From the
Liverpool School of Tropical Medicine,
Pembroke Place, Liverpool L35 QA and the ||Centre
for Cell Imaging, School of Biological Sciences, The University of Liverpool,
Liverpool L69 7ZB, United Kingdom
Received for publication, April 22, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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By using live-cell confocal laser scanning microscopy and a judicious choice of Ca2+ probes, we have uncovered a novel function for this unique organelle. We show that the food vacuole of the malaria parasite is involved in dynamic Ca2+ storage. Evidence is presented showing that Ca2+ storage by the digestive food vacuole is linked directly to parasite cell signaling and asexual development. This novel discovery raises many new questions about the functions, physiology, and regulation of this critical malarial parasite organelle. As the target for the actions of a number of drugs, the requirement for tightly regulated Ca2+ movement and storage within this organelle may represent a new chemotherapeutic target.
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EXPERIMENTAL PROCEDURES |
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Effect of FPIX on the Fluorescence Quantum Yield of Ca2+ IndicatorsFluorescence emission spectra for pentapotassium salts (1 µM) of Fura-2 (excitation 340 nm) and Fluo-4 (excitation 488 nm) in 39 µM free Ca2+ buffer (10 mM CaEGTA in 100 mM KCl, 30 mM MOPS, pH 7.2) with 0, 10, 20, and 50 µM ferriprotoporphyrin IX was performed in a Shimadzu RF-5001PC spectrofluorophotometer.
Real Time Ca2+ Confocal Laser Scanning MicroscopyFor experimentation, suspensions (1%) of infected erythrocytes in HEPES-buffered RPMI medium (no serum) were loaded with Fluo-4/AM (3 µM, Molecular Probes BV) for 20 min at 37 °C. For imaging, malaria parasite-infected erythrocytes were immobilized using polylysine-coated coverslips in a Bioptechs FCS2 perfusion chamber and maintained at 37 °C in growth medium (no serum). Inhibitors were added to the perfusate, and the Ca2+-dependent fluorescence responses were monitored in real time. Fluorescence signals from Fluo-4 were collected on a Zeiss LSM510 confocal microscope through a Plan-Apochromat 63x 1.4 N.A. oil objective using the "tracking mode." Excitation of Fluo-4 was performed using an argon ion laser at 488 nm. Emitted light was reflected through a 505550-nm band pass filter from a 540-nm dichroic mirror. Photobleaching (the irreversible damage of Fluo-4/AM producing a less fluorescent species) was assessed by continuous exposure (5 min) of loaded cells to laser illumination. For each experiment, the laser illumination setting that gave a minimal reduction in cytosolic and food vacuole fluorescence was used (this varied according to dye loading and confocal laser scanning microscopy (CLSM) settings). Data capture and extraction was carried out with LSM510 version 3 software (Zeiss). Cytosolic and vacuolar [Ca2+] was calibrated in situ by the addition of known amounts of [Ca2+]free in the presence of nigericin (0.8 µM) and the Ca2+ ionophore A23187 [GenBank] (1 µM). Using Calcium Calibration Buffer Kit 1 solutions (Molecular Probes), the [Ca2+]free was calculated from the Kd of EGTA for Ca2+ (107.9 nM at 37 °C (12)) using the equation [Ca2+]free = KdEGTA x [CaEGTA/K2EGTA].
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RESULTS |
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The Digestive Food Vacuole of P. falciparum Contains Elevated Free
Ca2+With the use of CLSM and the
cell-permeant AM ester of Fluo-4 to probe for Ca2+ in
trophozoites of infected red blood cells, we observed a strong fluorescence
signal deriving from the digestive food vacuole (e.g.
Fig. 3e). Some
Ca2+ indicators have a tendency to compartmentalize, and
differences in apparent Ca2+ affinities in different
compartments/organelles within a cell can sometimes lead to the formation of
artifactual Ca2+ gradients
(13). To overcome these
possible artifacts, free Ca2+ was calibrated
independently in situ in both the parasite cytosolic and food vacuole
compartments (Fig. 2). The
differing slopes (and fluorescence quantum yields in response to
Ca2+) of the parasite cytosol and food vacuole
calibration curves revealed that there was a difference in the respective
apparent Ca2+ binding affinities, probably resulting
from differences in the Ca2+ buffering capacity of the
different intracellular milieus and possibly from a degree of dye
compartmentalization. However, taking into account the differences in Fluo-4
Ca2+ affinities (by using the respective in
situ calibration curves), the digestive food vacuole was measured
nevertheless as having elevated free Ca2+ relative to
the cytosol, with levels ranging between 250 and 300 nM (n
= 27), some 5 to 6 times higher than the cytosol (measured at 50
nM, n = 27).
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Intracellular Distribution of Free Ca2+ during the Intraerythrocytic Development of the Malaria ParasiteThe distribution of free Ca2+ in the malaria parasite was observed to change during asexual development. As described, early trophozoites were observed as having a Ca2+ gradient between the digestive food vacuole and cytoplasmic compartments (Fig. 3e). However, as parasites underwent schizogony, free Ca2+ was observed to leave the digestive food vacuole and move to the intermembrane space surrounding each segment (Fig. 3, f and g). On rupture and release of merozoites from the erythrocytic host, there was a sustained elevation in free Ca2+; however the resolution of the CLSM did not allow us to determine the specific intracellular distribution of the signal (Fig. 3h). This increase was lost shortly after reinvasion of new red blood cells (i.e. the early ring stage), which may imply an important role for elevated merozoite free Ca2+ in the invasion process.
Spatiotemporal Dynamics of Free Ca2+ in
the Malaria Parasite and Digestive Food VacuoleFluo-4/AM is a
BAPTA
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid)-based indicator with a large dynamic range
(Rf 50200
(14)), making it very suitable
for the monitoring of both elementary and global Ca2+
signals (13). Using this
indicator, we were able to determine spatiotemporal Ca2+
fluctuations in malaria parasite-infected erythrocytes that were immobilized
using polylysine coated coverslips in a Bioptechs FCS2 perfusion chamber, in
response to a variety of inhibitors and agents. As described, trophozoites
were shown to posses elevated Ca2+ in the digestive
vacuoles (Fig. 3e).
The addition of thapsigargin (a plant sesquiterpene lactone that interacts
irreversibly with sarcoplasmic reticulum-like Ca2+
ATPases (15)) to trophozoites
caused a rapid depletion of vacuolar Ca2+ (Figs.
4A (and
Fig. 4B, QuickTime
movie) and 5A). The
addition of cyclopiazonic acid, another known selective irreversible inhibitor
of sarcoplasmic reticulum-like Ca2+-ATPases
(16), also caused a rapid
reduction in vacuolar free Ca2+ (Figs.
4C (and
Fig. 4D, QuickTime
movie) and 5B). Unlike
the case of thapsigargin, however, the reduction of food vacuolar
Ca2+ upon addition of cyclopiazonic acid was accompanied
by a conspicuous transient increase in cytosolic Ca2+.
Maneuvers that caused a perturbation of the trans-vacuolar membrane pH
gradient (the food vacuole is acidic relative to the cytosol), such as the
addition of the selective V-type ATPase inhibitor, bafilomycin A1
(Fig. 5C), and of
NH4Cl (QuickTime movie, Figs.
4E and
5C) to the perfusate,
also caused a rapid reduction of vacuolar Ca2+. A
transient rise in cytosolic Ca2+ was also evident upon
addition of NH4Cl to the perfusate
(Fig. 5C). The
membrane integrity of the digestive food vacuole was maintained on the
addition of the irreversible inhibitors thapsigargin, cyclopiazonic acid, and
bafilomycin A1 (see QuickTime movies,
Fig. 4, B, D, and
E) and the malaria pigment crystals were observed to
continue to move inside the vacuoles. Washing infected erythrocytes with
HEPES-buffered RPMI (pH 7.4) or Ringer solution after treatment with
NH4Cl restored the food vacuole Ca2+ gradient
of the cells (data not shown).
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As the digestive food vacuole of the malaria parasite contains a P-glycoprotein pump (PGH1), which can affect the compartmentalization of some fluorescence indicators, verapamil, a known PGH1 modulator (17), was added to the perfusate of the single cell chamber system. Verapamil (10 µM) was not shown to effect any change in the fluorescence signal deriving from either the food vacuole or the cytoplasm, suggesting that there was no interference from PGH1 in our system.
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DISCUSSION |
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With the use of CLSM and Fluo-4/AM (which is less susceptible to FPIX fluorescence quenching and has a good dynamic range) to probe for Ca2+ in mature trophozoites of infected red blood cells, we observed a free Ca2+ pool in the digestive food vacuole (Fig 3e) generating a Ca2+ gradient some 56-fold greater than that measured in the cytoplasm (at rest).
It was clearly observed that the sequestration of free Ca2+ altered during the asexual intraerythrocytic development of the malaria parasite (Fig 3). For example, as parasites underwent schizogony, free Ca2+ was observed to leave the digestive food vacuole and move to the intermembrane space surrounding each segment (Fig. 3, f and g) producing "wagon wheel"-looking Ca2+ distributions. It has been accepted for more than 20 years that Ca2+ in the culture medium is essential for the complete asexual development of the malaria parasite in red blood cells in culture (23). The results presented here lend further support to a role for Ca2+-dependent processes in the asexual development of the parasite.
Our findings showing Ca2+ storage by the digestive
food vacuole have allowed us to shed light on a recent controversy relating to
the pharmacology of the sarco/endoplasmic reticulum
Ca2+-ATPases of P. falciparum. A recent study
demonstrated that the malaria parasite contains an endoplasmic reticulum-like
Ca2+ store that is controlled by a
thapsigargin-insensitive but cyclopiazonic acid-sensitive
Ca2+-pump
(20), a finding that was at
odds with previous studies indicating a thapsigargin-sensitive mechanism
(19,
24). It is now apparent that
both of these studies adopted methods that were ineffective for the
measurement of vacuolar Ca2+ and therefore that their
fluorescence signals originated from cytosolic Ca2+
fluctuations. Using our live cell CLSM system to monitor Fluo-4-loaded
infected red blood cells, we observed that upon addition of thapsigargin (1
µM) or cyclopiazonic acid (10 µM) to the
perfusate, Ca2+ from the digestive food vacuole was
rapidly (60 s) released (Fig. 4,
A and C, and
Fig. 5, A and
B (QuickTime movies,
Fig. 4, B and
D)). In the case of cyclopiazonic acid (but undetectable
for thapsigargin), the vacuolar release of Ca2+ was
accompanied by a small transient increase in cytosolic
Ca2+ (Figs.
4C and
5B). These results
help explain previous observations of transient elevations of
Ca2+ upon addition of cyclopiazonic acid
(20). In addition these
results suggest the operation of more than one P-type
Ca2+ ATPase, one that is sensitive to both thapsigargin
and cyclopiazonic acid and one that is only sensitive to cyclopiazonic acid.
Significantly, and in support of our findings, the recent functional
characterization of P. falciparum transporters expressed in
Xenopus laevis oocytes have revealed the presence of both a
cyclopiazonic-sensitive Ca2+-ATPase (PfATPase4
(25)) and a
thapsigargin-sensitive Ca2+-ATPase
(PfATPase6).2 At this
early stage in the pharmacological analysis of intracellular
Ca2+ signaling in the malaria parasite, however, it
should be noted that thapsigargin and cyclopiazonic acid have previously been
shown to have different additional nonselective effects on
Ca2+ signaling pathway components, e.g.
capacitative Ca2+ entry
(26). However, it is unlikely
that capacitative Ca2+ entry is responsible for the
observed increase in cytoplasmic Ca2+ on treatment with
cyclopiazonic acid, as this effect was observed by Alleva and Kirk
(20) to occur in freed P.
falciparum parasites suspended in Ca2+-free
solutions.
The generation of a H+-gradient across the parasite vacuolar membrane by the action of V-type H+ ATPases and H+-pumping pyrophosphatases (8) suggests that, as with plant tonoplasts and acidocalcisomes of some parasites, the digestive food vacuole of the malaria parasite may also drive Ca2+ accumulation by H+-coupled transport mechanisms. This hypothesis is supported by our findings that a collapse of the transmembrane H+ gradient by the addition of NH4 (Fig. 5C (QuickTime movie, Fig. 4E)) or bafilomycin A1 (a selective V-type H+-ATPase inhibitor; Fig. 5C) results in a rapid discharge of vacuolar Ca2+. Given the essential role of Ca2+ as a secondary messenger molecule and the fact that physiological studies in plants have established a role for vacuolar (tonoplast) H+/Ca2+ exchange activity in ion transport, Ca2+ homeostasis, and signal transduction (27), further elucidation of the analogous H+-dependent mechanisms underlying Ca2+ homeostasis in the malaria parasite will be of significant importance.
Here we add another critical transport function to the malarial parasites repertoire of vacuolar transport mechanisms (Fig. 6). It will be important to determine how the interplay of Ca2+ transport and H+ transport via either the H+-pyrophosphatase or the V-type H+-ATPase contributes to organelle function. The rapid redistribution of vacuolar Ca2+ at schizogony (asexual multiplication) must implicate communication between the parasite nucleus and the food vacuole. In addition this organelle harbors transporters implicated in drug resistance (notably PfCRT and PGH1 (reviewed in Ref. 4)). The potential for proton-coupled Ca2+ transport to influence the function of these resistance transporters could have significant implications for malaria chemotherapy in the age of the quinoline-resistant parasite.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains QuickTime movies.
¶ Supported by a grant from the Wellcome Trust.
To whom correspondence may be addressed: Liverpool School of Tropical
Medicine, Pembroke Place, Liverpool L35 QA, United Kingdom. Tel.:
44-151-705-3151; Fax: 44-151-705-3371; E-mail:
Saward{at}liv.ac.uk
or
Biagini{at}liv.ac.uk.
1 The abbreviations used are: FPIX, ferriprotoporphyrin IX; MOPS,
4-morpholinepropanesulfonic acid; CLSM, confocal laser scanning microscopy;
AM, acetomethyl ester.
2 S. Krishna, personal communication.
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REFERENCES |
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