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

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Protist, Vol. 150, 283-295, October 1999 © Urban & Fischer Verlag



The Plastid in Plasmodium falciparum Asexual Blood Stages: a Three-Dimensional Ultrastructural Analysis John Hopkinsa,b, Ruth Fowler"·b, Sanjeev Krishna c , lain Wilson d , Graham Mitchellb, and Lawrence Bannister"·1 Department of Anatomy and Cell Biology, Guy's, King's College and St. Thomas's Hospitals' Medical and Dental Schools, 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, UK d Parasitology Division, National Institute for Medical Research, Mill Hill, London NW7 1M, UK


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

The plastid in Plasmodium fa/ciparum asexual stages is a tubular structure measuring about 0.5 IJm x 0.15 IJm in the merozoite, and 1.6 x 0.35 IJm in trophozoites. Each parasite contains a single plastid until 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 complex in 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-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids 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) of apicomplexan 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 9554915 e-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 et al. 1997). The morphological identity of the plastid was established finally by Kohler et al. (1997) who localized the characteristic plastid DNA in Toxoplasma gondii to a membranous organelle bounded 1434-4610/99/150/03-283 $ 12.00/0


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by four membranes, long known to ultrastructural investigators (e.g. the 'corps multilamellaire' of Vivier and Petitprez 1972). Ultrastructural profiles resembling the Toxoplasma organelle have also been reported for Plasmodium (see Aikawa 1971; Aikawa et al. 1967; Hepler et al. 1966). Aikawa et al. (1967) described it in merozoites of Plasmodium e/ongatum as a rounded structure (hence the name 'spherical body') bounded by membranes of 'undetermined number' and situated in an indentation of the mitochondrion, an association which they suggested implies metabolic interactions between these two organelles. The number of membranes surrounding apicomplexan plastids has generated some evolutionary speculation. The quadruple membranes in Toxoplasma and some other non-malarial apicomplexans resemble those of the plastids of chromistan algae (McFadden and Gilson 1995), but phylogenetic analyses of nuclear 18S rDNAs provide strong evidence that apicomplexans, dinoflagellates and ciliates form a separate monophyletic clade (Cavalier-Smith 1993). That plastids in dinoflagellates are surrounded by only three membranes may be evidence that they were independently acquired (Palmer and Delwiche 1996), or were modified during the evolutionary divergence of dinoflagellates and apicomplexans. In the present paper we describe the detailed structure of the plastid in Plasmodium falciparum. A preliminary study showed us that it has a complex organization, requiring three-dimensional information for an adequate analysis. We have therefore serially sectioned different asexual erythrocytic stages of the parasite, and reconstructed the organization of the plastid and its relations to other organelles within the parasite. This approach, which has only occasionally been used for Plasmodium (e.g. Siomianny and Prensier 1986, to study the mitochondrion in P. falciparum trophozoites), has enabled us to address a series of questions, including the number of membranes forming its wall, its association with the mitochondrion, and relation to other organelles within the parasite.

Results The Plastid in the Ring, Trophozoite and Early Schizont Stages In all specimens examined up to the mid-schizont stage, a single plastid was present in each parasite (Figs. 1-4A-C), but thereafter the number increased up t016 prior to the allocation of individual plastids

to budding merozoites (details of plastid replication will be given in another paper). In all stages the plastid is approximately cylindrical, with one or more bulges along its length which become more pronounced in trophozoites (Fig. 4C). As the intraerythrocytic cycle progresses the plastid lengthens considerably; in typical reconstructions the plastid in a merozoite is 0.5 mm long and in a young trophozoite 1.6 mm. In schizonts it increases considerably more than this, although we have yet to complete reconstructions of this stage. The merozoite plastid has an approximately uniform diameter of about 0.15 mm (Figs 4A, 5, 6), whereas in trophozoites and early schizonts the width varies from 0.25 mm to 0.35 mm depending on the region measured (Figs. 4,7-21).

Detailed Structure of the Plastid The internal morphology of the plastid (Figs. 9-17) is essentially similar throughout the different stages, although there are some changes in the details of membrane arrangements. We have studied the plastid most thoroughly in young trophozoites where it is still quite short, and therefore relatively easy to reconstruct from serial sections (Figs. 4A, 9-17), and where it is likely to be fully functional. The description 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 of the larger number of the plastid's membranes and its somewhat denser internal matrix. For its entire length the plastid wall is composed of three membranes (Figs. 2, 3, 9, 16, 18, 20) designated here as outer, middle and inner, but in certain regions of the plastid there are also, variably, more numerous membranes (see below). The basic three membranes have a characteristic intermembrane spacing, the two inner leaflets being very close to or touching each other, while the outermost is separated from the middle membrane by a wide though variable (20-40 nm) space. The outer membrane has occasional long leaf-like extensions into the surrounding cytoplasm (Fig.7, 8, 14, 15). The profiles of all three wall membranes, but especially the inner two, are typically very irregular compared with the generally smooth membranes of the parasite's mitochondrion (Fig. 2), a feature which initially made it difficult for us to be sure of the number of membranes composing it. Although many micrographs clearly showed three membranes, others suggested four or five, often appar-

Plasmodium Plastid Ultrastructure


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 budding merozoite. 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 in the thickness of the section (except when complex infold-

ings were present, as described below). This finding was confirmed in numerous sections of different stages, and so we are confident that for much of its length the plastid is lined by three membranes, contrasting with the four-fold wall found in Toxoplasma (see Discussion).


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Figure 4. Reconstructions of the arrangement of the plastid and mitochondrion in a budding merozoite (A), young ring (8) and young trophozoite (C), made from sets of serial electron microscopic sections. Figure 4A shows the pairing 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 attached at 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 the plastid has a greater diameter (Fig. 9). Here the usual three membranes are accompanied by vesicular or looped membranes, their complexity and numbers increasing with parasite development. Since the other membranous structures of the parasite are well preserved, these are clearly part of the plastid organization rather than fixation artefacts. The additional membranes occur in two regions: (1) an outer membrane complex between the outer and middle membranes, and (2) an inner membrane complex within the lumen of the plastid. A selection of transverse sections from the plastid of an early trophozoite (Figs. 11-15, see also Figs. 7-9,19) illustrates the arrangement and position of these membrane complexes. The outer membrane complex consists of coiled tubular and vesicular clusters and flattened cisternae which extend over a limited region 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. The inner membrane complex is composed of tubular whorls of up to 6 membranes. Stereoscopically viewed micrographs of tilted sections (Figs. 14, 15) indicate that the inner complex whorls are derived at least 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 membrane complexes are present in a minimal form in merozoites (Figs. 5, 6), but clearly increase in size and complexity in trophozoites. In some instances, the vesicles of the outer membrane complex are quite dense in appearance, and when clustered closely, they give the appearance of a dense body lodged within that space (Figs. 7, 8, 19). The Central Matrix of the Plastid

Within the central lumen of the plastid there is a heterogenous 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 irregular patches throughout the interior space. Individual granules about 12 nm across (of ribosomal dimensions and appearance) occur in small numbers and are dispersed throughout the plastid lumen (Figs.2, 3, 7, 8). Linking these structures is a 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 of finely granular material was occasionally found in the central lumen, towards one end of the plastid (Fig. 21); the significance of this is unknown (but see Discussion).

Plasmodium Plastid Ultrastructure


Figures 5 and 6. A stereopair (tilted 12°) showing a plastid (p) and mitochondrion (m) in transverse section in a merozoite. The plastid is anchored to a pair of subpellicular microtubules (arrows). The three membranes of the plastid wall are indicated by arrowheads. Outer and inner membrane complexes (oc and ic respectively) are also visible 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 outer and 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 of continuity (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.


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Positions of transverse sections shown in Figs 10 - 17










14 15







9 Figure 9. Reconstruction of a plastid from a series of 16 sections of the trophozoite shown in Figure 4C, showing the 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 indents the wall of a mitochondrion, only a short segment of which is depicted here. The positions of transverse sections shown in Figures 10-17 are indicated by arrows.

Association Between the Plastid and Other Organelles With the Mitochondrion The position of the plastid varies considerably within rings, trophozoites and early schizonts, but in all stages including merozoites there is always a close association between plastid and mitochondrion (Figs. 2, 4-6, 9). In merozoites (Figs. 4A, 5, 6) the plastid and mitochondrion lie side by side along their length, a small gap of about 5 nm separating the two, the mitochondrion being rather longer (0.8-1.3 I-Im compared with with the plastid's 0.4-0.65 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 plastid remains in contact, either with one end of the mitochondrion or some other region, the two organelles often being diametrically apposed to each other or subtending a large angle of divergence, the tip of the plastid deeply embedded in the mitochondrial surface (Figs. 9, 10).

With Other Pigment Vacuole, Nucleus and Endoplasmic Reticulum Another characteristic association in trophozoites and early schizonts is between the free end of the plastid 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 schizonts this end of the plastid is often very expanded and has an extensive area of contact with the vacuolar membrane. A further association often observed is between some region of the plastid and the nuclear envelope (Figs. 18, 19) and in some micrographs, clearly defined dense material connects the two organelles (Fig. 19). However, about one-third of reconstructed parasites did not show a clear plastid-nuclear relationship, indicating that it may represent only a transient contact. Serial sections sections also show rough and smooth endoplasmic reticulum cisternae situated close to the margins of the plastid (Figs. 16, 17) and in places these may be continuous with the plastid's outer wall membrane (Figs. 7, 8).

Plasmodium Plastid Ultrastructure


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 is just 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.


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Figures 14-17. A further set of transverse sections from the plastid shown in Figure 9. Figures 14 and 15 are a stereopair (tilted 12°) in which the inner membrane complex (ic) is seen to be a rolled loop of membrane, and the outer membrane complex (oc) a system of coiled tubules and vesicles. The outer wall membrane is also extended into a cisterna-like structure (arrow). Figures 16 and 17 are sections close to the end of the plastid, and show rough endoplasmic reticulum cisternae (c) adjacent to the plastid (ribosomes indicated by arrowheads). Scale bars are all 0.1 IJm.

Plasmodium Plastid Ultrastructure


Figures 18 and 19. Transverse sections through plastids in close proximity to the envelope of the nucleus (n); in Figure 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 white regions 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 three layers of the plastid wall and a dense finely granular region (d) within the internal matrix. Scale bar = 0.1 ~m.


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Figure 22. Transverse section through a plastid and mitochondrion in a Plasmodium knowlesi merozoite, showing the 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 in the middle third of the merozoite's length, close to the pellicle (FigsAA, 5-6) and immediately beneath the band of 2 or 3 subpellicular microtubules running from the merozoite's apex towards its base. Between the subpellicular microtubules and the plastid is a longitudinal band of dense finely granular material (seen only in tannic acid-glutaraldehyde fixed specimens). Short cross bridges and other filamentous structures were occasionally seen between the plastid and the microtubules; a detailed account of this complex structure will be given elsewhere. Comparison with Other Species of Plasmodium Examination of sections of merozoites of two other malaria parasites, P. know/esi (Fig. 22) and P. berghei (Fig. 23), showed that their plastids have a similar trilaminar wall, and each is closely associated with a mitochondrion. Interestingly, in both of these species , the mitochondrion is partially wrapped around the plastid so that the intimate contact between the two organelles is more extensive than in P. falciparum (see especially Fig. 22).

Discussion This study demonstrates that unlike Toxoplasma (see Kohler et al. 1997), the plastid in Plasmodium has a triple-membraned wall, and also possesses two localized sets of more elaborate membranes which increase in complexity during the intraerythrocytic life cycle. The phylogenetic implications of this difference are unclear, as only a few apicomplexan plastids have been studied, and it is possible that Plasmodium has lost one of the membranes during its evolutionary history, or that the extra membrane of Toxoplasma is vestigial within the complex whorls described in this paper. A much wider range of apicomplexans needs to be examined before any phylogenetic conclusion can be drawn from plastid membrane numbers, bearing in mind the potential difficulty of interpreting sections of irregularly folded membranes, as described in the present paper. It is clear from our results that the concept of a simple sac-like organelle enclosed in a set of three or four membranes has to be modified, as the outer and inner membrane complexes contribute substantially to the total membrane of the plastid. The inner membrane complex can be interpreted most

Plasmodium Plastid Ultrastructure

simply as a rolled-up myelin-like invagination of the inner wall membrane. The outer membrane complex is not so obviously continuous with either the outer or middle membranes, and its vesicles and tubules could be derived by budding from either the middle or outer membrane. Recently, Waller et al. (1998) have demonstrated that the plastids of P. fa/ciparum and Toxoplasma gondii are active in membrane lipid synthesis (as are the chloroplasts of higher plants which can export lipids to other cell structures: see the review by Joyard et al. 1998). Combining this information with the structural data we describe here, an interesting possibility emerges, i.e. that the apicomplexan plastid is essentially a membrane lipid factory for the parasite, budding off lipid in vesicular form between the inner and outer membranes and transporting it to adjacent organelles via extensions of its surface. The enlargement of the plastid and the increasing complexity of its internal membranes in trophozoites and early schizont stages may therefore denote an increasing demand for membrane lipids as other membranous organelles of the parasite increase in size and activity, although of course some membrane generation would also be needed for plastid replication in the schizont stage. If this view of plastid function is correct, plastids in other apicomplexan genera are likely to have similar membrane configurations, and a detailed analysis of plastid structure in representative genera of apicomplexans would be of much interest. Turning to the association between the plastid and mitochondrion, first described in Plasmodium elongatum merozoites by Aikawa et al. (1967), we have shown that in Plasmodium falciparum the two organelles invariably adhere to each other, with varying degrees of contact throughout the asexual cycle. As suggested by Aikawa et al. (1967), this duality may infer a metabolic interaction between the two structures. The attachment may also have a mechanical role, ensuring paired allocations of the two organelles to budding merozoites at the end of schizogony. The association between plastid and mitochondrion is particularly striking in merozoites, where in P. falciparum the position of both are specified by the plastid's connection to the band of subpellicular microtubules running along one side of this stage (see Bannister and Mitchell 1995; Read et al. 1993). Microtubules have been shown in other organisms to be responsible for mitochondrial movements (see e.g. Baumann and Murphy 1995; Knabe and Kuhn 1996), and could be the means of propelling plastids, mitochondria and other organelles into merozoites during


merozoite assembly (see Bannister and Mitchell 1995; Tilney and Tilney 1996). However, experiments in our laboratory with anti-microtubule drugs have as yet failed to show inhibition of mitochondrial placement during the formation of merozoites (Fowler et al. 1998), and the deeply placed location of the plastid in P. berghei and P. knowlesi suggest that they may not be anchored to microtubules in the same way in these species. Further work is in progress to clarify this issue. There are three other plastid associations which may 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 and these structures. As proposed above, the plastid may deliver membrane lipid to such organelles (and also perhaps to the mitochondrion), but in the instances of the nucleus and endoplasmic reticulum, the traffic may consist of nuclear-encoded proteins moving in the opposite direction. i.e. into the 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 plastid outer membrane, including its leaf-like extensions, with these cisternae could afford a route by which such proteins are conveyed to the plastid. The inner matrix of the plastid also deserves comment. The 12 nm granules are clearly good candidates for a ribosomal identity on the basis of their size and appearance. The fine filaments of the matrix are approximately the width of DNA strands, but the results of Waller et al. (1998) from fluorescence labeling indicate that the DNA is concentrated in a restricted region of the plastid. The dense material we show in Figure 21 may be relevant to this finding. Further work is in progress to localise DNA ultrastructurally within the plastid.

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


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2.5% glutaraldehyde in 0.075M sodium cacodylate buffer (pH 7.2) at room temperature, in some cases with the addition of 1% tannic acid (final pH 7.2). Cells were washed x3 in cacodylate buffer, then concentrated as above into pellets which were postfixed in 1% osmium tetroxide (1 h), briefly washed in buffer, then block stained for 1 h in 1% aqueous uranyl acetate. Pellets were then dehydrated in an acetone series and embedded in Medium TAAB resin. Serial sections were cut at a thickness of 85 nm on a Reichert Ultracut E ultramicrotome, mounted on slotted grids covered with a thin film of pioloform, and contrasted with uranyl acetate and lead citrate. Depending on orientation and external shape, 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 at x12,000 and printed at x53,000 for survey reconstructions, and at x40,000 (printed at x120,000) for more detailed reconstructions. Micrographs were traced on to acetate sheets, and reconstructed graphically from these. For detailed morphology, thinner (50 nm) sections were also prepared and examined. In some cases serial sections and thinner single sections were tilted using a goniometer stage and photographed at 0° and 12° at x100,000, then viewed with a stereoscope, to analyse complex membrane configurations and other internal details. For comparison with Plasmodium fa/ciparum, electron microscopy was carried out on specimens of Plasmodium knowlesi prepared for a previous study (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 for St.Thomas' Hospital for supporting this study. Sanjeev Krishna is a Wellcome Trust Senior Research Fellow in Clinical Science. The authors are also indebted to the Electron Microscope Unit at UMDS (Guy's site) for photographic assistance, and to the Medical Research Council of the UK and Professor B. Boycott for provision of serial sectioning facilities.

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