The Plastid In Plasmodium Falciparum Asexual Blood Stages: a Three-Dimensional Ultrastructural Analysis

Protist, Vol. 150, 283-295, October 1999 © Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/protist

ORIGINAL PAPER

The Plastid in Plasmodium falciparumAsexual Blood Stages:a Three-Dimensional Ultrastructural Analysis

Protist

John Hopkinsa,b, Ruth Fowler"·b, Sanjeev Krishnac, lain Wilsond, Graham Mitchellb, and

Lawrence Bannister"·1

a Department of Anatomy and Cell Biology, Guy's, King's College and St. Thomas's Hospitals' Medical and DentalSchools, Guy's Campus, Guy's Hospital London SE1 9RT, UK

b Department of Immunobiology, Guy's, King's College and St. Thomas's Hospitals' Medical and Dental Schools,Guy's Campus, Guy's Hospital London SE1 9RT, UK

c Department of Infectious Diseases, St.George's Hospital Medical School, Tooting, London SW17 OQT, UKd Parasitology Division, National Institute for Medical Research, Mill Hill, London NW7 1M, UK

Submitted May 6, 1999; Accepted July 28, 1999Monitoring Editor: Larry Simpson

The plastid in Plasmodium fa/ciparum asexual stages is a tubular structure measuring about 0.5 IJm x0.15 IJm in the merozoite, and 1.6 x 0.35 IJm in trophozoites. Each parasite contains a single plastiduntil this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochon­drion, along its whole length in merozoites and early rings, but only at one end in later stages. Re­gions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplas­mic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Re­constructions show the plastid wall is characteristically three membranes thick, with regions of addi­tional, complex membranes. These include inner and outer membrane complexes. The inner complexin the interior lumen is probably a rolled invagination of the plastid's inner membrane. The outer com­plex lies between the outer and middle wall membranes. The interior matrix contains ribosome-likegranules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possessplastids similar in structure to those of P. fa/ciparum. A model is proposed for the transfer of mem­brane lipid from the plastid to other organelles in the parasite.

Introduction

Recent interest in the plastids (or apicoplasts) ofapicomplexan parasites has centred on the prokary­ote-like nature of their DNA, RNA and ribosomes,and the implications of this for novel forms of

1Corresponding author;fax 44 171 9554915e-mail [email protected]

chemotherapy (for recent reviews see Feagin 1994;Fichera and Roos 1997; Jeffries and Johnson 1996;McFadden and Waller 1997; Williamson et al. 1994,1996; Wilson and Williamson 1997; Wilson et al.1994, 1996; see also Clough et al. 1997; Rogers etal. 1997). The morphological identity of the plastidwas established finally by Kohler et al. (1997) wholocalized the characteristic plastid DNA in Toxo­plasma gondii to a membranous organelle bounded

1434-4610/99/150/03-283 $ 12.00/0

284 J. Hopkins et al.

by four membranes, long known to ultrastructuralinvestigators (e.g. the 'corps multilamellaire' ofVivier and Petitprez 1972). Ultrastructural profilesresembling the Toxoplasma organelle have alsobeen reported for Plasmodium (see Aikawa 1971;Aikawa et al. 1967; Hepler et al. 1966). Aikawa et al.(1967) described it in merozoites of Plasmodiume/ongatum as a rounded structure (hence the name'spherical body') bounded by membranes of 'unde­termined number' and situated in an indentation ofthe mitochondrion, an association which they sug­gested implies metabolic interactions betweenthese two organelles.

The number of membranes surrounding apicom­plexan plastids has generated some evolutionaryspeculation. The quadruple membranes in Toxo­plasma and some other non-malarial apicomplex­ans resemble those of the plastids of chromistanalgae (McFadden and Gilson 1995), but phyloge­netic analyses of nuclear 18S rDNAs provide strongevidence that apicomplexans, dinoflagellates andciliates form a separate monophyletic clade (Cava­lier-Smith 1993). That plastids in dinoflagellates aresurrounded by only three membranes may be evi­dence that they were independently acquired(Palmer and Delwiche 1996), or were modified dur­ing the evolutionary divergence of dinoflagellatesand apicomplexans.

In the present paper we describe the detailedstructure of the plastid in Plasmodium falciparum. Apreliminary study showed us that it has a complexorganization, requiring three-dimensional informa­tion for an adequate analysis. We have therefore se­rially sectioned different asexual erythrocytic stagesof the parasite, and reconstructed the organizationof the plastid and its relations to other organelleswithin the parasite. This approach, which has onlyoccasionally been used for Plasmodium (e.g. Siomi­anny and Prensier 1986, to study the mitochondrionin P. falciparum trophozoites), has enabled us to ad­dress a series of questions, including the number ofmembranes forming its wall, its association with themitochondrion, and relation to other organelleswithin the parasite.

Results

The Plastid in the Ring, Trophozoite andEarly Schizont Stages

In all specimens examined up to the mid-schizontstage, a single plastid was present in each parasite(Figs. 1-4A-C), but thereafter the number increasedup t016 prior to the allocation of individual plastids

to budding merozoites (details of plastid replicationwill be given in another paper). In all stages the plas­tid is approximately cylindrical, with one or morebulges along its length which become more pro­nounced in trophozoites (Fig. 4C). As the intraery­throcytic cycle progresses the plastid lengthensconsiderably; in typical reconstructions the plastidin a merozoite is 0.5 mm long and in a young tropho­zoite 1.6 mm. In schizonts it increases considerablymore than this, although we have yet to completereconstructions of this stage. The merozoite plastidhas an approximately uniform diameter of about0.15 mm (Figs 4A, 5, 6), whereas in trophozoites andearly schizonts the width varies from 0.25 mm to0.35 mm depending on the region measured (Figs.4,7-21).

Detailed Structure of the Plastid

The internal morphology of the plastid (Figs. 9-17) isessentially similar throughout the different stages,although there are some changes in the details ofmembrane arrangements. We have studied theplastid most thoroughly in young trophozoiteswhere it is still quite short, and therefore relativelyeasy to reconstruct from serial sections (Figs. 4A,9-17), and where it is likely to be fully functional. Thedescription given here is based mainly on se­quences taken from this stage.

Membrane number

Throughout the asexual cycle the plastid and mito­chondrion are clearly distinct in structure because ofthe larger number of the plastid's membranes andits somewhat denser internal matrix. For its entirelength the plastid wall is composed of three mem­branes (Figs. 2, 3, 9, 16, 18, 20) designated here asouter, middle and inner, but in certain regions of theplastid there are also, variably, more numerousmembranes (see below). The basic three mem­branes have a characteristic intermembrane spac­ing, the two inner leaflets being very close to ortouching each other, while the outermost is sepa­rated from the middle membrane by a wide thoughvariable (20-40 nm) space. The outer membrane hasoccasional long leaf-like extensions into the sur­rounding cytoplasm (Fig.7, 8, 14, 15).

The profiles of all three wall membranes, but especiallythe inner two, are typically very irregular compared withthe generally smooth membranes of the parasite's mito­chondrion (Fig. 2), a feature which initially made it difficultfor us to be sure of the number of membranes composingit. Although many micrographs clearly showed threemembranes, others suggested four or five, often appar-

Plasmodium Plastid Ultrastructure 285

Figure 1. A section through a young schizont within a red cell, showing part of a plastid (p) between two nuclei (n).Mitochondrial profiles (m) are also visible. Scale bar = 0.5IJm.Figure 2. Transverse section through a plastid (p) closely associated with a mitochondrion (m) in an early buddingmerozoite. Note the three membranes of the plastid wall and the two membranes of the mitochondrion. Scale bar =0.1 IJm.Figure 3. Longitudinal section through part of a plastid, showing the wrinkled nature of its membranes, the granu­lar and filamentous interior, and the close proximity to the pigment vacuole (pv). Scale bar = 0.1 IJm.

ently varying in number around the organelle. When mi­crographs of tilted sections were viewed stereoscopically(Figs. 7, 8) the extra membranes could be seen to be wrin­kles in one or more of the basic three, viewed edge-on inthe thickness of the section (except when complex infold-

ings were present, as described below). This finding wasconfirmed in numerous sections of different stages, andso we are confident that for much of its length the plastidis lined by three membranes, contrasting with the four-foldwall found in Toxoplasma (see Discussion).

286 J. Hopkins et al.

A 1IJm B c 1 pm

Figure 4. Reconstructions of the arrangement of the plastid and mitochondrion in a budding merozoite (A), youngring (8) and young trophozoite (C), made from sets of serial electron microscopic sections. Figure 4A shows thepairing of plastid (p) and mitochondrion (m), and the association between the two subpellicular microtubules (mt)which extend basally from the polar rings of the merozoite apex. The position of the nucleus (n) is also shown. Fig­ure 48 shows that the parallel arrangement of plastid and mitochondrion is maintained in the early ring, while in Fig­ure 4C, a young trophozoite contains a plastid set at right-angles to the coiled mitochondrion, remaining attachedat one point. Abbreviations for 8 and C as in A.

In addition to this basic triple membrane condi­tion, plastids have more complex membrane config­urations at one or more restricted regions where theplastid has a greater diameter (Fig. 9). Here theusual three membranes are accompanied by vesicu­lar or looped membranes, their complexity andnumbers increasing with parasite development.Since the other membranous structures of the para­site are well preserved, these are clearly part of theplastid organization rather than fixation artefacts.The additional membranes occur in two regions: (1)an outer membrane complex between the outer andmiddle membranes, and (2) an inner membranecomplex within the lumen of the plastid. A selectionof transverse sections from the plastid of an earlytrophozoite (Figs. 11-15, see also Figs. 7-9,19) il­lustrates the arrangement and position of thesemembrane complexes. The outer membrane com­plex consists of coiled tubular and vesicular clustersand flattened cisternae which extend over a limitedregion on one side near the middle of the plastid.Because of the complicated configuration of this as­sembly it is not possible at present to assign its ori­gin to either the outer or middle membranes. Theinner membrane complex is composed of tubularwhorls of up to 6 membranes. Stereoscopicallyviewed micrographs of tilted sections (Figs. 14, 15)indicate that the inner complex whorls are derived atleast in part from an invagination of the inner plastid

wall membrane, rolled up into a myelin-like struc­ture. (see Fig. 9). Both outer and inner membranecomplexes are present in a minimal form in mero­zoites (Figs. 5, 6), but clearly increase in size andcomplexity in trophozoites. In some instances, thevesicles of the outer membrane complex are quitedense in appearance, and when clustered closely,they give the appearance of a dense body lodgedwithin that space (Figs. 7, 8, 19).

The Central Matrix of the Plastid

Within the central lumen of the plastid there is aheterogenous collection of granules and fine fila­ments (e.g. Figs. 3, 7-9, 11, 16). Clumps of granu­lar material are associated with the luminal sur­face of the inner membrane and form irregularpatches throughout the interior space. Individualgranules about 12 nm across (of ribosomal di­mensions and appearance) occur in small num­bers and are dispersed throughout the plastidlumen (Figs.2, 3, 7, 8). Linking these structures isa delicate branched network of well-defined thin(4 nm) filaments which fills the lumen (Figs. 7-9).In addition to these, a relatively rounded mass offinely granular material was occasionally found inthe central lumen, towards one end of the plastid(Fig. 21); the significance of this is unknown (butsee Discussion).

Plasmodium Plastid Ultrastructure 287

Figures 5 and 6. A stereopair (tilted 12°) showing a plastid (p) and mitochondrion (m) in transverse section in amerozoite. The plastid is anchored to a pair of subpellicular microtubules (arrows). The three membranes of theplastid wall are indicated by arrowheads. Outer and inner membrane complexes (oc and ic respectively) are alsovisible within the plastid. Scale bar = 0.1 ~m.

Figures 7 and 8. A stereopair (tilted 12°) depicting a transversely sectioned plastid in a late trophozoite/early sch­izont. A dense aggregate of membrane vesicles denoting the outer membrane complex (oc) lies between the outerand middle membranes of the plastid wall. Visible in the interior of the plastid are the network of branched fila­ments and clumps of granules (e.g. arrowhead), and on the right (shown more clearly in Fig. 8) is an indication ofcontinuity (open arrow) between the plastid's outer membrane and an adjacent endoplasmic reticulum cisterna (c).Note again the three membranes of the plastid wall. Scale bar =0.1 ~m.

288 J. Hopkins et al.

17

!16

113 14 15

t !11 12

! 1

Positions of transverse sections shown in Figs 10 - 17

10

!

mitochondrion

I

9

Figure 9. Reconstruction of a plastid from a series of 16 sections of the trophozoite shown in Figure 4C, showingthe arrangement of the three wall membranes, and the outer and inner membrane complexes (see text for details).The interior shows the granular structure and system of branched filaments. One end of the plastid deeply indentsthe wall of a mitochondrion, only a short segment of which is depicted here. The positions of transverse sectionsshown in Figures 10-17 are indicated by arrows.

Association Between the Plastid and OtherOrganelles

With the Mitochondrion

The position of the plastid varies considerably withinrings, trophozoites and early schizonts, but in allstages including merozoites there is always a closeassociation between plastid and mitochondrion(Figs. 2, 4-6, 9). In merozoites (Figs. 4A, 5, 6) theplastid and mitochondrion lie side by side alongtheir length, a small gap of about 5 nm separatingthe two, the mitochondrion being rather longer(0.8-1.3 I-Im compared with with the plastid's0.4-0.65 I-Im) and extending beyond it at both ends.This parallel arrangement is maintained in early rings(Fig. 48) but in more mature rings, trophozoites (Fig.4C) and early schizonts, only one end of the plastidremains in contact, either with one end of the mito­chondrion or some other region, the two organellesoften being diametrically apposed to each other orsubtending a large angle of divergence, the tip of theplastid deeply embedded in the mitochondrial sur­face (Figs. 9, 10).

With Other Pigment Vacuole, Nucleus and Endo­plasmic Reticulum

Another characteristic association in trophozoitesand early schizonts is between the free end of theplastid and the pigment vacuole, the outer mem­brane of the plastid being applied closely to the pig­ment vacuole membrane or deeply invaginating it(Figs. 3, 20). In late trophozoites and early schizontsthis end of the plastid is often very expanded andhas an extensive area of contact with the vacuolarmembrane.

A further association often observed is betweensome region of the plastid and the nuclear envelope(Figs. 18, 19) and in some micrographs, clearly defineddense material connects the two organelles (Fig. 19).However, about one-third of reconstructed parasitesdid not show a clear plastid-nuclear relationship, indi­cating that it may represent only a transient contact.

Serial sections sections also show rough andsmooth endoplasmic reticulum cisternae situatedclose to the margins of the plastid (Figs. 16, 17) and inplaces these may be continuous with the plastid'souter wall membrane (Figs. 7, 8).

Plasmodium Plastid Ultrastructure 289

Figures 10-13. Transverse section of the plastid shown in Figure 9. In Figure 10, one end of the plastid (p) is em­bedded in a depression in the wall of a mitochondrion (m); in Figure 11 the end of the inner membrane complex isjust visible (ic), and the plastid matrix shows ribosome-like granules. In Figure 12 the inner membrane complex (ic)becomes more elaborate, and in Fig.13 it appears as a set of concentric laminae, whilst the separate outer mem­brane complex is visible (oc). Scale bar =0.1 \-1m.

290 J. Hopkins et al.

Figures 14-17. A further set of transverse sections from the plastid shown in Figure 9. Figures 14 and 15 are astereopair (tilted 12°) in which the inner membrane complex (ic) is seen to be a rolled loop of membrane, and theouter membrane complex (oc) a system of coiled tubules and vesicles. The outer wall membrane is also extendedinto a cisterna-like structure (arrow). Figures 16 and 17 are sections close to the end of the plastid, and show roughendoplasmic reticulum cisternae (c) adjacent to the plastid (ribosomes indicated by arrowheads). Scale bars are all0.1 IJm.

Plasmodium Plastid Ultrastructure 291

Figures 18 and 19. Transverse sections through plastids in close proximity to the envelope of the nucleus (n); inFigure 19 dense material (arrow) forms an attachment between the two structures in the region of the outer mem­brane complex (oc). Scale bar = 0.1 ~m.

Figure 20. Transverse section of a plastid (p) closely related to a pigment vacuole (pv). The pigment vacuole mem­brane and a loop of the plastid wall outer membrane are highly infolded into the vacuole interior. The two whiteregions of the pigment vacuole are holes where two pigment crystals have fallen out of the section. Scale bar =0.1 ~m.

Figure 21. Longitudinal section through part of a plastid in a late trophozoite/young schizont showing the threelayers of the plastid wall and a dense finely granular region (d) within the internal matrix. Scale bar = 0.1 ~m.

292 J. Hopkins et al.

Figure 22. Transverse section through a plastid and mitochondrion in a Plasmodium knowlesi merozoite, showingthe close apposition of the two organelles, the typical trilaminar wall of the plastid (p), contrasting with the bilami­nar mitochondrion (m). Scale bar = 0.1 ~m.

Figure 23. Transverse section through a plastid (p) and mitochondrion (m) in a Plasmodium berghei merozoite,showing an arrangement similar to that in Plasmodium knowlesi depicted in Fig.22. Scale bar = 0.1 ~m.

With Microtubules in the Merozoite

Characteristically, the plastid lies longitudinally inthe middle third of the merozoite's length, close tothe pellicle (FigsAA, 5-6) and immediately beneaththe band of 2 or 3 subpellicular microtubules run­ning from the merozoite's apex towards its base.Between the subpellicular microtubules and theplastid is a longitudinal band of dense finely granularmaterial (seen only in tannic acid-glutaraldehydefixed specimens). Short cross bridges and other fila­mentous structures were occasionally seen be­tween the plastid and the microtubules; a detailedaccount of this complex structure will be given else­where.

Comparison with Other Species of Plasmodium

Examination of sections of merozoites of two othermalaria parasites, P. know/esi (Fig. 22) and P.berghei (Fig. 23), showed that their plastids have asimilar trilaminar wall, and each is closely associ­ated with a mitochondrion. Interestingly, in both ofthese species , the mitochondrion is partiallywrapped around the plastid so that the intimate con­tact between the two organelles is more extensivethan in P. falciparum (see especially Fig. 22).

Discussion

This study demonstrates that unlike Toxoplasma(see Kohler et al. 1997), the plastid in Plasmodiumhas a triple-membraned wall, and also possessestwo localized sets of more elaborate membraneswhich increase in complexity during the intraery­throcytic life cycle. The phylogenetic implications ofthis difference are unclear, as only a few apicom­plexan plastids have been studied, and it is possiblethat Plasmodium has lost one of the membranesduring its evolutionary history, or that the extramembrane of Toxoplasma is vestigial within thecomplex whorls described in this paper. A muchwider range of apicomplexans needs to be exam­ined before any phylogenetic conclusion can bedrawn from plastid membrane numbers, bearing inmind the potential difficulty of interpreting sectionsof irregularly folded membranes, as described in thepresent paper.

It is clear from our results that the concept of asimple sac-like organelle enclosed in a set of threeor four membranes has to be modified, as the outerand inner membrane complexes contribute sub­stantially to the total membrane of the plastid. Theinner membrane complex can be interpreted most

simply as a rolled-up myelin-like invagination of theinner wall membrane. The outer membrane complexis not so obviously continuous with either the outeror middle membranes, and its vesicles and tubulescould be derived by budding from either the middleor outer membrane.

Recently, Waller et al. (1998) have demonstratedthat the plastids of P. fa/ciparum and Toxoplasmagondii are active in membrane lipid synthesis (as arethe chloroplasts of higher plants which can exportlipids to other cell structures: see the review by Jo­yard et al. 1998). Combining this information withthe structural data we describe here, an interestingpossibility emerges, i.e. that the apicomplexan plas­tid is essentially a membrane lipid factory for theparasite, budding off lipid in vesicular form betweenthe inner and outer membranes and transporting itto adjacent organelles via extensions of its surface.The enlargement of the plastid and the increasingcomplexity of its internal membranes in trophozoitesand early schizont stages may therefore denote anincreasing demand for membrane lipids as othermembranous organelles of the parasite increase insize and activity, although of course some mem­brane generation would also be needed for plastidreplication in the schizont stage. If this view of plas­tid function is correct, plastids in other apicom­plexan genera are likely to have similar membraneconfigurations, and a detailed analysis of plastidstructure in representative genera of apicomplexanswould be of much interest.

Turning to the association between the plastidand mitochondrion, first described in Plasmodiumelongatum merozoites by Aikawa et al. (1967), wehave shown that in Plasmodium falciparum thetwo organelles invariably adhere to each other,with varying degrees of contact throughout theasexual cycle. As suggested by Aikawa et al.(1967), this duality may infer a metabolic interac­tion between the two structures. The attachmentmay also have a mechanical role, ensuring pairedallocations of the two organelles to buddingmerozoites at the end of schizogony.

The association between plastid and mito­chondrion is particularly striking in merozoites,where in P. falciparum the position of both arespecified by the plastid's connection to the bandof subpellicular microtubules running along oneside of this stage (see Bannister and Mitchell1995; Read et al. 1993). Microtubules have beenshown in other organisms to be responsible formitochondrial movements (see e.g. Baumann andMurphy 1995; Knabe and Kuhn 1996), and couldbe the means of propelling plastids, mitochondriaand other organelles into merozoites during

Plasmodium Plastid Ultrastructure 293

merozoite assembly (see Bannister and Mitchell1995; Tilney and Tilney 1996). However, experi­ments in our laboratory with anti-microtubuledrugs have as yet failed to show inhibition of mi­tochondrial placement during the formation ofmerozoites (Fowler et al. 1998), and the deeplyplaced location of the plastid in P. berghei and P.knowlesi suggest that they may not be anchoredto microtubules in the same way in these species.Further work is in progress to clarify this issue.

There are three other plastid associations whichmay be significant, i.e. with the pigment vacuole,the nuclear membrane and endoplasmic reticulum.Although proximity does not prove a functional re­lationship, there are clear signs of structural con­nection or interaction between the plastid andthese structures. As proposed above, the plastidmay deliver membrane lipid to such organelles(and also perhaps to the mitochondrion), but in theinstances of the nucleus and endoplasmic reticu­lum, the traffic may consist of nuclear-encodedproteins moving in the opposite direction. i.e. intothe plastid from their sites of synthesis in the endo­plasmic reticulum and cisterna of the nuclear en­velope, as shown biochemically by Waller et al.(1998). Intermittent fusion between the plastidouter membrane, including its leaf-like extensions,with these cisternae could afford a route by whichsuch proteins are conveyed to the plastid.

The inner matrix of the plastid also deservescomment. The 12 nm granules are clearly goodcandidates for a ribosomal identity on the basis oftheir size and appearance. The fine filaments ofthe matrix are approximately the width of DNAstrands, but the results of Waller et al. (1998) fromfluorescence labeling indicate that the DNA isconcentrated in a restricted region of the plastid.The dense material we show in Figure 21 may berelevant to this finding. Further work is in progressto localise DNA ultrastructurally within the plastid.

Methods

Cultures of Plasmodium falciparum (IT04 strain)were grown in continuous culture and loosely syn­chronised, maturing schizonts were concentratedon Percoll cushions by standard techniques (seee.g. Fowler et al. 1998). After collection, the cellswere incubated for a further 2h at 37°C. Thereafterthe cells were processed for electron microscopyaccording to a modification of the protocol de­scribed by Bannister and Mitchell (1989). Briefly, theparasites were concentrated again by brief centrifu­gation at 6000xg and fixed in suspension for 2 hrs in

294 J. Hopkins et al.

2.5% glutaraldehyde in 0.075M sodium cacodylatebuffer (pH 7.2) at room temperature, in some caseswith the addition of 1% tannic acid (final pH 7.2).Cells were washed x3 in cacodylate buffer, thenconcentrated as above into pellets which were post­fixed in 1% osmium tetroxide (1 h), briefly washed inbuffer, then block stained for 1 h in 1% aqueousuranyl acetate. Pellets were then dehydrated in anacetone series and embedded in Medium TAABresin. Serial sections were cut at a thickness of 85nm on a Reichert Ultracut E ultramicrotome,mounted on slotted grids covered with a thin film ofpioloform, and contrasted with uranyl acetate andlead citrate. Depending on orientation and externalshape, rings, trophozoites and schizonts each re­quired up to 75 serial sections for complete recon­struction, while merozoites needed up to 32 sec­tions. Sectioned cells were photographed atx12,000 and printed at x53,000 for survey recon­structions, and at x40,000 (printed at x120,000) formore detailed reconstructions. Micrographs weretraced on to acetate sheets, and reconstructedgraphically from these. For detailed morphology,thinner (50 nm) sections were also prepared and ex­amined. In some cases serial sections and thinnersingle sections were tilted using a goniometer stageand photographed at 0° and 12° at x100,000, thenviewed with a stereoscope, to analyse complexmembrane configurations and other internal details.

For comparison with Plasmodium fa/ciparum,electron microscopy was carried out on specimensof Plasmodium knowlesi prepared for a previousstudy (Bannister and Mitchell 1989), and on Plas­modium berghei kindly provided by Dr. A.w. Thomas(Primate Biomedical Research Centre, Rijkswijk,Netherlands), prepared as in Kocken et al. (1998).

Acknowledgements

The authors thank the Wellcome Trust (Grants No.037082 and 048244) and the Special Trustees forSt.Thomas' Hospital for supporting this study. San­jeev Krishna is a Wellcome Trust Senior ResearchFellow in Clinical Science. The authors are also in­debted to the Electron Microscope Unit at UMDS(Guy's site) for photographic assistance, and to theMedical Research Council of the UK and ProfessorB. Boycott for provision of serial sectioning facilities.

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