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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02673

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Re-defining the Golgi complex in Plasmodium falciparum using the novel Golgi marker PfGRASP

¹ Bernhard Nocht Institute for Tropical Medicine, Malaria II, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany
² The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne 3050, Australia

View larger version (43K): [in a new window]   Fig. 1. Structure of PfGRASP. (A) Schematic of the putative domain structure of PfGRASP, which comprises a N-terminal myristoylation motif (red), a well-conserved N-terminal GRASP domain (grey) and an unconserved C-terminus implicated in phosphorylation (blue, P-domain). (B) Comparison of PfGRASP with rat GRASP55 (RnGRASP55; GenBank AF110267), human GRASP55 (HsGRASP55; GenBank AAH07770) and Toxoplasma gondii GRASP55 (TgGRASP, ToxoDB TgTwinScan_6910). Asterisks indicate identical residues and colons indicate conserved residues. Proline and serine residues are highlighted in blue in the conserved P-domain; a cross-species conserved glycine is red.

View larger version (36K): [in a new window]   Fig. 2. Expression of PfGRASP in the asexual blood stages. (A) Transcription of Pfgrasp (red) was analysed by real-time RT-PCR using total RNA extracted from tightly synchronised parasites every 8 hours. A stage-specific control was performed using the early transcribed etramp gene (grey). Relative gene expression is shown in bar graphs. This ratio was calculated by comparing the average transcription of Pfgrasp and etramp with transcription of the housekeeping gene actin, which was set to 1. Relative quantification through real-time RT-PCR showed no stage-specific gene regulation for Pfgrasp. (B) Immunoblot analysis of wild-type parasites (3D7). Proteins from synchronised parasite cultures from samples taken every 8 hours, were separated by SDS-PAGE on a 10% gel under reducing conditions. Approximately equal amounts of parasite protein were loaded. Using anti-PfGRASP-specific antibodies, one major 70 kDa band can be detected throughout the asexual life cycle. Positions of molecular size markers (in kDa) are indicated.

View larger version (14K): [in a new window]   Fig. 3. Expression of PfGRASP-GFP in transgenic parasites. (A) Immunoblot using GFP-specific antibodies on wild-type (WT) and PfGRASP-GFP expressing parasites (GRASP-GFP). A band of 100 kDa, representing the GFP-fusion protein, is recognized by GFP-specific antibodies in the transgenic, but not in the WT parasite line. In addition, two smaller bands, possibly GFP breakdown products can be detected. (B) Anti-PfGRASP-specific antibodies recognize an 100 kDa PfGRASP-GFP fusion protein in addition to the endogenous PfGRASP protein of 70 kDa in PfGRASP-GFP-expressing parasites.

View larger version (36K): [in a new window]   Fig. 4. Localisation of PfGRASP by confocal and fluorescence microscopy in trophozoites (<24 hours post invasion). (A) Full-length PfGRASP is expressed as a GFP-fusion protein. Using fluorescence of the GFP reporter protein in live cells, PfGRASP-GFP distribution (green) is restricted to two compartments within the parasite (a) (see also supplementary material Movie 1). These compartments are in close proximity to the nucleus (b, blue). Merge with bright-field image (c). (B) Fixed WT parasites were incubated with PfGRASP-specific antibodies. PfGRASP-specific antibodies (a, red) show a similar fluorescence pattern in fixed cells compared with PfGRASP-GFP expressing parasites. However, additional unspecific staining can be detected. Merge with DNA-specific stain (b, blue). Merge with bright-field image (c). Bar, 2 µm.

View larger version (51K): [in a new window]   Fig. 5. Spatial organisation of the PfGRASP-GFP defined compartment by fluorescence microscopy on fixed parasites. (A) PfGRASP-GFP colocalises with antiPfGRASP-specific antibodies. PfGRASP-GFP is tightly confined to two compartments (a, green) near the parasite nucleus (a, blue). AntiPfGRASP-specific antibodies show a similar staining pattern (b, red with nucleus in blue). Merged image shows the colocalisation of the compartments defined by either PfGRASP-specific antibodies or PfGRASP-GFP-expressing parasites (c, yellow). (B) PfGRASP-GFP colocalises with the cis-Golgi marker ERD2. PfGRASP-GFP (a, green) accumulates in two discrete compartments in close proximity to the nucleus (a, blue). Anti-PfERD2 antibodies recognize similar structures (b, red with nucleus in blue). Merged image shows colocalisation of compartments (c, yellow). (C) PfGRASP-GFP defines a compartment that is distinct from the ER (see also supplementary material Movie 2). At the early stages of the parasite life cycle (<16 hours post invasion) PfGRASP is restricted to one compartment (a, green) juxtapose to the nucleus (a, blue). The ER is visualised by anti-PfBiP-specific antibodies (b, red). The membranous system of the ER forms an envelope around the nucleus (b, blue) with one protrusion (indicated by arrow). Merged image shows no colocalisation of the two compartments (c). (D) PfGRASP-GFP does not colocalise with the trans-Golgi marker PfRab6. PfGRASP accumulates in two discrete foci (a, green) adjacent to the nucleus (a, blue). Antibodies against PfRab6 visualise two distinct sites within the parasite (b, red with nucleus in blue). Merged image shows no colocalisation of the PfGRASP defined compartment with PfRab6 (c) (see also supplementary material Movie 3). All panels labelled d in A-D are merges of fluorescent and bright-field images. Bar, 2 µm.

View larger version (34K): [in a new window]   Fig. 6. Golgi dynamics throughout the asexual life cycle of Plasmodium. Live images of transgenic parasites expressing PfGRASP-GFP were visualised by confocal microscopy. (a-b) Images 8-16 hours post invasion. In ring-stage parasites PfGRASP-GFP is restricted to one compartment in close proximity to the nucleus (blue). (c) Images 24 hours post invasion. A second Golgi is generated prior to nuclear division. (d-e) Cells 32-40 hours post invasion. As the parasite matures, nuclear division commences and is accompanied by multiplication of the Golgi. (f) Cells 46 hours post invasion. The parasite has nearly reached the final stage of schizogony where each forming merozoite will be equipped with one Golgi and nucleus. (g) Released parasites at 0 hours. Each merozoite has inherited one Golgi. Bar, 2 µm (a-f); 1 µm (g).

View larger version (22K): [in a new window]   Fig. 7. Localisation of PfGRASP depends on a functional N-terminal myristoylation motif. Fluorescence microscopy was performed on live parasites. (a) In parasites expressing PfGRASP-GFP the protein is restricted to two tightly defined compartments (green). (b) Merge with bright-field image. (c) Mutation of the putative N-terminal myristoylation site (from glycine to alanine) abolishes targeting of PfGRASP-GFP and results in a cytoplasmic distribution of the fusion protein. (d) Merge with bright-field image. Bar, 2 µm.

View larger version (72K): [in a new window]   Fig. 8. Effect of Brefeldin A on the distribution of PfGRASP. (a) Parasites were incubated with BFA for 24 hours. Localisation of PfGRASP-GFP in live parasites is focused to one compartment within the parasite (green). (b) Merge with bright-field image. (c) As a control, cultures were incubated with ethanol to ensure normal growth and morphology. The image displays a mature parasite (>32 hours) with multiple fluorescing foci (green). (d) Merge with bright-field image. Bar, 2 µm.

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