Research Article
441
Digestive-vacuole genesis and endocytic processes in
the early intraerythrocytic stages of Plasmodium
falciparum
Nurhidanatasha Abu Bakar1,*, Nectarios Klonis1,2,*, Eric Hanssen1,2,*, Cherrine Chan1 and Leann Tilley1,2,‡
1
Department 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 ([email protected])
Journal of Cell Science
Accepted 2 November 2009
Journal of Cell Science 123, 441-450 Published by The Company of Biologists 2010
doi:10.1242/jcs.061499
Summary
The 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 haemoglobin
from the host cytoplasm are still debated. Here, we use live-cell imaging and photobleaching to monitor the uptake of the pH-sensitive
fluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in ring-stage and trophozoite-stage parasites. We compare these
results with electron tomography of serial sections of parasites at different stages of growth. We show that uptake of erythrocyte
cytoplasm is initiated in mid-ring-stage parasites. The host cytoplasm is internalised via cytostome-derived invaginations and concentrated
into several acidified peripheral structures. Haemoglobin digestion and haemozoin formation take place in these vesicles. The ringstage 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-containing
vesicles. There is also evidence for haemoglobin degradation in compartments outside the digestive vacuole. The work has implications
for the stage specificity of quinoline and endoperoxide antimalarials.
Key words: Malaria, Plasmodium falciparum, SNARF-1-dextran, pH, Protein trafficking, Plasmepsin, Digestive vacuole
Introduction
Plasmodium falciparum causes ~1 million malaria-related deaths
annually (Snow et al., 2005). Disease pathology is associated with
the parasite’s life cycle within a parasitophorous vacuole (PV) inside
the red blood cells (RBCs) of its human host. As it develops, the
intraerythrocytic 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 for
growth, as well as generating osmolytes that prevent premature host
cell lysis (Lew et al., 2003).
Early studies of the feeding process in P. falciparum-infected
RBCs indicated that uptake of host cytoplasm is most active in
trophozoites and involves morphologically distinct endocytic
structures, termed cytostomes, at the parasite surface (Aikawa et
al., 1966). The resulting endocytic invaginations are surrounded
by both the parasite plasma membrane (PPM) and the PV
membrane (PVM) (Slomianny, 1990). The double-membranebound vesicles that bud from the cytostome were proposed to
migrate to and fuse with an acidic digestive vacuole (DV). Singlemembrane-bound vesicles were assumed to be delivered into the
DV lumen, followed by release and degradation of the haemoglobin
(Yayon et al., 1984).
Recent studies have re-examined the process of haemoglobin
uptake in infected RBCs but have led to somewhat conflicting
conclusions. A study using serial thin-section electron microscopy
(EM) (Elliott et al., 2008) described four distinct pathways for
haemoglobin uptake by P. falciparum. These authors proposed that
ring-stage parasites fold around a ‘gulp’ of host cell cytoplasm as
the first step in the biogenesis of the DV. They suggested that small
cytostome-derived haemoglobin-containing vesicles and tubules
continue the uptake of haemoglobin as the parasite matures. In more
mature parasites they also described additional cytostomeindependent endocytic structures called phagosomes (Elliott et al.,
2008).
Another study examined the ultrastructure of the endocytic
apparatus and concluded that haemoglobin uptake occurs by a
vesicle-independent process (Lazarus et al., 2008). These authors
reported that cytostomal invaginations elongate to form tubes that
appose the DV but remain connected to the parasite surface and
open to the RBC cytoplasm. They suggested that the tubules pinch
off from the parasite surface and simultaneously undergo fusion
with the DV to release their contents into the DV lumen (Lazarus
et al., 2008).
Another area of debate is the point at which haemoglobin
digestion is initiated. Some authors have argued that haemoglobin
digestion commences only after cytostomal vesicles fuse with the
DV (Olliaro and Goldberg, 1995; Slomianny and Prensier, 1990).
Others suggest that haemoglobin digestion is initiated en route to
the DV, and that the DV is merely a dumping site for haemozoin
crystals (Hempelmann et al., 2003).
In this work we have re-examined DV genesis and estimated the
pH of the DV and the endocytic compartments by following the
uptake of pH-sensitive probes. We have used live-cell imaging and
photobleaching to examine the dynamics and connectivity of
different compartments. We also used electron tomography to
generate high-resolution images of sections of parasite-infected
RBCs. We have combined this with serial sectioning to obtain
tomographic reconstructions of whole parasites at different stages
of development. We compared the data from these two imaging
modalities to provide novel insights into the feeding process.
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Journal of Cell Science 123 (3)
Results
Journal of Cell Science
Characterisation of RBCs resealed to contain SNARF-1dextran
Human RBCs that have been lysed and resealed at high
haematocrit retain the ability to support the growth of P.
falciparum (Dluzewski et al., 1983) and can be used to trap
fluorescent 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 a
relatively homogenous population of resealed RBCs containing
the pH-sensitive probe, SNARF-1-dextran (Whitaker et al.,
1991). The SNARF-1 chromophore undergoes an acid-base
transition from a yellow- to a red-emitting form that can be
monitored using a single excitation wavelength (Bassnett et al.,
1990; Seksek and Bolard, 1996; van Erp et al., 1991). The
resealed RBCs retain 20-45% of their haemoglobin
(corresponding to 4-9 mM monomeric haemoglobin) and contain
0.1-0.2 mol% of the fluorescent dextran with respect to
haemoglobin, corresponding to an intracellular concentration of
10-20 mM and a trapping efficiency of 30-60%. The resealed
RBCs were invaded with 54±5% of the efficiency of control
RBCs, in agreement with previous data (Frankland et al., 2006).
Parasites developing in resealed RBCs complete the cycle and
form merozoites that are capable of reinvading further resealed
RBCs, although the efficiency of invasion in the second round
is reduced (data not shown).
We constructed a SNARF-1 pH calibration curve by suspending
resealed RBC in buffers of different pH in the presence of the
ionophore, carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
(Allen and Kirk, 2004; Klonis et al., 2007). CCCP has previously
been used to equilibrate compartments in P. falciparum-infected
RBCs to external buffers of known pH (Allen and Kirk, 2004; Klonis
et al., 2007). Fluorescence images were collected for resealed RBCs
with different levels of trapped fluorophore on three separate
occasions, and the ratio Rry (red/yellow fluorescence intensity) was
used as an indicator of pH (Fig. 1A). The relatively small standard
deviation (s.d.) values indicate that any differences in SNARF-1
uptake and haemoglobin levels do not affect the Rry values.
SNARF-1-dextran in buffered solutions gave a similar calibration
curve (data not shown). The decrease in Rry values with decreasing
pH is adequately described by a simple acid-base transition with
an 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 neutral
compartments, but cannot provide accurate values at pH values less
than 6 (Rry<0.9).
In order to visualise the spatial variation of Rry values (and hence
pH) within a cell, we generated Rry images in HSB colour space,
where the Rry value at each pixel is mapped to a particular colour
in the hue channel and its corresponding total intensity (Ir + Iy) is
placed in the brightness channel. This generated the look-up table
shown 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 images
of resealed RBC resuspended at pH 5.5 and 7.5 in the presence of
CCCP (Fig. 1B). We examined the effect of CCCP treatment on
SNARF-1 that has been accumulated into the DV of trophozoitestage parasites (Fig. 1C). In untreated cells, the DV appears green,
consistent with a DV pH value <6 (Fig. 1Ci), whereas in cells
suspended in PBS at pH 7.5 in the presence of CCCP, the DV
appears purple (Fig. 1Cii).
Fig. 1. Characterisation of SNARF-1-dextran as a pH sensor for live-cell
imaging. (A)RBCs resealed in the presence of 50mM SNARF-1-dextran were
resuspended in saline buffer containing 10mM CCCP and imaged at 37°C. Rry
values ± s.d. were determined for individual cells (n>30 for each point; three
experiments). The curve represents the best fit to the data using a simple acidbase transition with a pKa of 7.2. (B)Rry images of resealed CCCP-treated
RBCs in buffers at pH 5.5 (i) and pH 7.5 (ii). The colour at each pixel reflects
the Rry value from the look-up table. (C)DIC (left) total intensity (middle) and
Rry images (right) of trophozoite-infected SNARF-1-dextran-labelled RBCs at
37°C in RPMI, pH 7.4 (i), and in PBS, pH 7.4, containing 10mM CCCP (ii).
Scale bars: 5mm.
Uptake of host RBC cytoplasm is initiated in mid-ringstage parasites
To examine the earliest events associated with the uptake of the
RBC cytoplasm, we purified tightly synchronised (~6 hour window)
parasites (D10 strain) at the schizont stage (40-48 hours) and used
them to invade resealed RBC containing SNARF-1-dextran. After
20 to 24 hours (i.e. 12-24 hours after invasion) we transferred them
to a microscope chamber that was gassed and maintained at 37°C
(Fig. 2). The cells appear to be viable for ~10 hours, however we
imaged them within 2 hours. By analysing differential interference
contrast (DIC) images, we chose parasites that had no visible
haemozoin (Fig. 2A-D). Based on the estimated time after invasion
and the absence of visible pigment, we define these as mid-ringstage parasites. In some infected RBCs (Fig. 2A), the parasite is
observed 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-section
projections taken through the parasite, confirming that this is not a
consequence of an internalised compartment being out of the plane
of focus.
Journal of Cell Science
Visualising P. falciparum digestion
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 at
37°C. Shown are DIC images and average projections of the total intensity and
Rry values from serial sections encompassing the region occupied by the
parasite. Regions of the RBC cytoplasm (indicated by ‘x’ in the pre-bleach
intensity image) were subjected to a high-intensity bleaching pulse (10
seconds) to deplete the host fluorescence before acquisition of post-bleach
data. Yellow arrows correspond to small individual or multiple punctate
peripheral compartments within the parasite. Green arrows correspond to
larger spherical features in the parasite cytoplasm that are apparent in the prebleach projections. (I)A cell was subjected to high-intensity illumination for
10 seconds (‘x’) to deplete the host cell fluorescence, then the arrowed
structure was pulsed for 1 second. Scale bar: 5mm.
In other mid-ring-stage parasites, punctate structures containing
SNARF-1-dextran were observed near the parasite periphery (Fig.
2B-D, yellow arrowheads). The SNARF-1 concentration within
443
these compartments is comparable with that in the host cell
cytoplasm, suggesting that they represent early endocytic events.
They are observed as regions of altered Rry values in the
ratiometric images (Fig. 2) indicating they are acidic in nature;
however, analysis of the fluorescence signal in this compartment
was complicated by contributions from the host cell. To better
observe these compartments, we selectively photobleached the
SNARF-1 fluorescence in the host compartment by parking the
laser in a region distal to the parasite (indicated by ‘x’) and
illuminating with an unattenuated laser for 10 seconds. This results
in the loss of fluorescence throughout this compartment (Fig. 2,
middle and right panels) (Klonis et al., 2002), without affecting
the fluorescence intensity of any SNARF-1 that has been
internalised by the parasite. This increases the contrast of the acidic
compartments and confirms that they are located within the
parasite and are not in direct communication with the RBC
cytoplasm. Indeed they were often revealed as multiple puncta
that appear green in the post-bleach Rry images. The average Rry
of these compartments was 0.85±0.07 (n24; four experiments)
corresponding to a pH of <6. In no examples did we observe
endocytic compartments that exhibited a neutral pH. By contrast,
the Rry values in the RBC cytoplasm of uninfected and infected
erythrocytes were 1.43±0.19 (n29; four experiments) and
1.40±0.16 (n42; four experiments), corresponding to pH values
of 7.2±0.3 and 7.14±0.25, respectively. We also analysed another
parasite strain (3D7) and examined parasites developing in
resealed RBCs containing TMR-dextran or recombinant GFP as
reporters (supplementary material Fig. S1). We observed
equivalent 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. 2BD, green arrows) and was also evident in the DIC images. The
fluorophore appeared to be at a similar concentration to the bulk
of the RBC cytoplasm and the Rry value (in the pre-bleach image)
was similar to that of the RBC cytoplasm. The structure could
correspond to a proposed macropinocytic structure call the ‘big
gulp’, which was recently described (Elliott et al., 2008).
Interestingly, photobleaching of the host cell fluorescence depleted
the fluorescence associated with these spherical structures to the
same degree (Fig. 2B-D, middle and right panels) demonstrating
that this structure remained connected to the RBC cytoplasm. We
analysed 100 infected RBCs before the appearance of haemozoin
pigment and found punctate peripheral structures in 94% of cells
and larger spherical structures in 63% of cells.
DV formation is consolidated in late-ring and early
trophozoite stages
DIC images were used to identify a second population of parasites
containing small dark puncta; these probably represent the first
detectable haemozoin particles (Fig. 2E-F). Based on this criterion,
we define these as late-ring-stage parasites. Many of the cells
possessed more than one pigment-containing structure or a
combination of pigmented and non-pigmented structures. These
compartments often showed brighter fluorescence than the host
cytoplasm, indicating a concentration event. The internalised
compartments were invariably acidic with a pH <6 (Rry0.86±0.09;
n28; four experiments). The low pH, the accumulation of the host
cytoplasmic components, and the presence of haemozoin suggest
that haemoglobin digestion is initiated in several independent preDV compartments in these early stage parasites. Application of a
laser pulse to one of the peripheral structures (Fig. 2I, yellow arrow)
Journal of Cell Science
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Journal of Cell Science 123 (3)
Fig. 3. Endocytic compartments in mature trophozoites. DIC images, Rry
images of single sections through the endocytic compartments of SNARF-1dextran-containing infected RBCs, and corresponding overlays, at 32-40 hours
post-invasion. The SNARF-1 fluorescence is associated with the DV (red
arrows) and with extra-DV compartments (yellow arrows) with no visible
haemozoin (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 to
deplete the fluorescence. Scale bar: 5mm.
It should be noted that the DV compartment is very susceptible
to photo-induced damage. At higher illumination levels or upon
continuous illumination the Rry value for this compartment increased
during imaging (data not shown). Illumination-induced changes in
DV pH have been reported previously for another pH-sensitive
probe (Wissing et al., 2002). Light-induced generation of damaging
hydroxyl radicals during bleaching might be exacerbated by the
presence of haemoglobin breakdown products in the DV. We used
the lowest possible levels of illumination and used highly sensitive
APD detectors to minimise any light-induced artefacts in the data
presented here. None the less, it is difficult to ascribe a reliable pH
to the DV compartment. By contrast, the extra-DV compartment is
less susceptible to photo-induced damage (as is the case for the
acidic compartments in ring-stage parasites). This permitted a more
reliable estimate of Rry (0.82±0.04; n125; 12 experiments),
corresponding to a pH <6. Application of a laser pulse to an extraDV compartment completely ablated the fluorescence associated
with this compartment but did not affect the intensity of the DV
(Fig. 3A) and vice versa (Fig. 3B,C). The same result was obtained
in a total of 75 cells, indicating that the lumens of these
compartments are not connected.
Plasmepsin II is delivered to early endocytic
compartments and the extra-DV compartment
results in selective photobleaching of this compartment, confirming
the presence of several independent compartments.
The cells shown in Fig. 2G,H contained more prominent pigment
in the DIC image and probably represent the next stage of DV
formation (i.e. the early trophozoite stage). The haemozoin was
confined to one region of the parasite and was associated with
internalised acidic structures that were considerably brighter than
the surrounding host cell cytoplasm. In Fig. 2G, the endocytic
compartments might be in the process of coalescing into a single
site within the parasite, whereas in Fig. 2H they appear to have
coalesced. Other larger features were observed within the parasites
at these stages (Fig. 2E-H, green arrows), which appear to represent
invaginations of the host cell cytoplasm. In all cases, photobleaching
measurements demonstrate that these features remain connected to
host cell cytoplasm.
Plasmepsin II is a protease involved in haemoglobin digestion and
represents a marker for compartments where haemoglobin digestion
is 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-IIGFP was co-located with TMR-dextran in the peripheral puncta
(Fig. 4A, yellow arrows), indicating that plasmepsin II is delivered
to endocytic compartments early in parasite development and would
be available to initiate haemoglobin digestion. By contrast, the larger
spherical profile structures (Fig. 4B, white arrow) had no associated
plasmepsin-II-GFP. In more mature-stage parasites, plasmepsin-IIGFP 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 peripheral
plasmepsin-II-GFP compartments that are not associated with
accumulation of TMR-dextran (Fig. 4C, blue arrows). These
structures might represent regions of the parasite surface where
cytostomal invaginations are forming, but which have not yet
budded from the PPM.
Trophozoite-stage parasites possess acidified DV and
extra-DV compartments
LysoSensor Blue labelling confirms the low pH of the
endocytic compartments
We examined the distribution of SNARF-1-dextran in the endocytic
compartments of mature parasitised RBCs (i.e. 32-40 hours after
invasion). In these cells, SNARF-1 was considerably more
concentrated within the parasite compared with the host cytoplasm.
The probe accumulated in the major haemozoin-containing
compartment 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 parasite
cytoplasm (Fig. 3, yellow arrows). These structures usually did not
contain any visible haemozoin, although in some cases (Fig. 3C),
a small amount of haemozoin was visible. The structures varied in
size but were always smaller than the DV. These could represent
endocytic structures en route to the DV, or separate digestive
compartments. Both the DV and the extra-DV compartments
appeared green in the Rry images, indicating that they are acidified.
We also imaged cells labelled with LysoSensor Blue, a weak base
that accumulates in acidic compartments (Lin et al., 2001; Wissing
et al., 2002). We found that low levels of the probe and low level
illumination were needed to prevent alkalinisation of smaller
compartments and to avoid photo-induced damage. Under optimised
imaging conditions, LysoSensor Blue accumulated in the punctate
peripheral 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 spherical
profile structures (Fig. 4E, arrow). In mature-stage parasites,
LysoSensor Blue accumulated in the DV where its fluorescence
overlapped with the SNARF-1 fluorescence (Fig. 4G,H). The
SNARF-1-containing extra-DV compartments were also labelled
with LysoSensor Blue, confirming that these compartments are
acidic (Fig. 4H, yellow arrow). Interestingly, there were also some
Visualising P. falciparum digestion
445
compartments in the cytoplasm of mature-stage parasites that were
weakly labelled with LysoSensor Blue, but not with SNARF-1 (Fig.
4H, blue arrow). These might represent additional endocytic or
secretory compartments (e.g. rhoptries) that are separate from the
haemoglobin-uptake pathway.
Journal of Cell Science
Electron tomography provides details of the genesis of
the DV in ring-stage parasites
Fig. 4. Identification of haemoglobinase-containing and LysoSensor-Bluelabelled compartments in infected RBCs. The panels are DIC or bright-field
images, GFP or SNARF-1 fluorescence (depicted in green) or TMR-dextran or
LysoSensor Blue fluorescence (depicted in red), and overlays of the red and
green images. (A-D)Plasmepsin-II-GFP-3D7 transfectants in TMR-dextrancontaining resealed RBCs. The pre-DV and DV compartments (yellow arrows)
and the extra-DV compartments (red arrowheads) contain both plasmepsin-IIGFP and TMR-dextran. Plasmepsin-II-GFP is also present in structures that
could represent nascent cytostomal invaginations (blue arrows). There is no
plasmepsin-II-GFP associated with a larger spherical profile structure (white
arrow). (E-H)In rings (E,F) and trophozoites (G,H) in SNARF-1-dextrancontaining RBCs, LysoSensor Blue is associated with peripheral structures and
the DV (arrowheads) and extra-DV structures (yellow arrows). The spherical
structure in ring-stage parasites (A, white arrow) is not labelled. Some
additional structures in the trophozoite-stage parasites are weakly labelled with
LysoSensor Blue, but do not contain SNARF-1-dextran (H, blue arrow). Scale
bars: 5mm.
Electron tomography is an invaluable tool for obtaining 3D
information from samples at high resolution (Hanssen et al., 2008)
and can be combined with serial sectioning techniques to permit
high-resolution imaging of organelles, and even whole cells (Lucic
et al., 2008; Noske et al., 2008). Sections are examined in an EM
operating at moderate to high voltage with a tiltable stage. The
images are aligned and tomographic reconstructions of the sample
are generated computationally using segmentation tool. We carried
out an electron tomographic analysis of serial sections (7-15
sections of ~300 nm) of infected RBCs (K1 strain) at six different
stages of development.
Individual virtual sections (7 nm) from within tomograms of
whole parasites at early development stages are presented in Fig.
5A,E,I,M. (See translations through entire data sets for Fig. 5E and
M in supplementary material Movies 1 and 2.) The parasite-limiting
membrane and selected features within the parasite were rendered
to reveal details of the 3D organisation. Rotations of the rendered
data for B,F,J,N are presented in supplementary material Movies
3-6. The parasite surface is depicted in translucent cream, the
cytostome as a yellow ring. Attached cytostomal invaginations are
represented in translucent yellow and the nucleus is in gold. The
surface is rendered in opaque gold in the right panels.
At the earliest stage examined (Fig. 5A-D; parasite volume, 2.60
fl), very few intracellular features were apparent. The parasite
exhibited a cupped-hand morphology with several finger-like
extensions (Fig. 5B, red arrows), but there was no evidence for
endocytic vesicles. Another parasite (Fig. 5E-H; parasite volume,
2.54 fl) exhibited a flatter shape and the characteristic cytostome
was associated with a cytostomal invagination (Fig. 5E,F, red
arrows). The cytostome comprised an electron-dense ring with an
internal diameter of ~90 nm (outer diameter ~190 nm) that encircled
the neck of invaginations of the PPM and PVM (membrane-limited
opening of ~55 nm). Additional sections through cytostomes (Fig.
5Q,R) reveal the complex ultrastructure of this feature. The
endocytic flasks remained connected to the RBC cytoplasm through
the open mouth of the cytostome ring. A number of small vesicular
structures near the parasite periphery (Fig. 5F-H, depicted in
orange) are likely to be derived from cytostomal invaginations.
These contained small crystals (Fig. 5E, orange; also see translation
through the tomogram in supplementary material Movie 1). This
indicates that haemoglobin digestion and haemozoin formation have
been initiated. These structures probably represent the acidic
peripheral compartments observed in SNARF-1-dextran-labelled
ring-stage parasites.
A slightly larger volume parasite (Fig. 5I-L; 2.65 fl) had a cupped
profile (Fig. 5L, supplementary material Movie 5). This cell
contained several vesicles with microcrystalline contents (Fig. 5I,
orange) that accumulated in one region of the cell. In a more mature
parasite (Fig. 5M-P; 3.0 fl), the cup-shape was even more
pronounced (see Fig. 5P). Two cytostomes were observed at the
surface of the parasite (Fig. 5O, arrowheads) and a series of vesicles
and a larger flattened structure containing small crystalline structures
were present in one region of the parasite (Fig. 5M-O, orange, blue).
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Journal of Cell Science 123 (3)
Journal of Cell Science
Fig. 5. 3D reconstruction of entire ring-stage parasites from
multi-section electron tomograms. (A-P)Serial sections were
collected through ring-infected RBCs (K1 strain). Left panels
show selected virtual sections (7 nm) from within some of the
physical sections. Vesicles and pre-DV compartments with
crystalline material are outlined in orange and blue; a cytostome
(in E) is indicated with a red arrow. Middle panels show
segmentation of individual structures. The parasite surface is
depicted in translucent cream, the cytostome as a yellow ring
and the attached cytostomal invaginations in translucent yellow;
the nucleus is gold, crystal-containing vesicles, orange, and preDVs, blue. Right panels show the parasite surface rendered in
opaque gold. Scale bars: 0.2mm (A,E,I,M); 500 nm (B,F,J,N).
(Q,R)Longitudinal and transverse sections through a cytostome
in a D10-infected RBC. Scale bars: 0.1mm. (S,T)Section from
a tomogram through a region of a late-ring-stage parasite and
rendered model of this region. Some crystal-containing vesicles
and a pre-DV are rendered in orange and blue. Another vesicle
in this region (pink) appears to contain haemoglobin and a
nascent crystal. Scale bars: 300 nm (S); 500 nm (T).
The larger compartment might represent the initial stage of
coalescence 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 be
inside the parasite, however, a global analysis revealed that this
invagination remained connected to the RBC cytoplasm (see
supplementary material Movies 2 and 6). A region of a late-ringto early trophozoite-infected RBC was examined (Fig. 5S,T). This
cell had a structure (depicted in pink) that contained haemoglobin,
as well as a crystalline feature (black arrowhead). This might
represent a haemoglobin-containing vesicle where haemozoin
formation is being initiated.
Electron tomographic analysis of the DV and endocytic
compartments in trophozoite-stage parasites
We also generated individual virtual sections (7 nm) and rendered
tomograms of trophozoite-infected RBCs (Fig. 6, supplementary
material Movies 7-9). In a mid-trophozoite parasite (Fig. 6A-D;
12.1 fl; two nuclei), a centrally located DV, containing large crystals
of haemozoin, was surrounded by a single membrane. The DV was
folded and wrapped around a region of parasite cytoplasm (Fig.
6A,C, blue outline/rendering). An additional compartment, adjacent
to the DV, contained microcrystallites of haemozoin (Fig. 6B-D,
orange outline, black arrow). This compartment is probably
equivalent to one of the acidified extra-DV compartments observed
in the SNARF-1-dextran-labelled trophozoites. The cell had three
cytostomal invaginations (Fig. 6C-D, yellow rings with attached
translucent yellow pouches), one of which was visible in the virtual
section (Fig. 6A, arrow). The trophozoite stage sees the genesis of
small compartments comprising a clear lumen around an electrondense body (Fig. 6, pink outline/rendering). These are likely to be
acidocalcisomes (Docampo et al., 2005).
In a more mature-stage parasite (21.7 fl, four nuclei), a large DV
was surrounded by a single membrane (Fig. 6E-H, blue).
Characteristic electron-dense cytostomes, again with an internal
diameter of ~90 nm, and associated endocytic flasks were observed
at the parasite surface (depicted in translucent yellow; yellow arrow
in Fig. 6E). A cytostome also constricted a bi-lobal haemoglobincontaining feature that appeared to be inside the parasite (Fig. 6F,G,
yellow arrows). This type of compartment might also contribute to
the extra-DV compartments observed in SNARF-1-dextran-labelled
trophozoites; however, the structure was opposed to the PPM at the
interface between two physical EM sections, so we cannot rule out
Visualising P. falciparum digestion
447
Journal of Cell Science
Fig. 6. 3D reconstruction of entire trophozoite-stage
parasites from multi-section electron tomograms.
Serial sections were collected through RBCs infected with
parasites at mid trophozoite (A-D) and early-schizont
(E-H) stages (K1 strain). Left panels show selected
virtual sections (7 nm) from within some of the physical
sections. The DV is outlined in blue, crystal-containing
extra-DV compartments in orange and a putative
acidocalcisome in pink. Cytostomes are indicated with
yellow arrows and a furrow in the DV in A with an
arrowhead. Right panels show individual structures that
were segmented and depicted with the colours described
in Fig. 5. An extra-DV compartment (B-D) is rendered in
orange (black arrow in C). The inset in E shows a
structure containing haemoglobin and haemozoin that
appears to be in the process of being engulfed by the DV.
Scale bars: 500 nm and 100 nm (inset in E).
the possibility that it retains some connectivity with the PPM.
Interestingly, we observed a haemoglobin-containing vesicle located
within the DV (Fig. 6E, square). A virtual section though a tilted
plane suggests that the DV is engulfing the haemoglobin-containing
vesicle (see inset). The vesicle contained an electron-lucent body
with the appearance of a haemozoin crystal.
Discussion
Haemoglobin uptake and degradation is initiated in early
stage parasites
Two recent studies of DV genesis and haemoglobin uptake processes
used serial thin section electron microscopy (Elliott et al., 2008;
Lazarus et al., 2008). However, because of the huge amount of work
involved in this very challenging technique, these studies were
restricted to reconstruction of a limited cell depth or of a few whole
cells. Moreover the resolution in the z-plane is determined by the
section thickness (i.e. ~70 nm), which limits the ultrastructural
information. In this work, we used electron tomography to study
the digestive apparatus. Electron tomography permits analysis of
300-400 nm sections with enhanced z-plane resolution and has been
used to identify novel features of the P. falciparum exomembrane
system (Hanssen et al., 2008; Henrich et al., 2009). We implemented
methods for collecting data from multiple sections and used
programs for tiling and stitching tomographic data (Hanssen et al.,
2009; Noske et al., 2008) to generate whole parasite images; this
allowed us to re-examine the 3D ultrastructure of the digestive
apparatus at different stages of parasite development.
We compared the electron tomography data with images of live
parasites in resealed RBCs containing a fluorescent tracer. Resealed
RBCs have been validated previously for use in studies of P.
falciparum invasion and growth (Dluzewski et al., 1985; Rangachari
et al., 1987) and for trapping inhibitors in the RBC cytoplasm
(Frankland et al., 2006; Tilley et al., 1990). In this work, we resealed
RBCs to contain SNARF-1-dextran, using it as a convenient,
chemically stable, non-toxic, pH-sensitive marker of endocytic
processes. We optimised temperature and gas control in the
microscope 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-labelled
RBCs showed no endocytic compartments; however, at the midring stage, the fluorescent marker was observed in acidified
peripheral compartments. Electron tomography revealed cytostomal
invaginations that remained connected to the RBC cytoplasm, as
well as several small peripherally located vesicular compartments
containing microcrystals of haemozoin. We suggest that these
cytostome-mediated endocytic events represent the first step in the
genesis of the haemoglobin-degrading apparatus of P. falciparum.
In late-ring-stage parasites, the SNARF-1 signal in the endocytic
structures was often more intense than in the RBC cytoplasm. As
SNARF-1 fluorescence intensity decreases at low pH (Bassnett et
al., 1990), this indicates that endocytosis of the host compartment
is followed by a process that concentrates the contents of the
endocytic compartment. Excess lipids in the cytostomal vesicles
might be consumed by internalisation and degradation, providing
a catalyst for haemozoin formation (Bendrat et al., 1995; Jackson
et al., 2004). Electron tomography confirms the accumulation of
haemozoin crystals in late-ring-stage parasites. Moreover, a GFP
chimera of plasmepsin II was delivered to the early endocytic
structures, indicating that conditions for initiation of haemoglobin
degradation are met. The data suggest that following budding of
cytostomal vesicles, the integrity of the inner membrane is rapidly
lost, allowing mixing of proteases and haemoglobin in an acidified
compartment; this would initiate haemoglobin digestion and
haemozoin formation. Indeed, we observed examples of
haemoglobin-containing endocytic compartments apparently
undergoing the first stages of crystal formation. Our observation of
haemoglobin digestion in mid-ring-stage parasites contrasts with
the often-quoted view that haemoglobin degradation is only initiated
in more mature-stage parasites.
It is interesting to consider how the pH of the ring-stage
endocytic structures might be controlled. The membrane potential
across the PPM is maintained by the electrogenic extrusion of
protons via a V-type proton pump (Allen and Kirk, 2004; Mikkelsen
et al., 1982). Acidification of the DV is thought to involve the same
V-type proton-ATPase, as well as a proton-pyrophosphatase (Saliba
et al., 2003). It appears likely that proton pumps are transferred to
the DV via the same endocytic process that drives haemoglobin
uptake. Thus, endocytosis of a region of the PPM would capture
proton pumps leading to the immediate acidification of the endocytic
compartments once it has budded from the PPM. The fact that we
did not observe any neutral compartments that are wholly within
the parasite is consistent with their rapid and obligate acidification.
A larger fluorescent feature, with a spherical appearance, that is
associated with most of the SNARF-1-labelled ring-stage parasites,
is likely to be equivalent to the recently described ‘big gulp’ (Elliott
et al., 2008). Our electron tomography studies reveal that the ring-
Journal of Cell Science
448
Journal of Cell Science 123 (3)
stage parasite adopts a pronounced cup shape, and we propose that
this invagination gives rise to the feature of spherical appearance
in the confocal microscopy images. Indeed, early ultrastructural
studies showed that shortly after invasion the ovoid-shaped
merozoite flattens into a thin disc, then invaginates to form a cup
shape (Bannister et al., 2000; Bannister et al., 2004; Langreth et
al., 1978). However, we found that this structure is not acidified
and remains connected to the RBC cytoplasm. Moreover it co-exists
with the peripheral structures in which haemoglobin digestion is
occurring. Taken together, our live-cell imaging and electron
tomography data argue against the ‘big gulp’ as the progenitor of
the DV and instead indicate the formation of several cytostomederived 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 the
ring parasites. Shortly after invasion, material is released from the
dense granules into the PV (Aikawa et al., 1990), followed by the
elaboration of different membrane structures in the RBC cytoplasm
(Lanzer et al., 2006; Tilley et al., 2008). The membrane vesicles
involved in protein export (Marti et al., 2004) would be expected
to expand the parasite surface; however, the high concentration of
haemoglobin in the RBC cytoplasm might prevent a significant
increase in parasite volume. So the cup shape might simply reflect
the need to increase surface area without a significant increase in
volume. The increased parasite surface area might also provide
material that can be endocytosed via the cytostomes. Indeed,
elongation of the endocytic flask to accommodate the expanding
surface area of the parasite could lead to strain at the constricted
neck of the flask and promote budding into the parasite cytoplasm.
Acidic DV and extra-DV compartments in trophozoitestage parasites
As the parasite matures the peripheral structures coalesce into a
central acidic DV that is marked by the presence of prominent
haemozoin crystals. Another recent study (Dluzewski et al., 2008)
also concluded that small crystal-containing vacuoles coalesce as
the parasite matures to form a single DV. The trophozoite-stage
parasites have 3–4 cytostomes with attached endocytic invaginations
that remain open to the RBC cytoplasm. A recent study suggested
that the cytostomal invaginations do not detach and migrate to the
DV but extend till they contact and are engulfed by the DV (Lazarus
et al., 2008). By contrast, we observed (in the SNARF-1-labelled
cells) acidified extra-DV compartments in the parasite cytoplasm
(~70% of the trophozoite-stage parasites examined). Many of these
were tightly juxtaposed to the DV and they might have been
assumed to be part of the DV in previous studies. However, selective
photobleaching experiments showed that they are indeed separate
structures. Plasmepsin-II-GFP was present in these acidified
structures and electron tomography revealed examples of
compartments adjacent to the DV that contained haemozoin crystals
or small haemozoin crystals in the presence of haemoglobin. These
structures are presumably derived from large cytostomal
invaginations and appear to represent relatively long-lived extraDV compartments. We also observed a vesicle that appeared to
contain both haemoglobin and an early haemozoin crystal being
engulfed by the DV. This suggests that the DV can phagocytose
haemoglobin-containing structures as well as fusing with them.
Taken together, our data suggest that following endocytosis,
haemoglobin digestion can be initiated before, during or after
delivery of the endocytic structures to the DV.
Our data provide clear evidence for the budding of cytostomederived vesicles into the parasite cytoplasm and support previous
studies showing that this is a major pathway for the uptake of
haemoglobin in both ring-stage and trophozoite-stage parasites. We
also observed a large invaginated structure bisected by a cytostome
that might represent an example of a phagosome (Elliott et al., 2008).
At this stage, we cannot exclude a role for an additional cytostomeindependent pathway in the trophozoite stage. Identification of the
protein components of the cytostome and generation of transfectants
expressing GFP chimeras of these proteins would make it feasible
to distinguish cytostomal invaginations from phagotrophs in live
cells and would help to resolve this question.
pH of the DV and endocytic compartments
We found that the pH of the pre-DV and extra-DV compartments
was less than 6. This is consistent with other recent estimates of
DV pH (Klonis et al., 2007; Kuhn et al., 2007). The pH optima for
the DV plasmepsins, histo-aspartic protease (HAP) and the
falcipains are in the range pH 5-6 (Banerjee et al., 2002; Goldberg
et al., 1990; Rosenthal, 2004) and the pH optimum for b-haematin
formation is between 3 and 6 (Egan et al., 2001; Slater and Cerami,
1992). Thus our data indicate that haemoglobin digestion and
haemozoin will occur in both ring-stage and trophozoite-stage
parasites, and in both the DV, extra-DV and pre-DV compartments.
In a previous study, we labelled plasmepsin-II-GFP transfectants
with LysoSensor Blue (Klonis et al., 2007) and found that a number
of plasmepsin-II-GFP-labelled features were not acidified. We
concluded that these were endocytic compartments that were not
acidified; however, our current data indicate that they are more likely
to represent nascent cytostomal invaginations that have not yet lost
the connection to the RBC cytoplasm. This is consistent with a
previous immuno-EM study showing plasmepsin II located at
nascent cytostomal invaginations (Klemba et al., 2004).
Implications for antimalarial drug action
Our finding that the pre-DV compartments in the ring-stage
parasites are acidified and that haemoglobin degradation is
initiated at this stage has implications for the action of quinoline
antimalarials. Quinoline antimalarials accumulate in acidic
compartments and inhibit haemozoin formation (Geary et al.,
1990; Hawley et al., 1996; Tilley et al., 2006). Our data suggest
that both ring and trophozoite stages would accumulate
chloroquine in acidic compartments. Although some studies
suggest maximal chloroquine sensitivity at the trophozoite stage
(Hoppe et al., 2004; Yayon et al., 1983), others find that ring stages
are the most sensitive (Orjih, 1997; Zhang et al., 1986); yet others
suggest that both stages are sensitive but that ring stages exhibit
a ‘delayed death’ effect (Gligorijevic et al., 2008; Yayon et al.,
1983). Our data indicate that chloroquine would accumulate in
ring-infected RBCs and would inhibit the early stages of
haemozoin formation.
The data also have implications for the action of endoperoxide
antimalarials. Endoperoxides are activated by the haem that is
released during haemoglobin breakdown [or by reduced iron that
is derived from haem (Krishna et al., 2004; Weissbuch and
Leiserowitz, 2008)]. Recent reports suggest that artemisinin targets
the 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 from
haemoglobin would be initiated in the ring stage of parasite growth
and would be available as a source of an iron-based activator. This
Visualising P. falciparum digestion
449
yellow (580-635 nm) and red (>635 nm) fluorescence detected using the avalanche
photodiode detectors (APDs) of a Confocor3 system. Spot photobleaching was
performed as described previously (Klonis et al., 2002). For pH calibration, resealed
cells were suspended in MES-buffer saline (20 mM MES and 150 mM NaCI, pH
5.5 and 6.0), PBS saline (20 mM PBS and 150 mM NaCl, pH 6.5, 7.0, 7.5, 8.0) and
TRIS saline (20 mM TRIS and –150 mM NaCl, pH 9.0) in the presence of carbonyl
cyanide m-chlorophenylhydrazone (CCCP; Sigma; 10 mM). GFP and TMR-dextran
were 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 for
20 minutes; Invitrogen) were imaged using the FITC and DAPI filter cubes of an
Olympus IX81 microscope.
Journal of Cell Science
Fig. 7. Model of digestive vacuole genesis and endocytic processes in the
different intraerythrocytic stages of P. falciparum. (A)In early- to mid-ringstage parasites, a cytostome becomes active. Cytostomal invaginations form
and endocytic vesicles pinch off to form acidified pre-DV compartments.
These compartments are supplied with haemoglobinases and the inner
membrane is degraded and haemoglobin digestion initiated. (B)Expansion of
the parasite surface area induces a cup-shaped morphology; however, the
parasite does not undergo macropinocytosis. As the parasite matures,
additional pre-DV compartments form then fuse to generate a single central
DV. (C)In the trophozoite stage, cytostomal invaginations continue to form
and bud into the parasite cytoplasm where they are rapidly acidified.
Cytostome-independent budding of regions of the parasite surface might also
contribute to uptake of host cytoplasm. In some cases, these endocytic
structures form longer-lived extra-DV structures where haemoglobin digestion
is initiated before they fuse with or are engulfed by the DV.
might well underlie the finding that artemisinin is highly active
against ring-stage parasites (Skinner et al., 1996).
The live-cell imaging and multi-section electron tomography
approaches used in this study represent important technical
advances. In combination with data from previous studies, these
approaches provide new insights and resolve some controversies
regarding the endocytic processes at the different stages of parasite
development. This is illustrated in the model presented in Fig. 7.
The data suggest that uptake of haemoglobin commences early in
the intraerythrocytic development of the parasite. Haemoglobin
digestion is initiated in multiple pre-DV compartments that coalesce
in the early trophozoite stage to form a central DV. The data show
that cytostome-dependent endocytosis is the mechanism for uptake
of haemoglobin the ring stage, and is probably the principle
pathway in the trophozoite stage. Following budding of endocytic
vesicles, haemoglobin digestion can occur either before, during or
after fusion with the DV. These findings are important in
understanding the mode of action of different classes of antimalarial
drugs.
Materials and Methods
Resealing RBCs and culturing parasites
RBCs were lysed in 2.5 volumes of ice-cold 5 mM phosphate, pH 7.5, 1 mM MgATP (Sigma) in the presence of 50 mM tetamethylrhodamine-dextran (TMR-dextran;
10 kDa; Invitrogen) or 5⬘(and 6⬘)-carboxy-10-dimethylamino-3-hydroxy-spiro[7Hbenzo[c]xanthene-7,1⬘(3H)-isobenzofuran]-3⬘-one (SNARF-1-dextran; 10 kDa;
Invitrogen) or recombinant His-tagged enhanced GFP (pEGFP-N1, Clontech) and
resealed as described previously (Frankland et al., 2006; Krogstad et al., 1985). The
levels of haemoglobin and incorporated SNARF-1-dextran were determined
spectrophotometrically. P. falciparum (D10, 3D7, K1 strains and plasmepsin-II-GFP
transfectants) was cultured and isolated as described previously (Klemba et al., 2004;
Klonis et al., 2007).
Fluorescence microscopy
Live-cell imaging of SNARF-1-dextran-containing resealed RBC was performed at
37°C using a ⫻100 oil immersion objective (1.4 NA) on a Zeiss LSM 510 inverted
confocal microscope equipped with an incubation chamber and heated stage under
an atmosphere of 5% CO2 in air. Glass coverslips (Menzel-Glaser, #1.5) were cleaned
with 10% NaOH, 60% ethanol, before being mounted in a Sykes-Moore culture
chamber (Bellco Glass, NJ). The chamber was coated with concanavalin A (0.05
mg/ml, Sigma) then rinsed with RPMI. SNARF-1 was excited at 514 nm and the
Image analysis
Images were analysed using NIH ImageJ (http://rsb.info.nih.gov/ij/). We define the
ratio Rry as Ir/Iy where the Ir and Iy correspond to the fluorescence intensities in the
red and yellow channels. Rry images were constructed in HSB colour space by mapping
the 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 adjusted
to increase the contrast. Pixels that are saturated (values of 255) are coloured cyan
in the Rry images and were not analysed when calculating Rry values. Quantification
of Rry values was performed using data from individual sections.
Electron microscopy
Infected RBCs were prepared for electron tomography as described previously
(Hanssen et al., 2009). For electron tomography, 300 nm sections were cut, and
collected as serial sections. Each section was layered with fiducials, stained with
uranyl acetate and lead citrate and observed on a Tecnai G2 F30 (FEI Company)
transmission EM (Bio21 Institute, Melbourne). Tilt series were acquired using Xplore
3D (FEI Company). Tomograms were recorded between –69 and +69° at 1.5° intervals
for 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 join
programs from the eTomo GUI interface of the IMOD package. The models were
corrected by a factor of 1.5 in the z direction to account for shrinkage of the resin
during exposure to the electron beam.
We thank the National Health and Medical Research Council of
Australia 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 at
http://jcs.biologists.org/cgi/content/full/123/3/441/DC1
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