Streptolysin O (SLO) was a kind gift from Sucharit Bhakdi (University of Mainz, Germany). Saponin was purchased from Fluka. Sequence-grade modified trypsin was purchased from Promega (Madison, WI, USA). All the other chemicals, including Brefeldin A (BFA), were of the highest available purity and were purchased from Sigma-Aldrich.
Parasite cultures and fractionation of iRBC
Parasites (FCBR strain of P. falciparum ) were continuously cultured in erythrocytes of blood group A+ in heat inactivated human serum as described previously . Cultures were synchronized and trophozoite infected erythrocytes (iRBC), 28-30 hours post infection, were enriched to a parasitaemia of more than 90% by gel flotation . IRBC were permeabilized with SLO as described previously . Briefly, 1 × 109 iRBC (in aliquots of 2 × 108 cells) were incubated with 3-4 haemolytic units of SLO in PBS pH 7.2 at room temperature for 6 min. Samples were centrifuged at 10,000 × g for 15 s. The pellet containing intact parasites, the vacuolar contents, and membranes was washed twice with 200 μl of PBS. The removal of haemoglobin was monitored spectrophotometrically as described previously . For saponin lysis, 1 × 109 iRBC (in aliquots of 2 × 108 cells) were incubated in 200 μl of 0.1% saponin in PBS pH 7.2 on ice for 5 min. The samples were centrifuged at 2,500 × g for 5 min. The pellet containing the intact parasite devoid of the host cytosol and PV contents was washed twice with 200 μl of PBS before processing for further analysis by SDS-PAGE and western blot. To prevent proteolysis by endogenous proteases, all the buffers used in this section contained a protease inhibitor cocktail (PIC) consisting of antipain, chymostatin, aprotinin, trypsin inhibitor, Na-EDTA, pepstatin, leupeptin, and elastatinal, each at a concentration of 1 μg.ml-1.
Stage specific expression experiments
Parasite cultures (FCBR strain) were synchronized and trophozoite-infected erythrocytes (iRBC), 28-30 hours post-infection, were enriched to a parasitaemia of more than 90% by gel flotation . Aliquots equivalent to 2 × 108 iRBC were used to initiate new 5 ml cultures. After reinvasion these cultures were synchronized by alanine treatment  to obtain tightly synchronized parasite stages. The parasites were harvested 6-12 hours, 18-24 hours, 30-36 hours and 38-44 hours post-invasion. 2 × 107 equivalent amounts of the parasites from each set time point were lysed in SDS-PAGE sample buffer before analysis by SDS-PAGE and western blot. All the buffers used in this section contained the protease inhibitor cocktail (PIC).
Recombinant p68 form and production of specific anti-p68 antibodies in mice
The region encoding the p68 form of PfA-M1 was amplified by using Forward 5'-ACG GAT CCT GTT AAA AAG AAC GAA CC-3' and Reverse 5'-ATG GAT CCA TTG TGC ATT TAC TGG TG-3' primers (BamHI site in bold and underlined) and inserted into the BamHI site of pET-15b (Novagen). The recombinant plasmid was sequenced to confirm the correct reading frame and the absence of mutation. It encodes a N-terminal (His)6-tagged protein, corresponding to residues 191 to 802 of PfA-M1. This (His)6-p68 recombinant protein was produced in BL21(DE3) Escherichia coli as described , but was not soluble. It was isolated, by using BugBuster™ (Novagen) in presence of 6 M urea according to manufacturer's instructions and purified on nickel-affinity columns, in presence of urea. The eluted fractions containing recombinant (His)6-p68 were dialyzed against 10 mM HEPES NaOH pH 8.0 to remove urea and concentrated on Centriprep10 (Ultrafiltration device, Millipore) prior to being used to immunize three mice. Mice were injected four times with the purified-recombinant (His)6-p68 at three weeks intervals, the first injection being performed with complete Freund's adjuvant and the next three injections with incomplete Freund's adjuvant. The three mice produced antibodies specifically labelled p68, but also p96 and p120, as determined by immunoblotting.
2D-gel electrophoresis for the comparative analysis of anti-MAP1 and anti-p68 antibodies
Parasites (FcB1 strain of P. falciparum) were cultured but not synchronized, and isolated by using 0.1% saponin as previously described . Purified parasites were lysed in 0.1 mM Tris-HCl pH 7.5 by three cycles of freezing/thawing in presence of protease inhibitors (Complete™ EDTA-free (Boehringer Mannheim) and 2 mM EDTA) as previously described , and separated into soluble and membrane fractions by ultra-centrifugation (100,000 × g for one hour). 100,000 g soluble extracts were concentrated by using kit-2D clean up (GE Healthcare), and identical amounts (70 μg proteins) were used to rehydrate two identical 3-10 strips (GE Healthcare) in the presence of 8.75 M urea, 2.5 M thiourea, 5% CHAPS, 3.5 mg.ml-1 DTT and 2% ampholytes (GE Healthcare). After electrofocusing, the two strips were equilibrated 15 minutes in 6 M urea, 50 mM Tris-HCl pH 8.8, 70 mM SDS, 34.5% Glycerol and 65 mM DTT then 15 minutes in 6 M urea, 50 mM Tris-HCl pH 8.8, 70 mM SDS, 34.5% Glycerol and 130 mM iodoacetamide, before being placed side by side on a single large 7%-SDS-PAGE. The SDS-PAGE was run and processed for immunoblot analysis, as described in the corresponding section.
Brefeldin A treatment of parasites
Parasites (FcB1 strain of P. falciparum) were cultured and synchronized as previously described  in the presence of 367 μM hypoxanthine to obtain a population of early rings (one to six hours post-invasion) that was divided into three identical cultures (B, E and O, with about 5 × 108 iRBC each). Brefeldin A (BFA) was added to B at a final concentration of 5 μM (about 1.5 μg.ml-1) from a 5 mg.ml-1 solution in ethanol as previously described . Control cultures received (E) or not (O) equivalent amount of ethanol to ensure that ethanol had no effect on growth and cell morphology. These three cultures were further incubated for 19 hours prior to parasite harvest and preparation by using 0.1% saponin as described . Purified parasites were lysed in 0.1 mM Tris-HCl pH 7.5 by three cycles of freezing/thawing and analysed by SDS-PAGE and immunoblotting as described in the corresponding section.
Immunofluorescence analysis of parasites
In parallel, aliquots of BFA-treated and control cultures were smeared on slides and fixed for 5 min in a mixture of methanol and acetone (1:4) at -20°C. Slides were washed in PBS, blocked at room temperature in non-fat milk (5% in PBS) then incubated for two hours at room temperature with anti-MAP1 antibodies as previously described . Slides were then washed again three times in PBS and incubated for two hours at room temperature with mouse anti-exp2  antibodies. After three further washes in PBS, slides were incubated with a mixture of anti-rabbit-Ig coupled to Alexa-fluor-568 and anti-mouse-Ig coupled to Alexa-fluor-488 secondary antibodies (Molecular Probes). Nuclei were labelled by 10 min incubation with 4 μg.ml-1 Hoechst 33342 (Molecular Probes). Slides were mounted in Vectashield medium (Vector laboratories) with a coverslip and viewed using a Nikon Eclipse TE 300 DV inverted microscope with a 100× (NA 1.3) oil objective mounted on a piezo electric device using appropriate fluorescence emission filters. Image acquisition was performed using a Coolsnap HQ camera (Roper Scientific, France) and images were finally processed by using Metamorph software.
Isolation of intact food vacuoles
To isolate intact food vacuoles parasites were fractionated as previously described with minor modifications [3, 20]. Briefly 109 iRBC were washed three times with PBS and permeabilized with SLO as described previously. The iRBC (in aliquots of 2 × 108 cells) were incubated with 3-4 haemolytic units of SLO in PBS pH 7.2 at room temperature for 6 min. Samples were centrifuged at 10,000 × g for 15 s. The pellet containing intact parasites, the vacuolar contents, and membranes was washed twice with 200 μl of PBS. To release the food vacuoles the pellet was lysed by resuspension in 10 volumes of ice-cold water, pH 4.5, and immediately triturated four times using a 27-G 1.2 cm needle. The mixture was centrifuged at 13,000 rpm for 10 min to obtain the soluble proteins (A) and a pellet containing membranes and vacuoles. The crude vacuole preparation was washed with 10 volumes of ice-cold water, pH 4.5 centrifuged at 13,000 rpm for 2 min and the supernatant was analysed (B) to monitor the distribution of marker proteins. The pellet was resuspended in 1 ml of uptake buffer (2 mM MgSO4, 100 mM KCl, 10 mM NaCl, 25 mM HEPES, 25 mM NaHCO3, and 5 mM sodium phosphate pH 7.4, containing 5 mg.ml-1 of DNase 1), and incubated at 37° C for 5 min followed by centrifugation for 2 min at 13,000 rpm. The supernatant was discarded and the pellet was again resuspended in 100 μL of ice-cold uptake buffer and mixed with 1.3 ml of ice-cold 42% Percoll™ containing 0.25 M sucrose and 1.5 mM MgSO4, pH 7.4. The suspension was triturated 2 times through a 27-G 1.2-cm needle before centrifugation at 13,000 rpm for 10 min at 4°C. The fractions C, D, E, and F were obtained after the centrifugation step. Purified vacuoles (E) were collected as a dark band at the bottom 50 μL of the gradient. The vacuoles were then resuspended in 1 mL of uptake buffer, and centrifuged at 13,000 rpm for 2 min to wash off the residual Percoll™ before proceeding with immunoblot analysis using an equivalent of 4 × 107 iRBC per lane. All the buffers used in this section contained the protease inhibitor cocktail (PIC).
Western blot analysis
Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes using standard procedures. To detect the presence of PfA-M1 [4, 6] and the other marker proteins (PfAldolase , PfBip , PfPV1 , PfPlasmepsin I , PfSERP ), the membranes were blocked with 3% BSA in PBS, pH 7.4, for 1 h at room temperature before overnight incubation at 4°C with the primary antibodies. After the overnight incubation the membranes were washed three times with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl before incubating with the secondary antibody, anti rabbit IgG conjugated with horseradish peroxidase (FCBR strain) or anti rabbit IgG conjugated with alkaline phosphatase (FcB1 strain) for one hour. The proteins bands were visualized using the ECL (FCBR strain) or nitroblue tetrazolium/5-bromo-4-chloro-3-indol phosphate (FcB1 strain).
Stable transfection of P. falciparum with a PfA-M1-GFP construct
The plasmid pPM2GT, used to stably transfect plasmepsin II-GFP into Plasmodium , kindly provided by Dr. Klemba, was modified as follows: the plasmepsin II gene segment encoding the C-terminal end of the protease was excised by using the AvrII and XhoI sites and was replaced by a 1024-bp segment encoding the C-terminal part of PfA-M1. To amplify this PfA-M1 gene segment, we used the Forward 5'-GCA CGC TCG AGT AAT TAT TAT TGA AAT ATG ATA GTG ATG C-3'(AvrII site in bold) and Reverse 5'-GCA CGC CTA GGT AAT TTA TTT GTT AAT CTT AAT AAA TAT TC-3'(XhoI site in bold) primers. The recombinant plasmid was sequenced to confirm the correct reading frame and the absence of mutation before transfection into the FcB1 strain of P. falciparum according to . Transfected parasites were selected using 10 nM WR99210 as described in  and the selective pressure was removed and applied again to isolate pseudo-clonal parasites named PfA-M1-GFP-51. These parasites, which are very similar to the PfA-M1-YFP parasites generated by  were analysed by southern blotting to confirm proper integration of the plasmid, immunoblotting to visualize the recombinant protein and fluorescence imaging to localize the GFP fluorescence. Live imaging was performed on parasites stained with 4 μg.ml-1Hoechst 33342, using the Nikon Eclipse TE 300 DV inverted microscope, the Coolsnap HQ camera and the Metamorph software for image processing, as described above for the immunofluorescence imaging.
Transient transfection of P. falciparum with a PfA-M1 [1-30]-GFP construct
The PfA-M1 [1-30]-GFP plasmid was constructed using Multisite Gateway™ technology described by van Dooren et al . Briefly, the Multisite Gateway™ is based on specific recombination of pENTR plasmids called promoter vector, gene vector and reporter vector with a destination vector. Promoter vector (PfHSP86), reporter vector (GFP mut2) and destination vector (hDHFR cassette conferring resistance to WR99210 ) were kind gifts of G. van Dooren and G. MacFadden. The gene vector specific for the first 30 amino acids of PfA-M1 was constructed by amplifying the corresponding segment (on P. falciparum genomic DNA) using Forward 5'-CAC CAT TAC AAA ATG AAA TTA ACA AAA GGC TG-3' and Reverse 5'-GCA CCT TTT TTT ATT ATC ATA AAG AAT-3'primers and inserting the segment into pENTR™D/Topo® (Invitrogen). The recombinant plasmid was produced following the Multisite Gateway™ technology (Invitrogen) as described  and sequenced to confirm the correct reading frame and the absence of mutation. P. falciparum (FcB1 strain) parasites were then transfected with this plasmid and transfectants were selected using 5 nM WR99210 as described . Transfectants were analysed by immunoblotting and live imaging as described above.