutungi

Yahata

Sakaguchi

et al. 2014

Parasitology International

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Host cell invasion by Apicomplexan parasites marks a crucial step in disease establishment and pathogenesis. The moving junction (MJ) is a conserved and essential feature among parasites of this phylum during host cell invasion, thus proteins that associate at this MJ are potential targets of drug and vaccine development. In both Toxoplasma gondii and Plasmodium falciparum, a micronemal protein, Apical Membrane Antigen 1 (AMA1), and Rhoptry Neck proteins (RONs; RON2 and RON4) form an essential complex at the MJ. A new RON member, RON5, was shown to be important to stabilize RON2 during development and to associate with the MJ complex in T. gondii and also to be immunoprecipitated by anti-AMA1 antibody in P. falciparum. However, the detailed molecular nature of RON5 in Plasmodium is not well understood. In this study, Plasmodium yoelii RON5 gene (pyron5) was identified as an ortholog of P. falciparum and Plasmodium berghei ron5. The pyron5 exon-intron structure was validated by comparing genomic DNA sequences and experimentally determining fulllength complementary DNA sequence. PyRON5 was detected in water-insoluble fractions but no reliable transmembrane domain(s) were predicted by transmembrane prediction algorithms. PyRON5 formed a complex with PyRON4, PyRON2, and PyAMA1 in late schizont protein extract. Taken together, we infer that these results suggest that PyRON5 associates with membrane indirectly via other MJ components. Indirect immunofluorescence assay and immunoelectron microscopy localized PyRON5 at the rhoptry neck of the late schizont merozoites and at the rhoptry of sporozoites. The two-stage expression of PyRON5 suggests that PyRON5 plays roles in invasion of not only erythrocytes, but also of mosquito salivary glands and/or mammalian hepatocytes. Graphic Abstract Highlights• PyRON5 forms a complex with RON2, RON4, and AMA1 in schizont extract.• PyRON5, a putative soluble protein, exists in the water-insoluble fractions.• These suggest that PyRON5 associates with membrane via other complex component(s).• PyRON5 is expressed in the rhoptry in both merozoite and sporozoite parasites.• Targeting PyRON5 may prevent cell invasion by both merozoite and sporozoite. Mutungi et al., 2

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“…Fixed PE were processed as previously described (Tonkin et al, 2004), except all washes and solutions used PIPES buffer. Anti-Tg actin antibody, used to visualize Pfactin, and the 677-1 antibody, which labels an epitope in the PVM/PPM (Gormley et al, 1992), were both used at a dilution of 1:700.…”

Section: Electron Microscopymentioning

confidence: 99%

A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum

Lazarus

Schneider

Taraschi

2008

Journal of Cell Science

108

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The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial thin sections of parasitized erythrocytes (PE) by electron microscopy. We find that actin dynamics play multiple, important roles in the hemoglobin transport pathway, and that hemoglobin delivery to the FV via the cytostomes might be required for parasite survival. Evidence is provided for a new model, in which hemoglobin transport to the FV occurs by a vesicle-independent process.

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Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method

Cited by 415 publications

References 36 publications

Identity of a Plasmodium lactate/H+ symporter structurally unrelated to human transporters

Identity of a Plasmodium lactate/H+ symporter structurally unrelated to human transporters

Expression and localization of rhoptry neck protein 5 in merozoites and sporozoites of Plasmodium yoelii

A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum

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