441Research Article

IntroductionPlasmodium falciparum causes ~1 million malaria-related deathsannually (Snow et al., 2005). Disease pathology is associated withthe parasite’s life cycle within a parasitophorous vacuole (PV) insidethe red blood cells (RBCs) of its human host. As it develops, theintraerythrocytic parasite degrades ~75% of the host haemoglobin(Francis et al., 1997; Loria et al., 1999; Rosenthal and Meshnick,1996). This provides a source of amino acids and creates space forgrowth, as well as generating osmolytes that prevent premature hostcell lysis (Lew et al., 2003).

Early studies of the feeding process in P. falciparum-infectedRBCs indicated that uptake of host cytoplasm is most active introphozoites and involves morphologically distinct endocyticstructures, termed cytostomes, at the parasite surface (Aikawa etal., 1966). The resulting endocytic invaginations are surroundedby both the parasite plasma membrane (PPM) and the PVmembrane (PVM) (Slomianny, 1990). The double-membrane-bound vesicles that bud from the cytostome were proposed tomigrate to and fuse with an acidic digestive vacuole (DV). Single-membrane-bound vesicles were assumed to be delivered into theDV lumen, followed by release and degradation of the haemoglobin(Yayon et al., 1984).

Recent studies have re-examined the process of haemoglobinuptake in infected RBCs but have led to somewhat conflictingconclusions. A study using serial thin-section electron microscopy(EM) (Elliott et al., 2008) described four distinct pathways forhaemoglobin uptake by P. falciparum. These authors proposed thatring-stage parasites fold around a ‘gulp’ of host cell cytoplasm asthe first step in the biogenesis of the DV. They suggested that smallcytostome-derived haemoglobin-containing vesicles and tubules

continue the uptake of haemoglobin as the parasite matures. In moremature parasites they also described additional cytostome-independent endocytic structures called phagosomes (Elliott et al.,2008).

Another study examined the ultrastructure of the endocyticapparatus and concluded that haemoglobin uptake occurs by avesicle-independent process (Lazarus et al., 2008). These authorsreported that cytostomal invaginations elongate to form tubes thatappose the DV but remain connected to the parasite surface andopen to the RBC cytoplasm. They suggested that the tubules pinchoff from the parasite surface and simultaneously undergo fusionwith the DV to release their contents into the DV lumen (Lazaruset al., 2008).

Another area of debate is the point at which haemoglobindigestion is initiated. Some authors have argued that haemoglobindigestion commences only after cytostomal vesicles fuse with theDV (Olliaro and Goldberg, 1995; Slomianny and Prensier, 1990).Others suggest that haemoglobin digestion is initiated en route tothe DV, and that the DV is merely a dumping site for haemozoincrystals (Hempelmann et al., 2003).

In this work we have re-examined DV genesis and estimated thepH of the DV and the endocytic compartments by following theuptake of pH-sensitive probes. We have used live-cell imaging andphotobleaching to examine the dynamics and connectivity ofdifferent compartments. We also used electron tomography togenerate high-resolution images of sections of parasite-infectedRBCs. We have combined this with serial sectioning to obtaintomographic reconstructions of whole parasites at different stagesof development. We compared the data from these two imagingmodalities to provide novel insights into the feeding process.

Digestive-vacuole genesis and endocytic processes inthe early intraerythrocytic stages of PlasmodiumfalciparumNurhidanatasha Abu Bakar1,*, Nectarios Klonis1,2,*, Eric Hanssen1,2,*, Cherrine Chan1 and Leann Tilley1,2,‡

1Department of Biochemistry and 2Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne, 3086, Australia*These authors contributed equally to this work‡Author for correspondence (L.Tilley@latrobe.edu.au)

Accepted 2 November 2009Journal of Cell Science 123, 441-450 Published by The Company of Biologists 2010doi:10.1242/jcs.061499

SummaryThe digestive vacuole of the malaria parasite Plasmodium falciparum is the site of haemoglobin digestion and haem detoxification,and is the target of chloroquine and other antimalarials. The mechanisms for genesis of the digestive vacuole and transfer of haemoglobinfrom the host cytoplasm are still debated. Here, we use live-cell imaging and photobleaching to monitor the uptake of the pH-sensitivefluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in ring-stage and trophozoite-stage parasites. We compare theseresults with electron tomography of serial sections of parasites at different stages of growth. We show that uptake of erythrocytecytoplasm is initiated in mid-ring-stage parasites. The host cytoplasm is internalised via cytostome-derived invaginations and concentratedinto several acidified peripheral structures. Haemoglobin digestion and haemozoin formation take place in these vesicles. The ring-stage parasites can adopt a deeply invaginated cup shape but do not take up haemoglobin via macropinocytosis. As the parasite matures,the haemozoin-containing compartments coalesce to form a single acidic digestive vacuole that is fed by haemoglobin-containingvesicles. There is also evidence for haemoglobin degradation in compartments outside the digestive vacuole. The work has implicationsfor the stage specificity of quinoline and endoperoxide antimalarials.

Key words: Malaria, Plasmodium falciparum, SNARF-1-dextran, pH, Protein trafficking, Plasmepsin, Digestive vacuole

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ResultsCharacterisation of RBCs resealed to contain SNARF-1-dextranHuman RBCs that have been lysed and resealed at highhaematocrit retain the ability to support the growth of P.falciparum (Dluzewski et al., 1983) and can be used to trapfluorescent markers (Goodyer et al., 1997; Krogstad et al., 1985).We used an optimised ratio of lysis buffer to RBC volume (2.5:1)to minimise the loss of RBC cytoplasm while achieving arelatively homogenous population of resealed RBCs containingthe pH-sensitive probe, SNARF-1-dextran (Whitaker et al.,1991). The SNARF-1 chromophore undergoes an acid-basetransition from a yellow- to a red-emitting form that can bemonitored using a single excitation wavelength (Bassnett et al.,1990; Seksek and Bolard, 1996; van Erp et al., 1991). Theresealed RBCs retain 20-45% of their haemoglobin(corresponding to 4-9 mM monomeric haemoglobin) and contain0.1-0.2 mol% of the fluorescent dextran with respect tohaemoglobin, corresponding to an intracellular concentration of10-20 mM and a trapping efficiency of 30-60%. The resealedRBCs were invaded with 54±5% of the efficiency of controlRBCs, in agreement with previous data (Frankland et al., 2006).Parasites developing in resealed RBCs complete the cycle andform merozoites that are capable of reinvading further resealedRBCs, although the efficiency of invasion in the second roundis reduced (data not shown).

We constructed a SNARF-1 pH calibration curve by suspendingresealed RBC in buffers of different pH in the presence of theionophore, carbonyl cyanide 3-chlorophenylhydrazone (CCCP)(Allen and Kirk, 2004; Klonis et al., 2007). CCCP has previouslybeen used to equilibrate compartments in P. falciparum-infectedRBCs to external buffers of known pH (Allen and Kirk, 2004; Kloniset al., 2007). Fluorescence images were collected for resealed RBCswith different levels of trapped fluorophore on three separateoccasions, and the ratio Rry (red/yellow fluorescence intensity) wasused as an indicator of pH (Fig. 1A). The relatively small standarddeviation (s.d.) values indicate that any differences in SNARF-1uptake and haemoglobin levels do not affect the Rry values.SNARF-1-dextran in buffered solutions gave a similar calibrationcurve (data not shown). The decrease in Rry values with decreasingpH is adequately described by a simple acid-base transition withan apparent pKa of 7.2, which is similar to the reported value of~7.5 (Owen, 1992; Whitaker et al., 1991). Given its response profile,SNARF-1 can be used to distinguish between acidic and neutralcompartments, but cannot provide accurate values at pH values lessthan 6 (Rry<0.9).

In order to visualise the spatial variation of Rry values (and hencepH) within a cell, we generated Rry images in HSB colour space,where the Rry value at each pixel is mapped to a particular colourin the hue channel and its corresponding total intensity (Ir + Iy) isplaced in the brightness channel. This generated the look-up tableshown in Fig. 1B, where neutral to basic compartments (Rry values>1.2) appear blue-purple whereas compartments with pH values <6(Rry values<0.8) appear green. This is illustrated by the Rry imagesof resealed RBC resuspended at pH 5.5 and 7.5 in the presence ofCCCP (Fig. 1B). We examined the effect of CCCP treatment onSNARF-1 that has been accumulated into the DV of trophozoite-stage parasites (Fig. 1C). In untreated cells, the DV appears green,consistent with a DV pH value <6 (Fig. 1Ci), whereas in cellssuspended in PBS at pH 7.5 in the presence of CCCP, the DVappears purple (Fig. 1Cii).

Uptake of host RBC cytoplasm is initiated in mid-ring-stage parasitesTo examine the earliest events associated with the uptake of theRBC cytoplasm, we purified tightly synchronised (~6 hour window)parasites (D10 strain) at the schizont stage (40-48 hours) and usedthem to invade resealed RBC containing SNARF-1-dextran. After20 to 24 hours (i.e. 12-24 hours after invasion) we transferred themto a microscope chamber that was gassed and maintained at 37°C(Fig. 2). The cells appear to be viable for ~10 hours, however weimaged them within 2 hours. By analysing differential interferencecontrast (DIC) images, we chose parasites that had no visiblehaemozoin (Fig. 2A-D). Based on the estimated time after invasionand the absence of visible pigment, we define these as mid-ring-stage parasites. In some infected RBCs (Fig. 2A), the parasite isobserved as a dark region in the fluorescence intensity images(second column), with no evidence for endocytic compartments.The images shown are average fluorescence intensity z-sectionprojections taken through the parasite, confirming that this is not aconsequence of an internalised compartment being out of the planeof focus.

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Fig. 1. Characterisation of SNARF-1-dextran as a pH sensor for live-cellimaging. (A)RBCs resealed in the presence of 50mM SNARF-1-dextran wereresuspended in saline buffer containing 10mM CCCP and imaged at 37°C. Rry

values ± s.d. were determined for individual cells (n>30 for each point; threeexperiments). The curve represents the best fit to the data using a simple acid-base transition with a pKa of 7.2. (B)Rry images of resealed CCCP-treatedRBCs in buffers at pH 5.5 (i) and pH 7.5 (ii). The colour at each pixel reflectsthe Rry value from the look-up table. (C)DIC (left) total intensity (middle) andRry images (right) of trophozoite-infected SNARF-1-dextran-labelled RBCs at37°C in RPMI, pH 7.4 (i), and in PBS, pH 7.4, containing 10mM CCCP (ii).Scale bars: 5mm.

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In other mid-ring-stage parasites, punctate structures containingSNARF-1-dextran were observed near the parasite periphery (Fig.2B-D, yellow arrowheads). The SNARF-1 concentration within

these compartments is comparable with that in the host cellcytoplasm, suggesting that they represent early endocytic events.They are observed as regions of altered Rry values in theratiometric images (Fig. 2) indicating they are acidic in nature;however, analysis of the fluorescence signal in this compartmentwas complicated by contributions from the host cell. To betterobserve these compartments, we selectively photobleached theSNARF-1 fluorescence in the host compartment by parking thelaser in a region distal to the parasite (indicated by ‘x’) andilluminating with an unattenuated laser for 10 seconds. This resultsin the loss of fluorescence throughout this compartment (Fig. 2,middle and right panels) (Klonis et al., 2002), without affectingthe fluorescence intensity of any SNARF-1 that has beeninternalised by the parasite. This increases the contrast of the acidiccompartments and confirms that they are located within theparasite and are not in direct communication with the RBCcytoplasm. Indeed they were often revealed as multiple punctathat appear green in the post-bleach Rry images. The average Rry

of these compartments was 0.85±0.07 (n24; four experiments)corresponding to a pH of <6. In no examples did we observeendocytic compartments that exhibited a neutral pH. By contrast,the Rry values in the RBC cytoplasm of uninfected and infectederythrocytes were 1.43±0.19 (n29; four experiments) and1.40±0.16 (n42; four experiments), corresponding to pH valuesof 7.2±0.3 and 7.14±0.25, respectively. We also analysed anotherparasite strain (3D7) and examined parasites developing inresealed RBCs containing TMR-dextran or recombinant GFP asreporters (supplementary material Fig. S1). We observedequivalent structures with these different reporters.

In many ring-stage parasites, a large fluorophore-labelled feature(1-2 mm in diameter) appeared to lie within the parasite (Fig. 2B-D, green arrows) and was also evident in the DIC images. Thefluorophore appeared to be at a similar concentration to the bulkof the RBC cytoplasm and the Rry value (in the pre-bleach image)was similar to that of the RBC cytoplasm. The structure couldcorrespond to a proposed macropinocytic structure call the ‘biggulp’, which was recently described (Elliott et al., 2008).Interestingly, photobleaching of the host cell fluorescence depletedthe fluorescence associated with these spherical structures to thesame degree (Fig. 2B-D, middle and right panels) demonstratingthat this structure remained connected to the RBC cytoplasm. Weanalysed 100 infected RBCs before the appearance of haemozoinpigment and found punctate peripheral structures in 94% of cellsand larger spherical structures in 63% of cells.

DV formation is consolidated in late-ring and earlytrophozoite stagesDIC images were used to identify a second population of parasitescontaining small dark puncta; these probably represent the firstdetectable haemozoin particles (Fig. 2E-F). Based on this criterion,we define these as late-ring-stage parasites. Many of the cellspossessed more than one pigment-containing structure or acombination of pigmented and non-pigmented structures. Thesecompartments often showed brighter fluorescence than the hostcytoplasm, indicating a concentration event. The internalisedcompartments were invariably acidic with a pH <6 (Rry0.86±0.09;n28; four experiments). The low pH, the accumulation of the hostcytoplasmic components, and the presence of haemozoin suggestthat haemoglobin digestion is initiated in several independent pre-DV compartments in these early stage parasites. Application of alaser pulse to one of the peripheral structures (Fig. 2I, yellow arrow)

Fig. 2. Endocytosis of RBC cytoplasm in ring and early trophozoite stages.Mid rings (A-D), late rings (E,F) and early trophozoite (G,H) parasites (D10)developing in SNARF-1-dextran-containing resealed RBCs were imaged at37°C. Shown are DIC images and average projections of the total intensity andRry values from serial sections encompassing the region occupied by theparasite. Regions of the RBC cytoplasm (indicated by ‘x’ in the pre-bleachintensity image) were subjected to a high-intensity bleaching pulse (10seconds) to deplete the host fluorescence before acquisition of post-bleachdata. Yellow arrows correspond to small individual or multiple punctateperipheral compartments within the parasite. Green arrows correspond tolarger spherical features in the parasite cytoplasm that are apparent in the pre-bleach projections. (I)A cell was subjected to high-intensity illumination for10 seconds (‘x’) to deplete the host cell fluorescence, then the arrowedstructure was pulsed for 1 second. Scale bar: 5mm.

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results in selective photobleaching of this compartment, confirmingthe presence of several independent compartments.

The cells shown in Fig. 2G,H contained more prominent pigmentin the DIC image and probably represent the next stage of DVformation (i.e. the early trophozoite stage). The haemozoin wasconfined to one region of the parasite and was associated withinternalised acidic structures that were considerably brighter thanthe surrounding host cell cytoplasm. In Fig. 2G, the endocyticcompartments might be in the process of coalescing into a singlesite within the parasite, whereas in Fig. 2H they appear to havecoalesced. Other larger features were observed within the parasitesat these stages (Fig. 2E-H, green arrows), which appear to representinvaginations of the host cell cytoplasm. In all cases, photobleachingmeasurements demonstrate that these features remain connected tohost cell cytoplasm.

Trophozoite-stage parasites possess acidified DV andextra-DV compartmentsWe examined the distribution of SNARF-1-dextran in the endocyticcompartments of mature parasitised RBCs (i.e. 32-40 hours afterinvasion). In these cells, SNARF-1 was considerably moreconcentrated within the parasite compared with the host cytoplasm.The probe accumulated in the major haemozoin-containingcompartment in the parasite cytoplasm, i.e. in the mature DV (Fig.3, red arrows). In approximately 70% of the mature-stage parasites,additional acidified compartments were observed within the parasitecytoplasm (Fig. 3, yellow arrows). These structures usually did notcontain any visible haemozoin, although in some cases (Fig. 3C),a small amount of haemozoin was visible. The structures varied insize but were always smaller than the DV. These could representendocytic structures en route to the DV, or separate digestivecompartments. Both the DV and the extra-DV compartmentsappeared green in the Rry images, indicating that they are acidified.

It should be noted that the DV compartment is very susceptibleto photo-induced damage. At higher illumination levels or uponcontinuous illumination the Rry value for this compartment increasedduring imaging (data not shown). Illumination-induced changes inDV pH have been reported previously for another pH-sensitiveprobe (Wissing et al., 2002). Light-induced generation of damaginghydroxyl radicals during bleaching might be exacerbated by thepresence of haemoglobin breakdown products in the DV. We usedthe lowest possible levels of illumination and used highly sensitiveAPD detectors to minimise any light-induced artefacts in the datapresented here. None the less, it is difficult to ascribe a reliable pHto the DV compartment. By contrast, the extra-DV compartment isless susceptible to photo-induced damage (as is the case for theacidic compartments in ring-stage parasites). This permitted a morereliable estimate of Rry (0.82±0.04; n125; 12 experiments),corresponding to a pH <6. Application of a laser pulse to an extra-DV compartment completely ablated the fluorescence associatedwith this compartment but did not affect the intensity of the DV(Fig. 3A) and vice versa (Fig. 3B,C). The same result was obtainedin a total of 75 cells, indicating that the lumens of thesecompartments are not connected.

Plasmepsin II is delivered to early endocyticcompartments and the extra-DV compartmentPlasmepsin II is a protease involved in haemoglobin digestion andrepresents a marker for compartments where haemoglobin digestionis occurring. We examined plasmepsin-II-GFP transfectants(Klemba et al., 2004) developing in TMR-dextran-containing RBCs(Fig. 4A-D). In mid-ring-stage parasites (Fig. 4A), plasmepsin-II-GFP was co-located with TMR-dextran in the peripheral puncta(Fig. 4A, yellow arrows), indicating that plasmepsin II is deliveredto endocytic compartments early in parasite development and wouldbe available to initiate haemoglobin digestion. By contrast, the largerspherical profile structures (Fig. 4B, white arrow) had no associatedplasmepsin-II-GFP. In more mature-stage parasites, plasmepsin-II-GFP and TMR-dextran were co-located in the DV (Fig. 4C,D,yellow arrows) and in some extra-DV and endocytic compartments(Fig. 4C,D, red arrowheads). However, there are some peripheralplasmepsin-II-GFP compartments that are not associated withaccumulation of TMR-dextran (Fig. 4C, blue arrows). Thesestructures might represent regions of the parasite surface wherecytostomal invaginations are forming, but which have not yetbudded from the PPM.

LysoSensor Blue labelling confirms the low pH of theendocytic compartmentsWe also imaged cells labelled with LysoSensor Blue, a weak basethat accumulates in acidic compartments (Lin et al., 2001; Wissinget al., 2002). We found that low levels of the probe and low levelillumination were needed to prevent alkalinisation of smallercompartments and to avoid photo-induced damage. Under optimisedimaging conditions, LysoSensor Blue accumulated in the punctateperipheral compartments in ring-stage-infected RBCs (Fig. 4E,F,depicted in red) and overlapped with the SNARF-1 fluorescence(Fig. 4E,F, depicted in green). It is not present in the larger sphericalprofile structures (Fig. 4E, arrow). In mature-stage parasites,LysoSensor Blue accumulated in the DV where its fluorescenceoverlapped with the SNARF-1 fluorescence (Fig. 4G,H). TheSNARF-1-containing extra-DV compartments were also labelledwith LysoSensor Blue, confirming that these compartments areacidic (Fig. 4H, yellow arrow). Interestingly, there were also some

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Fig. 3. Endocytic compartments in mature trophozoites. DIC images, Rry

images of single sections through the endocytic compartments of SNARF-1-dextran-containing infected RBCs, and corresponding overlays, at 32-40 hourspost-invasion. The SNARF-1 fluorescence is associated with the DV (redarrows) and with extra-DV compartments (yellow arrows) with no visiblehaemozoin (A,B) or a small pigment punctum (C). High-intensity illumination(2 seconds) was applied to the extra-DV (A) or the DV (B,C) compartments todeplete the fluorescence. Scale bar: 5mm.

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compartments in the cytoplasm of mature-stage parasites that wereweakly labelled with LysoSensor Blue, but not with SNARF-1 (Fig.4H, blue arrow). These might represent additional endocytic orsecretory compartments (e.g. rhoptries) that are separate from thehaemoglobin-uptake pathway.

Electron tomography provides details of the genesis ofthe DV in ring-stage parasitesElectron tomography is an invaluable tool for obtaining 3Dinformation from samples at high resolution (Hanssen et al., 2008)and can be combined with serial sectioning techniques to permithigh-resolution imaging of organelles, and even whole cells (Lucicet al., 2008; Noske et al., 2008). Sections are examined in an EMoperating at moderate to high voltage with a tiltable stage. Theimages are aligned and tomographic reconstructions of the sampleare generated computationally using segmentation tool. We carriedout an electron tomographic analysis of serial sections (7-15sections of ~300 nm) of infected RBCs (K1 strain) at six differentstages of development.

Individual virtual sections (7 nm) from within tomograms ofwhole parasites at early development stages are presented in Fig.5A,E,I,M. (See translations through entire data sets for Fig. 5E andM in supplementary material Movies 1 and 2.) The parasite-limitingmembrane and selected features within the parasite were renderedto reveal details of the 3D organisation. Rotations of the rendereddata for B,F,J,N are presented in supplementary material Movies3-6. The parasite surface is depicted in translucent cream, thecytostome as a yellow ring. Attached cytostomal invaginations arerepresented in translucent yellow and the nucleus is in gold. Thesurface is rendered in opaque gold in the right panels.

At the earliest stage examined (Fig. 5A-D; parasite volume, 2.60fl), very few intracellular features were apparent. The parasiteexhibited a cupped-hand morphology with several finger-likeextensions (Fig. 5B, red arrows), but there was no evidence forendocytic vesicles. Another parasite (Fig. 5E-H; parasite volume,2.54 fl) exhibited a flatter shape and the characteristic cytostomewas associated with a cytostomal invagination (Fig. 5E,F, redarrows). The cytostome comprised an electron-dense ring with aninternal diameter of ~90 nm (outer diameter ~190 nm) that encircledthe neck of invaginations of the PPM and PVM (membrane-limitedopening of ~55 nm). Additional sections through cytostomes (Fig.5Q,R) reveal the complex ultrastructure of this feature. Theendocytic flasks remained connected to the RBC cytoplasm throughthe open mouth of the cytostome ring. A number of small vesicularstructures near the parasite periphery (Fig. 5F-H, depicted inorange) are likely to be derived from cytostomal invaginations.These contained small crystals (Fig. 5E, orange; also see translationthrough the tomogram in supplementary material Movie 1). Thisindicates that haemoglobin digestion and haemozoin formation havebeen initiated. These structures probably represent the acidicperipheral compartments observed in SNARF-1-dextran-labelledring-stage parasites.

A slightly larger volume parasite (Fig. 5I-L; 2.65 fl) had a cuppedprofile (Fig. 5L, supplementary material Movie 5). This cellcontained several vesicles with microcrystalline contents (Fig. 5I,orange) that accumulated in one region of the cell. In a more matureparasite (Fig. 5M-P; 3.0 fl), the cup-shape was even morepronounced (see Fig. 5P). Two cytostomes were observed at thesurface of the parasite (Fig. 5O, arrowheads) and a series of vesiclesand a larger flattened structure containing small crystalline structureswere present in one region of the parasite (Fig. 5M-O, orange, blue).

Fig. 4. Identification of haemoglobinase-containing and LysoSensor-Blue-labelled compartments in infected RBCs. The panels are DIC or bright-fieldimages, GFP or SNARF-1 fluorescence (depicted in green) or TMR-dextran orLysoSensor Blue fluorescence (depicted in red), and overlays of the red andgreen images. (A-D)Plasmepsin-II-GFP-3D7 transfectants in TMR-dextran-containing resealed RBCs. The pre-DV and DV compartments (yellow arrows)and the extra-DV compartments (red arrowheads) contain both plasmepsin-II-GFP and TMR-dextran. Plasmepsin-II-GFP is also present in structures thatcould represent nascent cytostomal invaginations (blue arrows). There is noplasmepsin-II-GFP associated with a larger spherical profile structure (whitearrow). (E-H)In rings (E,F) and trophozoites (G,H) in SNARF-1-dextran-containing RBCs, LysoSensor Blue is associated with peripheral structures andthe DV (arrowheads) and extra-DV structures (yellow arrows). The sphericalstructure in ring-stage parasites (A, white arrow) is not labelled. Someadditional structures in the trophozoite-stage parasites are weakly labelled withLysoSensor Blue, but do not contain SNARF-1-dextran (H, blue arrow). Scalebars: 5mm.

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The larger compartment might represent the initial stage ofcoalescence of the pigment-containing vesicles into a single DV.The double-membrane-bound haemoglobin-containing structure(asterisk) is likely to be equivalent to structure referred to as the‘big gulp’ (Elliott et al., 2008). In single sections it appeared to beinside the parasite, however, a global analysis revealed that thisinvagination remained connected to the RBC cytoplasm (seesupplementary material Movies 2 and 6). A region of a late-ring-to early trophozoite-infected RBC was examined (Fig. 5S,T). Thiscell had a structure (depicted in pink) that contained haemoglobin,as well as a crystalline feature (black arrowhead). This mightrepresent a haemoglobin-containing vesicle where haemozoinformation is being initiated.

Electron tomographic analysis of the DV and endocyticcompartments in trophozoite-stage parasitesWe also generated individual virtual sections (7 nm) and renderedtomograms of trophozoite-infected RBCs (Fig. 6, supplementarymaterial Movies 7-9). In a mid-trophozoite parasite (Fig. 6A-D;12.1 fl; two nuclei), a centrally located DV, containing large crystalsof haemozoin, was surrounded by a single membrane. The DV wasfolded and wrapped around a region of parasite cytoplasm (Fig.

6A,C, blue outline/rendering). An additional compartment, adjacentto the DV, contained microcrystallites of haemozoin (Fig. 6B-D,orange outline, black arrow). This compartment is probablyequivalent to one of the acidified extra-DV compartments observedin the SNARF-1-dextran-labelled trophozoites. The cell had threecytostomal invaginations (Fig. 6C-D, yellow rings with attachedtranslucent yellow pouches), one of which was visible in the virtualsection (Fig. 6A, arrow). The trophozoite stage sees the genesis ofsmall compartments comprising a clear lumen around an electron-dense body (Fig. 6, pink outline/rendering). These are likely to beacidocalcisomes (Docampo et al., 2005).

In a more mature-stage parasite (21.7 fl, four nuclei), a large DVwas surrounded by a single membrane (Fig. 6E-H, blue).Characteristic electron-dense cytostomes, again with an internaldiameter of ~90 nm, and associated endocytic flasks were observedat the parasite surface (depicted in translucent yellow; yellow arrowin Fig. 6E). A cytostome also constricted a bi-lobal haemoglobin-containing feature that appeared to be inside the parasite (Fig. 6F,G,yellow arrows). This type of compartment might also contribute tothe extra-DV compartments observed in SNARF-1-dextran-labelledtrophozoites; however, the structure was opposed to the PPM at theinterface between two physical EM sections, so we cannot rule out

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Fig. 5. 3D reconstruction of entire ring-stage parasites frommulti-section electron tomograms. (A-P)Serial sections werecollected through ring-infected RBCs (K1 strain). Left panelsshow selected virtual sections (7 nm) from within some of thephysical sections. Vesicles and pre-DV compartments withcrystalline material are outlined in orange and blue; a cytostome(in E) is indicated with a red arrow. Middle panels showsegmentation of individual structures. The parasite surface isdepicted in translucent cream, the cytostome as a yellow ringand the attached cytostomal invaginations in translucent yellow;the nucleus is gold, crystal-containing vesicles, orange, and pre-DVs, blue. Right panels show the parasite surface rendered inopaque gold. Scale bars: 0.2mm (A,E,I,M); 500 nm (B,F,J,N).(Q,R)Longitudinal and transverse sections through a cytostomein a D10-infected RBC. Scale bars: 0.1mm. (S,T)Section froma tomogram through a region of a late-ring-stage parasite andrendered model of this region. Some crystal-containing vesiclesand a pre-DV are rendered in orange and blue. Another vesiclein this region (pink) appears to contain haemoglobin and anascent crystal. Scale bars: 300 nm (S); 500 nm (T).

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the possibility that it retains some connectivity with the PPM.Interestingly, we observed a haemoglobin-containing vesicle locatedwithin the DV (Fig. 6E, square). A virtual section though a tiltedplane suggests that the DV is engulfing the haemoglobin-containingvesicle (see inset). The vesicle contained an electron-lucent bodywith the appearance of a haemozoin crystal.

DiscussionHaemoglobin uptake and degradation is initiated in earlystage parasitesTwo recent studies of DV genesis and haemoglobin uptake processesused serial thin section electron microscopy (Elliott et al., 2008;Lazarus et al., 2008). However, because of the huge amount of workinvolved in this very challenging technique, these studies wererestricted to reconstruction of a limited cell depth or of a few wholecells. Moreover the resolution in the z-plane is determined by thesection thickness (i.e. ~70 nm), which limits the ultrastructuralinformation. In this work, we used electron tomography to studythe digestive apparatus. Electron tomography permits analysis of300-400 nm sections with enhanced z-plane resolution and has beenused to identify novel features of the P. falciparum exomembranesystem (Hanssen et al., 2008; Henrich et al., 2009). We implementedmethods for collecting data from multiple sections and usedprograms for tiling and stitching tomographic data (Hanssen et al.,2009; Noske et al., 2008) to generate whole parasite images; thisallowed us to re-examine the 3D ultrastructure of the digestiveapparatus at different stages of parasite development.

We compared the electron tomography data with images of liveparasites in resealed RBCs containing a fluorescent tracer. ResealedRBCs have been validated previously for use in studies of P.falciparum invasion and growth (Dluzewski et al., 1985; Rangachariet al., 1987) and for trapping inhibitors in the RBC cytoplasm(Frankland et al., 2006; Tilley et al., 1990). In this work, we resealedRBCs to contain SNARF-1-dextran, using it as a convenient,chemically stable, non-toxic, pH-sensitive marker of endocyticprocesses. We optimised temperature and gas control in themicroscope sample chamber to allow imaging of viable parasites.This assured us that any observations have physiological relevance.

Very early stage parasites developing in SNARF-1-labelledRBCs showed no endocytic compartments; however, at the mid-ring stage, the fluorescent marker was observed in acidifiedperipheral compartments. Electron tomography revealed cytostomalinvaginations that remained connected to the RBC cytoplasm, as

well as several small peripherally located vesicular compartmentscontaining microcrystals of haemozoin. We suggest that thesecytostome-mediated endocytic events represent the first step in thegenesis of the haemoglobin-degrading apparatus of P. falciparum.

In late-ring-stage parasites, the SNARF-1 signal in the endocyticstructures was often more intense than in the RBC cytoplasm. AsSNARF-1 fluorescence intensity decreases at low pH (Bassnett etal., 1990), this indicates that endocytosis of the host compartmentis followed by a process that concentrates the contents of theendocytic compartment. Excess lipids in the cytostomal vesiclesmight be consumed by internalisation and degradation, providinga catalyst for haemozoin formation (Bendrat et al., 1995; Jacksonet al., 2004). Electron tomography confirms the accumulation ofhaemozoin crystals in late-ring-stage parasites. Moreover, a GFPchimera of plasmepsin II was delivered to the early endocyticstructures, indicating that conditions for initiation of haemoglobindegradation are met. The data suggest that following budding ofcytostomal vesicles, the integrity of the inner membrane is rapidlylost, allowing mixing of proteases and haemoglobin in an acidifiedcompartment; this would initiate haemoglobin digestion andhaemozoin formation. Indeed, we observed examples ofhaemoglobin-containing endocytic compartments apparentlyundergoing the first stages of crystal formation. Our observation ofhaemoglobin digestion in mid-ring-stage parasites contrasts withthe often-quoted view that haemoglobin degradation is only initiatedin more mature-stage parasites.

It is interesting to consider how the pH of the ring-stageendocytic structures might be controlled. The membrane potentialacross the PPM is maintained by the electrogenic extrusion ofprotons via a V-type proton pump (Allen and Kirk, 2004; Mikkelsenet al., 1982). Acidification of the DV is thought to involve the sameV-type proton-ATPase, as well as a proton-pyrophosphatase (Salibaet al., 2003). It appears likely that proton pumps are transferred tothe DV via the same endocytic process that drives haemoglobinuptake. Thus, endocytosis of a region of the PPM would captureproton pumps leading to the immediate acidification of the endocyticcompartments once it has budded from the PPM. The fact that wedid not observe any neutral compartments that are wholly withinthe parasite is consistent with their rapid and obligate acidification.

A larger fluorescent feature, with a spherical appearance, that isassociated with most of the SNARF-1-labelled ring-stage parasites,is likely to be equivalent to the recently described ‘big gulp’ (Elliottet al., 2008). Our electron tomography studies reveal that the ring-

Fig. 6. 3D reconstruction of entire trophozoite-stageparasites from multi-section electron tomograms.Serial sections were collected through RBCs infected withparasites at mid trophozoite (A-D) and early-schizont(E-H) stages (K1 strain). Left panels show selectedvirtual sections (7 nm) from within some of the physicalsections. The DV is outlined in blue, crystal-containingextra-DV compartments in orange and a putativeacidocalcisome in pink. Cytostomes are indicated withyellow arrows and a furrow in the DV in A with anarrowhead. Right panels show individual structures thatwere segmented and depicted with the colours describedin Fig. 5. An extra-DV compartment (B-D) is rendered inorange (black arrow in C). The inset in E shows astructure containing haemoglobin and haemozoin thatappears to be in the process of being engulfed by the DV.Scale bars: 500 nm and 100 nm (inset in E).

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stage parasite adopts a pronounced cup shape, and we propose thatthis invagination gives rise to the feature of spherical appearancein the confocal microscopy images. Indeed, early ultrastructuralstudies showed that shortly after invasion the ovoid-shapedmerozoite flattens into a thin disc, then invaginates to form a cupshape (Bannister et al., 2000; Bannister et al., 2004; Langreth etal., 1978). However, we found that this structure is not acidifiedand remains connected to the RBC cytoplasm. Moreover it co-existswith the peripheral structures in which haemoglobin digestion isoccurring. Taken together, our live-cell imaging and electrontomography data argue against the ‘big gulp’ as the progenitor ofthe DV and instead indicate the formation of several cytostome-derived acidified compartments as the earliest event in DV genesis,as originally postulated by Aikawa and colleagues (Aikawa et al.,1966).

An interesting question is what determines the cup shape of thering parasites. Shortly after invasion, material is released from thedense granules into the PV (Aikawa et al., 1990), followed by theelaboration of different membrane structures in the RBC cytoplasm(Lanzer et al., 2006; Tilley et al., 2008). The membrane vesiclesinvolved in protein export (Marti et al., 2004) would be expectedto expand the parasite surface; however, the high concentration ofhaemoglobin in the RBC cytoplasm might prevent a significantincrease in parasite volume. So the cup shape might simply reflectthe need to increase surface area without a significant increase involume. The increased parasite surface area might also providematerial that can be endocytosed via the cytostomes. Indeed,elongation of the endocytic flask to accommodate the expandingsurface area of the parasite could lead to strain at the constrictedneck of the flask and promote budding into the parasite cytoplasm.

Acidic DV and extra-DV compartments in trophozoite-stage parasitesAs the parasite matures the peripheral structures coalesce into acentral acidic DV that is marked by the presence of prominenthaemozoin crystals. Another recent study (Dluzewski et al., 2008)also concluded that small crystal-containing vacuoles coalesce asthe parasite matures to form a single DV. The trophozoite-stageparasites have 3–4 cytostomes with attached endocytic invaginationsthat remain open to the RBC cytoplasm. A recent study suggestedthat the cytostomal invaginations do not detach and migrate to theDV but extend till they contact and are engulfed by the DV (Lazaruset al., 2008). By contrast, we observed (in the SNARF-1-labelledcells) acidified extra-DV compartments in the parasite cytoplasm(~70% of the trophozoite-stage parasites examined). Many of thesewere tightly juxtaposed to the DV and they might have beenassumed to be part of the DV in previous studies. However, selectivephotobleaching experiments showed that they are indeed separatestructures. Plasmepsin-II-GFP was present in these acidifiedstructures and electron tomography revealed examples ofcompartments adjacent to the DV that contained haemozoin crystalsor small haemozoin crystals in the presence of haemoglobin. Thesestructures are presumably derived from large cytostomalinvaginations and appear to represent relatively long-lived extra-DV compartments. We also observed a vesicle that appeared tocontain both haemoglobin and an early haemozoin crystal beingengulfed by the DV. This suggests that the DV can phagocytosehaemoglobin-containing structures as well as fusing with them.Taken together, our data suggest that following endocytosis,haemoglobin digestion can be initiated before, during or afterdelivery of the endocytic structures to the DV.

Our data provide clear evidence for the budding of cytostome-derived vesicles into the parasite cytoplasm and support previousstudies showing that this is a major pathway for the uptake ofhaemoglobin in both ring-stage and trophozoite-stage parasites. Wealso observed a large invaginated structure bisected by a cytostomethat might represent an example of a phagosome (Elliott et al., 2008).At this stage, we cannot exclude a role for an additional cytostome-independent pathway in the trophozoite stage. Identification of theprotein components of the cytostome and generation of transfectantsexpressing GFP chimeras of these proteins would make it feasibleto distinguish cytostomal invaginations from phagotrophs in livecells and would help to resolve this question.

pH of the DV and endocytic compartmentsWe found that the pH of the pre-DV and extra-DV compartmentswas less than 6. This is consistent with other recent estimates ofDV pH (Klonis et al., 2007; Kuhn et al., 2007). The pH optima forthe DV plasmepsins, histo-aspartic protease (HAP) and thefalcipains are in the range pH 5-6 (Banerjee et al., 2002; Goldberget al., 1990; Rosenthal, 2004) and the pH optimum for b-haematinformation is between 3 and 6 (Egan et al., 2001; Slater and Cerami,1992). Thus our data indicate that haemoglobin digestion andhaemozoin will occur in both ring-stage and trophozoite-stageparasites, and in both the DV, extra-DV and pre-DV compartments.

In a previous study, we labelled plasmepsin-II-GFP transfectantswith LysoSensor Blue (Klonis et al., 2007) and found that a numberof plasmepsin-II-GFP-labelled features were not acidified. Weconcluded that these were endocytic compartments that were notacidified; however, our current data indicate that they are more likelyto represent nascent cytostomal invaginations that have not yet lostthe connection to the RBC cytoplasm. This is consistent with aprevious immuno-EM study showing plasmepsin II located atnascent cytostomal invaginations (Klemba et al., 2004).

Implications for antimalarial drug actionOur finding that the pre-DV compartments in the ring-stageparasites are acidified and that haemoglobin degradation isinitiated at this stage has implications for the action of quinolineantimalarials. Quinoline antimalarials accumulate in acidiccompartments and inhibit haemozoin formation (Geary et al.,1990; Hawley et al., 1996; Tilley et al., 2006). Our data suggestthat both ring and trophozoite stages would accumulatechloroquine in acidic compartments. Although some studiessuggest maximal chloroquine sensitivity at the trophozoite stage(Hoppe et al., 2004; Yayon et al., 1983), others find that ring stagesare the most sensitive (Orjih, 1997; Zhang et al., 1986); yet otherssuggest that both stages are sensitive but that ring stages exhibita ‘delayed death’ effect (Gligorijevic et al., 2008; Yayon et al.,1983). Our data indicate that chloroquine would accumulate inring-infected RBCs and would inhibit the early stages ofhaemozoin formation.

The data also have implications for the action of endoperoxideantimalarials. Endoperoxides are activated by the haem that isreleased during haemoglobin breakdown [or by reduced iron thatis derived from haem (Krishna et al., 2004; Weissbuch andLeiserowitz, 2008)]. Recent reports suggest that artemisinin targetsthe DV and associated lipid bodies (del Pilar Crespo et al., 2008;Hartwig et al., 2009) and also inhibits endocytosis of haemoglobin(Hoppe et al., 2004). Our data suggest that the release of haem fromhaemoglobin would be initiated in the ring stage of parasite growthand would be available as a source of an iron-based activator. This

Journal of Cell Science 123 (3)

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might well underlie the finding that artemisinin is highly activeagainst ring-stage parasites (Skinner et al., 1996).

The live-cell imaging and multi-section electron tomographyapproaches used in this study represent important technicaladvances. In combination with data from previous studies, theseapproaches provide new insights and resolve some controversiesregarding the endocytic processes at the different stages of parasitedevelopment. This is illustrated in the model presented in Fig. 7.The data suggest that uptake of haemoglobin commences early inthe intraerythrocytic development of the parasite. Haemoglobindigestion is initiated in multiple pre-DV compartments that coalescein the early trophozoite stage to form a central DV. The data showthat cytostome-dependent endocytosis is the mechanism for uptakeof haemoglobin the ring stage, and is probably the principlepathway in the trophozoite stage. Following budding of endocyticvesicles, haemoglobin digestion can occur either before, during orafter fusion with the DV. These findings are important inunderstanding the mode of action of different classes of antimalarialdrugs.

Materials and MethodsResealing RBCs and culturing parasitesRBCs were lysed in 2.5 volumes of ice-cold 5 mM phosphate, pH 7.5, 1 mM Mg-ATP (Sigma) in the presence of 50 mM tetamethylrhodamine-dextran (TMR-dextran;10 kDa; Invitrogen) or 5�(and 6�)-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1�(3H)-isobenzofuran]-3�-one (SNARF-1-dextran; 10 kDa;Invitrogen) or recombinant His-tagged enhanced GFP (pEGFP-N1, Clontech) andresealed as described previously (Frankland et al., 2006; Krogstad et al., 1985). Thelevels of haemoglobin and incorporated SNARF-1-dextran were determinedspectrophotometrically. P. falciparum (D10, 3D7, K1 strains and plasmepsin-II-GFPtransfectants) was cultured and isolated as described previously (Klemba et al., 2004;Klonis et al., 2007).

Fluorescence microscopyLive-cell imaging of SNARF-1-dextran-containing resealed RBC was performed at37°C using a �100 oil immersion objective (1.4 NA) on a Zeiss LSM 510 invertedconfocal microscope equipped with an incubation chamber and heated stage underan atmosphere of 5% CO2 in air. Glass coverslips (Menzel-Glaser, #1.5) were cleanedwith 10% NaOH, 60% ethanol, before being mounted in a Sykes-Moore culturechamber (Bellco Glass, NJ). The chamber was coated with concanavalin A (0.05mg/ml, Sigma) then rinsed with RPMI. SNARF-1 was excited at 514 nm and the

yellow (580-635 nm) and red (>635 nm) fluorescence detected using the avalanchephotodiode detectors (APDs) of a Confocor3 system. Spot photobleaching wasperformed as described previously (Klonis et al., 2002). For pH calibration, resealedcells were suspended in MES-buffer saline (20 mM MES and 150 mM NaCI, pH5.5 and 6.0), PBS saline (20 mM PBS and 150 mM NaCl, pH 6.5, 7.0, 7.5, 8.0) andTRIS saline (20 mM TRIS and –150 mM NaCl, pH 9.0) in the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP; Sigma; 10 mM). GFP and TMR-dextranwere excited at 488 nm and 543 nm using a Leica TCS SP2 confocal microscope,while cells co-labelled with SNARF-1 and LysoSensor Blue-192 (1 mM at 37°C for20 minutes; Invitrogen) were imaged using the FITC and DAPI filter cubes of anOlympus IX81 microscope.

Image analysisImages were analysed using NIH ImageJ (http://rsb.info.nih.gov/ij/). We define theratio Rry as Ir/Iy where the Ir and Iy correspond to the fluorescence intensities in thered and yellow channels. Rry images were constructed in HSB colour space by mappingthe Rry value at each pixel to a particular hue value and placing the total intensity (Ir

+ Iy) in the brightness channel. The brightness channel was often linearly adjustedto increase the contrast. Pixels that are saturated (values of 255) are coloured cyanin the Rry images and were not analysed when calculating Rry values. Quantificationof Rry values was performed using data from individual sections.

Electron microscopyInfected RBCs were prepared for electron tomography as described previously(Hanssen et al., 2009). For electron tomography, 300 nm sections were cut, andcollected as serial sections. Each section was layered with fiducials, stained withuranyl acetate and lead citrate and observed on a Tecnai G2 F30 (FEI Company)transmission EM (Bio21 Institute, Melbourne). Tilt series were acquired using Xplore3D (FEI Company). Tomograms were recorded between –69 and +69° at 1.5° intervalsfor the first axis and 3° for the second axis and aligned with IMOD (Kremer et al.,1996; Mastronarde, 1997). Segmentation models were generated with 3dmod(http://bio3d.colorado.edu/). Individual tomograms were stacked using the joinprograms from the eTomo GUI interface of the IMOD package. The models werecorrected by a factor of 1.5 in the z direction to account for shrinkage of the resinduring exposure to the electron beam.

We thank the National Health and Medical Research Council ofAustralia and the Australian Research Council. We thank Mike Klemba,Virginia Tech, USA, for supplying the plasmepsin-II-GFP transfectants.We thank Samantha Deed for expert technical assistance.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/3/441/DC1

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