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Home , Dactylosoma, Piroplasmida, Syncytium, Theileria, Theileria parva

Journal of Science 101, 109-123 (1992) 109 Printed in Great Britain © The Company of Biologists Limited 1992

How individual cells develop from a syncytium: merogony in pan/a ()

MICHAEL K. SHAW and LEWIS G. TILNEY*

International Laboratory for Research on Animal Disease, P. O. Box 30709, Nairobi, Kenya

*Present address: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

Summary

The central problem for during mero- development of an orderly array of tubules that gony is how to form numerous individual, uninucleate originate from the apical end and progressively form a merozoites from a syncytial schizont so that each longitudinal basket enclosing first the complex, merozoite contains a single nucleus and a prescribed then the mitochondria and , and finally the assortment of organelles. The way T. parva packages all nucleus. the requisite organelles into free merozoites is by binding The process of merogony is compared to sporogony these organelles to the nuclear envelope, which in turn within the salivary gland and with the differen- becomes associated, both directly and through the tiation of the intra-erythrocytic piroplasm stage. Be- rhoptry complex, with the schizont plasma membrane. cause all three processes occur by a morphologically Formation of the merozoites occurs in a synchronous similar mechanism, the possibility that the parasite uses manner by a budding process. The merozoites develop a single cassette of genes to perform each of these similar with the rhoptry complex at the apical end by the processes is discussed. progressive, outward evagination of the schizont plasma membrane. This evagination of the plasma membrane is Key words: Theileria parva, merogony, merozoite associated with, and presumably induced by, the formation, from a syncytium.

Introduction daughter cell acquires at least one copy of each organelle, there are examples of unequal cell division In general eukaryotic cells cannot form cell organelles (e.g. during early embryonic differentiation) or cell de novo and require the presence of pre-existing copies divisions in which certain cellular components become within the cells either to act as templates or from which selectively segregated into only one daughter cell (e.g. new organelles can develop by growth and . In the preferential localization of P granules into only one the case of specialized organelles (e.g. secretory daughter cell during early embryogenesis in Caenorhab- granules or specialized invasive organelles such as ditis elegans, see Strome and Wood, 1983). It is thought , microspheres etc.) that appear to be made de that this asymmetrical segregation of cell organelles novo, the cell must, however, contain the requisite involves the cytoskeleton. organelles from which to synthesize these specialized There is a third category, which is the antithesis of the cellular inclusions. Thus, the mechanism(s) whereby an unequal distribution of cellular components and is of organism distributes its cell organelles into daughter particular relevance to the present study. This is the cells during cell division is a fundamental problem in problem of how organisms are able to produce cells cell biology. In higher eukaryotic cells there appear to which contain either a fixed number of cell organelles or be enough copies of most cell organelles so that random a selected range of organelles (e.g. of many distribution alone should ensure that at least one copy organisms). A particularly good example is the sperm of each organelle would be present in each daughter cell of where ribosomes are after . In the case of organelles occurring in excluded from the mature sperm by a particular gene low copy numbers such as the Golgi complex and the product (see Ward, 1986). Of particular interest to us is , these organelles fragment, the problem of how syncytia become cellularized and prior to cell division, into numerous vesicular struc- produce large numbers of individual cells containing a tures, which then become randomly distributed be- precise and identical set of organelles. tween the daughter cells. Even though there may be no Theileria parva is a tick-borne protozoan parasite that special mechanism(s) required to ensure that each causes East Coast fever, an acute and often fatal disease 110 M. K. Shaw and L. G. Tilney

must be the appearance of different molecules on the Sporogony in cell surface, related to the specificity of the subsequent Tick saBvary Merogony in host cell. gland Lymphoblasts In the present study we have described in detail the process of merogony in T. parva to determine how Sporozoites Lymphocyte uninucleate cells containing a fixed complement of organelles are formed from a syncytium. We will then relate these observations to what is known about the Schlzont similar and related processes of sporogony within the tick salivary gland (Fawcett et al. 1982, 1985) and the generally more limited development of the intra- Sporoblast erythrocytic piroplasm stage (Conrad et al. 1985, 1986; Fawcett et al. 1987). Until now there is only one brief Merozoltes and rather incomplete report describing the process of merogony in Theileria (see Schein et al. 1978).

Materials and methods

Erythroeyte Parasites and cells Piroplasm Theileria parva (Muguga stock) and the tick, differ errtiation appendiculatus maintained in the Tick Unit at ILRAD, were used. were infected as nymphs by feeding them on cattle Fig. 1. A highly simplified drawing of the life cycle of infected with ground up tick stabilate (3087), after which the Theileria parva. There are three stages (at sporogony, engorged nymphs were maintained at 23-25°C and 80% merogony and at the piroplasm stage) in which the parasite relative humidity and allowed to moult to the adult stage. undergoes karyokinesis without cytokinesis resulting in the Most of our observations were made on samples of formation of a syncytium. To continue its life peripheral blood and lymph node cells collected from cycle the parasite has to form uninucleate cells from these stabilate-infected cattle. These cattle had very high piroplasm syncytia. For a detailed account of the life cycle of T. parasitemia (up to 70%) and low (<20%) packed cell parva, including the sexual stages within the tick gut, see volumes. Small samples (e.g. 5-10 f.A) of peripheral blood Norval et al. (1991). collected into heparin were fixed immediately for electron microsocpy as described below. Lymph node cells were aspirated from the prescapular lymph node into either RPMI in cattle, which is endemic in large areas of East and 1640 culture medium or directly into one of the fixatives Central Africa (see Norval et al. 1991). The life cycle of described below. T. parva is shown diagrammatically in Fig. 1. The Observations were also made on in vitro cultures of parasite is transmitted to cattle when the tick, Rhipi- schizont-infected lymphoblastoid cells. Bovine peripheral cephalus appendiculatus, feeds on a susceptible host. blood lymphocytes (PBL) were obtained from the defibri- Within the life cycle of T. parva there are three stages at nated blood of uninfected cattle (Lalor et al. 1986), isolated which the parasite undergoes a period of extensive on Ficoll-Hypaque (Pharmacia, Sweden) gradients, washed growth involving nuclear division without cytokinesis, three times in Alsever's solution and resuspended in RPMI prior to sporogony, merogony and to a lesser extent, at 1640 culture medium with 10 mM Hepes buffer supplemented the piroplasm stage (Fig. 1). This results in all three with 10% heat-inactivated foetal bovine serum (Gibco, stages in the formation of a multinucleate syncytium Paisley, Scotland, UK). Bovine PBLs were infected in vitro (Fig. 1). From these syncytia the parasite, in order to with T. parva by incubation with sporozoites derived from salivary glands of infected adult ticks fed on a rabbit for 4 days continue its life cycle, needs to form uninucleate cells (Brown, 1987). The infected cells were maintained in RPMI (the sporozoites and merozoites) each of which contain, 1640 culture medium with 10 mM Hepes buffer supplemented as a minimum, a nucleus and nuclear envelope, at least with 10% heat-inactivated foetal bovine serum, 5xlO~5 M one , ribosomes and the appropriate 2-mercaptoethanol, 2 mM L-glutamine and 50 ng ml"1 secretory organelles required by the parasite to invade gentamycin. the next host cell. Merogony was observed in in vitro infected PBLs as early as Until now individual investigators have studied day 10 post-infection and low levels (10-20%) of merogony separately either sporogony (Fawcett et al. 1982,1985), were observed throughout the period of observation to day merogony (Schein et al. 1978) or the piroplasm stage 48. (Conrad et al. 1985, 1986; Fawcett et al. 1987) in Theileria, and no one has taken an overview of all three Electron microscopy stages. As T. parva undergoes the same or a very similar Samples were fixed in suspension by the addition of an equal process three times during its life cycle it would seem volume of either (Method 1) a mixture of 2.5% glutaralde- hyde, 2% formaldehyde and 0.01% picric acid in 0.1 M reasonable to predict that the parasite might use the phosphate or cacodylate buffer (pH 7.2), or (Method 2) in a same mechanism to develop uninucleated cells contain- freshly prepared solution containing 1% glutaraldehyde (from ing a similar set of organelles from a syncytium. The an 8% stock solution supplied by Electron Microscope only major difference between these life cycle stages Sciences, Fort Washington, PA, USA), 1% OsO4 and 0.05% Cellularization in Theileria parva 111

M phosphate buffer (pH 6.2). In the case of the first fixative, bers of membrane-free ribosomes and some small samples were fixed at room temperature (22°C) for 2 h, clusters of polysomes. These ribosomes measure be- pelleted, post-fixed in buffered, 1% OSO4 for 60 min, washed, tween 20 and 25 nm in diameter and are, therefore, and "en bloc" stained with aqueous 0.5% uranyl acetate for 6- somewhat smaller than those of the host cell. In the 16 h. In the case of the low ionic strength glutaraldehyde- osmium tetroxide combined fixative (Method 2), fixation was early stage of merogony, ribosomes of comparable size carried out on ice (0-4°C) for 40-45 min after which the to host cell ribosomes become attached to the newly samples were pelleted, washed several times in distilled water formed, rough endoplasmic reticulum. Thus the differ- to remove excess phosphate and then "en bloc" stained with ence in size of the schizont and host cell ribosomes may uranyl acetate as described above. be due to the schizont ribosomes existing as ribosomal The samples were dehydrated with either ethanol or subunits and not fully assembled ribosomes. If this is acetone and embedded in an Epon-Araldite mixture. the case, then it seems reasonable to conclude that the Ultrathin sections (50-70 nm thick) were collected on parasite is engaged in only a minimal amount of protein uncoated copper grids, double stained with aqueous uranyl synthesis, possibly involved in the production of more acetate and lead citrate, and examined in a Zeiss EM 10A ribosomal subunits, and is relying on the host cell for electron microscope. the majority of its metabolic and synthetic require- ments. Apart from numerous ribosomes and occasion- Results ally some small membrane-bounded vesicles, the schizont contains very few other organelles. The purpose of merogony is the production, from a In particular, no smooth or rough endoplasmic reticula multinucleate syncytial schizont, of a large number of or anything resembling a were ob- individual merozoites, each of which contains a precise served in the schizont cytoplasm. collection of organelles. The following description of merogony in T. parva is based on the examination of a Early events in merogony large number of sections of both in vitro cultured The initial events in merogony are characterized by a material and material obtained from the lymph nodes of series of structural and organizational changes within cattle having very high piroplasm counts. Material fixed the schizont that apparently occur either simultaneously with a hypotonic fixative (Method 2) did not provide or in rapid succession. These changes include the optimal preservation of the material, but proved most extensive elaboration of the nuclear envelope and the revealing because much of the cytoplasmic matrix was appearance of endoplasmic reticulum, the formation of extracted revealing many details which are not observ- an obvious external coat on the outer surface of the able following more conventional fixation (Method 1). schizont plasma membrane, changes in the schizont From a wide range of images of both conventionally and nucleus, the appearance of rhoptries and the associ- hypotonically fixed material we have reconstructed the ation of mitochondria with the schizont nuclei. stages of merogony that culminate in free merozoites. Appearance and formation of endoplasmic The schizont prior to the onset of merogony reticulum The schizont lies free within the host cell cytoplasm and A major change in the schizont cytoplasm is the is surrounded by a plasma membrane that has no development and elaboration of both rough (RER) and obvious outer surface coat (Fig. 2A,B). Micropores, smooth (SER) endoplasmic reticulum (Fig. 3A). In thought by earlier investigators to be involved in the particular, the appearance of stacks or parallel arrays of uptake of host cell components, are present in the outer RER was commonly observed in schizonts in the initial plasma membrane at frequent intervals around the phase(s) of merogony. Because in the same section we schizont surface. The micropores measure about 50-80 often see extensive elaboration of the outer membrane nm in diameter and up to 150 nm in depth. of the schizont nuclear envelope (Fig. 3B), it seems The schizont nuclei are scattered randomly through- reasonable to assume that these elaborations give rise out the cytoplasm, have a uniform, granular appear- to the endoplasmic reticulum. At this stage significantly ance and exhibit no differentiation into areas of more ribosomes are attached to the outer nuclear dispersed and condensed chromatin. The nuclei are membrane than were seen prior to the onset of surrounded by a typical nuclear envelope (Fig. 2C) with merogony. nuclear pores. The nuclear pores measure between 60 The appearance of both RER and SER within the and 70 nm in diameter and are therefore slightly smaller schizont at this early stage of merogony is indicative of in diameter than the nuclear pores present in the host an increased synthetic activity by the schizont that is in cell nuclear envelope (85-95 nm in diameter). Occasion- keeping with the formation of a schizont surface coat ally ribosomes were found attached to the cytoplasmic and accessory secretory organelles. face of the outer nuclear membrane. The schizont mitochondria are usually found in pairs Surface coat and occur randomly throughout the cytoplasm. They The outer surface of the schizont becomes covered by a consist of an outer and inner membrane but lack cristae. prominent, 20-25 nm thick, coat (Fig. 3C). This coat In the normal schizont the nuclei and mitochondria are covers the entire surface of the schizont, persists not associated. throughout merogony and is present on the surface of The schizont cytoplasm contains conspicuous num- the mature merozoites. Although the exact appearance 112 M. K. Shaw and L. G. Tilney

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Fig. 2. (A) Low power electron micrograph of part of a 7". porva-infected lymphocyte showing the presence of a multinucleate schizont (Sc). HN, host . (B) High magnification micrograph of the schizont plasma membrane showing the absence of any obvious surface coat. Sc, schizont cytoplasm. HC, host cell cytoplasm. (C) Part of a T. parva schizont showing a schizont nucleus (N) and a mitochondrion (M). The schizont nucleus is surrounded by a typical nuclear envelope with ribosomes attached to the cytoplasmic face of the outer membrane (arrows). Note that the nuclear contents have a uniform, granular appearance, and that the mitochondrion (M) consists of an outer and inner membrane without any cristae. Cellularization in Theileria parva 113

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Fig. 3. (A) Low magnification micrograph of a section through a T. parva schizont at an early stage in merogony. The early stages of merogony are characterized by: (i) the appearance of extensive arrays of rough and smooth endoplasmic reticulum (RER and SER respectively), (ii) the condensation of the nuclear chromatin and the localization of nuclei (N) at the periphery of the schizont, (iii) the appearance within the schizont cytoplasm of electron dense rhoptry-like inclusions (arrows). (B) Schizont nucleus showing the apparent formation of smooth endoplasmic reticulum (SER) from the outer nuclear membrane. (C) A pair of opposed schizont plasma membranes showing the presence of a 20-25 nm thick surface coat. Sc, schizont cytoplasm.

varies depending upon the fixatives used, this coat condensation of the chromatin and the close apposition material often has a distinct "peg-like" substructure (cf. of the membranes of the nuclear envelope around part Bannister et al. 1986). of the nucleus. In many schizont nuclei the condensed chromatin also became localized along the inner Nuclear changes nuclear membrane in the region where the nuclear The initial changes in the schizont nuclei are a membranes were closely apposed (Fig. 4A). This close 114 M. K. Shaw and L. G. Tilney

Fig. 4. (A) Schizont nucleus showing the condensation and marginalization of chromatin and the close apposition of the two nuclear envelope membranes around part of the nucleus. In this micrograph the nucleus is now located close to the outer schizont plasma membrane with the specialized portion of the nuclear envelope adjacent to the schizont membrane. Note also the presence of two schizont mitochondria (M) closely associated with the non-specialized part of the nuclear envelope. (B) High magnification micrograph of the specialized portion of the schizont nuclear envelope. In this region the apposed leaflets of the individual nuclear envelope membranes fuse to produce a pentalaminate structure. This specialized portion of the nuclear envelope is separated from the schizont plasma membrane by a 15-20 nm gap. Note also the presence of the surface coat on the outer face of the schizont plasma membrane (arrows). apposition of the nuclear envelope membranes was and there appears to be no morphological specializ- most apparent in hypotonically fixed material (Method ation^) associated with the outer plasma membrane in 2). It is almost certainly not an artifact because even in the regions where the nuclei attach. sections of material showing extensive swelling of the remaining nuclear envelope and looking extremely Development of the rhoptry complex "poorly fixed", the two membranes at the point of Concurrent with the above changes, electron dense apposition still remained closely apposed. rhoptry-like structures appear within the schizont We initially observed nuclei with both condensed cytoplasm. The site of origin of the rhoptries is unclear. nuclear material and a modified nuclear envelope In a small number of schizonts, profiles of SER within the central regions of the schizonts. However, it containing material of similar density to the contents of is apparent that concomitant with, or immediately mature rhoptries were observed (Fig. 5A). Arising following, these initial changes some nuclei become from the SER were numerous smaller, flask-shaped relocated to contact the periphery of the schizont (Fig. structures that resembled the rhoptries seen in mature 4A). Here the nuclei are invariably orientated with the merozoites. Thus the rhoptries appear to arise from the modified portion of the nuclear envelope closely SER. associated with the schizont plasma membrane (Fig. Whereas newly formed individual rhoptries could be 4B). Although the nuclear envelope does not fuse with found throughout the schizont cytoplasm, they were the outer plasma membrane, the association of the most frequently observed associated in small clusters nucleus with the outer membrane is intimate, with the with one margin of the schizont nuclei in close membranes being separated by a uniform gap of 15-20 proximity to a dense plaque structure present in the run. Around the remaining regions of the schizont nuclear envelope (Fig. 5B). These dense plaques, which nuclear envelope the two component membranes are span the nuclear envelope, closely resemble the spindle separated by a variable and much wider spacing (Fig. pole bodies described by Schein et al. (1978) and 4A). Fawcett et al. (1985). These bodies are thought to play a The process whereby the schizont nuclei migrate to role in nuclear division. Prior to the onset of merogony the periphery of the schizont and become preferentially the spindle pole bodies often have orientated with respect to the outer plasma membrane associated with them. is unclear. We have been unable to find any obvious When the nuclei are close to, or have formed their structural entities that could account for the movement tight association with, the schizont plasma membrane, Cellularization in Theileria parva 115

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Fig. 5. (A) Part of a conventionally fixed (Method 1) T. parva schizont showing profiles of smooth endoplasmic reticulum (SER) containing material of a similar electron density and consistency to the mature merozoite rhoptries. (B) A schizont nucleus located close to the schizont plasma membrane showing the presence of rhoptries (R) associated with both the nuclear envelope and with fibrous material that projects towards the schizont membrane. Note that the specialized portion of the schizont nuclear envelope is adjacent to the schizont plasma membrane (arrowheads). (C) High magnification micrograph of a portion of the schizont nucleus showing the attachment of the rhoptry complex with the spindle pole body (SP) and through a fibrous structure with a dense structural specialization associated with the schizont plasma membrane (arrows). we can find fine fibrous structures that join the nuclear apical complex of the merozoite, develop beneath the dense plaques to structural specializations which de- outer plasma membrane and consist of a plate-like velop beneath and are usually associated with the structure composed of a central, inwardly projecting plasma membrane (Fig. 5C). The rhoptries are usually peg, and a number of concentric rings of either denser associated with these fibrous structures. In a single material or membranous tubules lying parallel to the section there can be two or even three of these rhoptry- schizont plasma membrane (Fig. 6). These membra- associated fibrous connections between the nuclei and nous tubules become flattened to form a slightly the plasma membrane. concave disc or annulus which, in favourable images, The structural specializations, that form the modified can be seen to be composed of two closely apposed 116 M. K. Shaw and L. G. Tilney

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Fig. 6. High magnification micrographs, in three different planes of section, of the structural specialization associated with the schizont plasma membrane to which the rhoptry complex ultimately attaches. (A) is a section cut perpendicular to the attachment complex and shows the central dense peg-like structure (arrow) which projects from the plasma membrane and to which the individual rhoptries attach, and a number of membranous tubules. (B) is an oblique, en face section through the complex showing that the structure is composed of a central dense peg and a series of concentric dense rings. (C) is a slightly oblique section showing a group of six rhoptries and the two closely apposed membranes which form part of the much reduced inner membrane complex (arrows). membranes and subsequently forms the very much schizonts and developing merozoites is so electron reduced inner membrane complex in the mature dense (Fig. 7A) that information relating to the merozoite. The fine fibrous material connecting the intermediate stages in merogony was extremely difficult rhoptries and the nuclear envelope with the schizont to discern. Accordingly, we have concentrated on plasma membrane attaches to the inwardly projecting examining material fixed at low ionic strength (Method peg in the central region of this subplasmalammel 2) which "washes out" much of the soluble, electron specializations. The whole of this structural specializ- dense cytoplasmic constituents (Fig. 7B). By examining ation is separated from the schizont plasma membrane large numbers of sections we have been able to by a uniform 10-20 nm gap. reconstruct the probable sequence of events occurring during the formation of merozoites from the syncytial Mitochondria schizont (Fig. 8). At some point during the early stages in merogony the The earliest identifiable protrusion from the surface mitochondria become closely associated with the outer of the schizont occurs at the point where the rhoptry membrane of the nuclear envelope, at a point where the complex attaches to the inwardly projecting peg membranes of the nuclear envelope, are not closely structure and ring of flattened membranous tubules apposed (Fig. 4A). The adjacent leaflets of the closely associated with the schizont plasma membrane. mitochondrial and nuclear membranes do not fuse, Just lateral to the tip of the protrusion, which contains however, and are separated by a small, 10-25 nm wide the rhoptry complex and the flattened membranous gap- tubules of the much reduced inner membrane complex, Thus, during the early events in merogony prior to appear some tubular structures (Fig. 8A,B), which the formation of the free mature merozoites, the form a regular arrangement radiating out from the rhoptries, nucleus, mitochondria and outer plasma point at which the rhoptry complex attaches to the membrane have become interconnected in an orderly plasma membrane. These tubules are sometimes flat- and reproducible manner. tened in cross section and measure between 30-65 nm in diameter, and are, therefore, somewhat larger in size Later events in merogony: the "budding" of than similar sub-plasmalammel microtubules described merozoites from the syncytial schizont in other Apicomplexa. As the tip of the developing Up to this point in merogony the schizont is still a merozoite protrudes further from the surface of the multinucleate syncytium, although the constituent or- schizont, these radiating tubules form a basket enclos- ganelles present in a mature merozoite are intimately ing the rhoptry complex. The lateral walls of the tubules associated with both themselves and with the schizont are closely applied to the membrane forming the plasma membrane. Thus, the next step in the process is protrusion. As the developing merozoite buds further the formation of individual unicellular merozoites from from the surface of the schizont the tubules enclose the the syncytial schizont. nucleus, passing between the nuclear envelope and the In conventionally fixed material the cytoplasm of the outer plasma membrane. High resolution images of Cellularization in Theileria parva 117

Fig. 7. (A) Micrograph of a conventionally fixed (Method 1) schizont showing merozoites in the process of budding from the main syncytial body. Note that the parasite cytoplasm is extremely electron dense and that it is therefore difficult to discern many of the structural details of the process of budding. (B) Micrograph of a schizont in the process of merozoite budding fixed with low ionic strength glutaraldehyde-osmium tetroxide combined fixative (Method 2). cross sections through these tubules show that they plasma membrane. Whether this is due to parasite- make a direct contact with the outer leaflet of the induced lysis or to the physical presence of large nuclear envelope but are separated from the schizont numbers of parasites within the cell is not known. plasma membrane by a small 10-20 nm gap (Fig. 8B). The free merozoite, which is covered by a distinct 20- Budding of the merozoite proceeds in an orderly 25 nm thick surface coat, measures between 1 and 2 /*m manner with the modified portion of the nuclear in diameter and contains a single eccentric nucleus, envelope invariably being orientated towards the basal between 3 and 6 rhoptries, one or two mitochondria, portion of the developing merozoite (Fig. 8C,D). A microspheres and some cytoplasm containing free possible reason for the close association of the tubule ribosomes. Unlike other genera of Apicomplexa, the with the modified portion of the nuclear envelope is that merozoites of T. parva have no clearly defined apical the basket of tubules, by following the contour of the complex and a conoid or similar apical structure is nucleus, ensures the formation of a rounded merozoite. absent. The rhoptries attach to an inwardly projected The tubules continue around the basal portion of the peg at the opposite pole to the nucleus, and are nucleus. At the last stage the ends of the tubules associated with a very much reduced inner membrane terminate in some electron dense material which in complex composed of two tightly apposed membranes. grazing section appears as a ring of dense material (Fig. No or micropore was found, even though 8E-G). It seems reasonable to suspect that this ring numerous are present in the schizont. We structure is involved in the final separation of the did not observe the tubular basket in the free, mature merozoite from the syncytial schizont. Thus the basket merozoites but because of the density of the merozoite of tubules that extends from the rhoptry complex to the cytoplasm it is difficult to be certain that this is true in all basal ring encloses a cytoplasmic volume beneath the mature merozoites. As we did not see any microspheres rhoptry complex that contains both the mitochondria or obvious microsphere precursors in the schizont still attached to the nuclear envelope and some free cytoplasm or in the earlier stages of merozoite budding ribosomes (Fig. 9). these organelles would seem to be formed at a very late While merozoite budding from the syncytial schizont stage in merozoite formation. Furthermore, the num- is a highly synchronous process it is our impression that ber of microspheres present in the mature merozoites two or more waves of budding may occur. We have was significantly less than observed in T. parva observed frequently free merozoites within a host cell in sporozoites. which newly budding merozoites are forming from the Whilst the above description was the most commonly schizont. At the end of merogony the host cell contains encountered method of merozoite budding, in some large numbers of mature merozoites and usually a schizonts merozoite budding had started prior to the residual schizont body which contains a number of final karyokinesis resulting in a number of merozoites nuclei, rhoptries, mitochondria and various amounts of budding from a central nuclear mass, a similar situation endoplasmic reticulum. Eventually the mature mero- to that described by Schein et al. (1978). The stages in zoites are liberated by the breakdown of the host cell merozoite budding are, however, essentially the same 118 M. K. Shaw and L. G. Tilney Cellularization in Theileria parva 119

Fig. 8. (A and B) High magnification electron micrographs sporozoites from the highly branched labyrinthine of the earliest stage in merozoite budding. (A) Beneath the sporoblast (see Fawcett et al. 1982, 1985). initial protrusion, which consists of the peg to which the rhoptries attach and the membranous cisternal structure (arrowheads) that forms the very much reduced inner membrane complex of the mature merozoite, are a series Discussion of tubular structures (arrows) that are closely associated with the schizont plasma membrane and extend from the Cellularization from a syncytium: the nucleus plays a apex around the lateral margins of the nucleus (B). (C and pivotal role D) As merozoite budding proceeds, the rhoptry complex The central problem for T. parva during merogony is (R) is always located at the apical end of the developing how to form many individual uninucleate cells from a merozoite. Arising from the apical region of the bud the syncytium. Moreover, because eukaryotic cells cannot tubular structures (arrows) extend basally forming a form organelles de novo, each resultant individual cell sublamellar basket progressively enclosing the rhoptry must contain all the organelles or organelle precursors complex, mitochondria and nucleus. The nucleus (N) is that the parasite requires, not only for the next stage, always situated at the base of the bud with the specialized portion of the nuclear envelope (arrowheads) associated but for all subsequent stages of its life cycle. For with the most basal portion of the budding merozoite. (E- example, although ribosomes will assemble spon- F) Final stages of merozoite budding. The tubular taneously from their component parts they cannot be structures continue around to the basal end of the nucleus made without the presence of pre-existing ribosomes to (arrows in E) and terminate in a ring structure at the most synthesize the required proteins. Thus, the mature basal constriction of the bud (arrowheads in F). merozoite, which has a distinctive surface coat, must (G) Grazing section through the basal region of a budding contain at a minimum a nucleus and nuclear envelope, a merozoite showing the ends of the tubules terminating in mitochondrion, ribosomes and the rhoptry complex to an electron dense ring structure (arrows). allow for entry and establishment in the next host cell. We have shown that the way T. parva controls what organelles are destined to be included in the merozoites is by coupling these organelles to the nuclear envelope, that in turn associates both directly and indirectly to the schizont plasma membrane. Thus, as shown diagram- matically in Fig. 10, a portion of the two membranes of the nuclear envelope become tightly apposed and in turn become associated with the schizont plasma membrane. The rhoptry complex associates with the nuclear spindle pole bodies and subsequently with a newly formed specialization situated immediately be- neath the plasma membrane. The mitochondria bind to an unspecialized portion of the nuclear envelope, to which ribosomes also attach. All that is now required is a mechanism to bud off individual nuclei with their associated array of organelles from the syncytial schizont. This appears to be accomplished by the progressive evagination of the plasma membrane in association with an array of tubules that originate from the apically situated rhoptry complex. These tubules elongate and enclose the nucleus with its attached mitochondria in a manner resembling an array of ribs. In retrospect, it seems obvious that in order to have a precise assortment of organelles sequestered into each Fig. 9. Three-dimensional drawing of a budding merozoite just prior to separation from the syncytium showing the merozoite, the organelles must self-associate in some basket of tubules enclosing the rhoptry complex (R), a way. Furthermore, since the nucleus dominates in size mitochondrion (Mito), ribosomes and the nucleus (N). and is limited by a membrane that has inserted in it at least one spindle pole body to which other objects could be attached, it would be a reasonable candidate to begin as described above. The only difference is that as the this self association. It is likely that a similar mechanism merozoite finally buds away the nucleus has to separate may exist in other cases where cellularization from a from the nuclear mass, which remains within the syncytium occurs, as will be discussed in the next schizont. section. Occasionally large, highly branched schizonts under- going merogony were observed in the in vitro cultures. How cellularization proceeds from syncytia in other In these cases the merozoites, that develop in an organisms: again the nucleus appears to play a identical manner as described above, bud from the ends pivotal role of the branches in a situation similar to the formation of Although cellularization from a syncytium is not a 120 M. K. Shaw and L. G. Tilney

Fig. 10. Diagrammatic summary of the process of merozoite formation in Theileria parva. 1. Schizont prior to the onset of merogony. 2. The earnest events in merogony include the appearance of both RER and SER, a prominent surface coat, rhoptries and changes in the appearance of the nuclei. 3. The schizont nuclear envelope membranes become closely apposed around part of the nucleus and this specialized portion becomes preferentially associated with the schizont plasma membrane (see insert). The bodies become associated with the nuclear spindle pole bodies and are attached to a sublamellar specialization beneath the schizont plasma membrane. 4-6. The merozoites bud from the surface in a synchronous manner with the rhoptry complex at the apical end. An array of tubular structures arising from the apical end forms a sublamellar basket enclosing the rhoptry complex, mitochondria and the basally located nucleus.

common phenomenon in biological systems, it never- this process of cellularization involves a complex theless occurs in, for example, insect embryogenesis, network of interacting cytoskeletal proteins (see Miller cellularization in and in spermato- et al. 1989). A similar process involving the cytoskel- genesis. In all cases, when cellularization occurs, a eton occurs in the process of cellularization of the common feature is the central role played by the endosperm of higher plants (Fineran et al. 1982). nucleus in orchestrating the non-random distribution of During spermatid differentiation from a syncytium, the organelles and other inclusions as it does in merogony nucleus with its limited but highly selective array of in T. parva and in other apicomplexan parasites. For associated organelles buds away from the mass of example, during insect embryogenesis there is a series cytoplasm leaving an anucleate residual mass (Baccetti of rapid nuclear divisions without cytokinesis giving rise and Afzelius, 1976). Again the nucleus plays a pivotal to the syncytial blastoderm in which a proportion of the role in determining what organelles and inclusions will nuclei migrate outwards in a stepwise manner to be present in the mature spermatid by binding to the become aligned beneath the syncytial surface mem- requisite organelles in a manner similar to that brane. Each peripherally located nucleus is associated described in merogony (Tilney, 1976). with a of structured cytoplasm. By the subsequent imagination of the outer plasma membrane Merogony, sporogonyand piroplasm differentiation in these nuclei with their associated cytoplasm form a T. parva all occur by a similar mechanism cellular layer (see Foe and Alberts, 1983). The whole of It is our contention that merogony, sporogony and Cellularization in Theileria parva 121 piroplasm differentiation, all processes involving cellu- in part to the difficulties of obtaining suitable material, larization from a syncytium, have or undergo similar if either because of the low densities of parasites obtained not identical intermediate steps resulting in mature from experimental animals or due to the lack of merozoites, sporozoites and piroplasms all of which alternative tissue culture systems. contain a similar set of organelles packaged in an This report not only describes the process of identical manner. In particular, the end products of merogony in T. parva in detail, but it also attempts for merogony, sporogony and piroplasm differentiation are the first time to relate ultrastructural events that occur all approximately 1-2 pcm in diameter. In each case, the at merogony, sporogony, and at the piroplasm stage; zoite nucleus is eccentrically located with a modified three stages that each involve cellularization from a portion of the nuclear envelope closely apposed to the syncytium. As already mentioned, there are remark- outer plasma membrane. The mitochondria are associ- able similarities in the structure of the merozoites, ated with the non-specialized portion of the nuclear sporozoites and piroplasms and in the process of their envelope, to which ribosomes also attach. At the pole formation from syncytia. Because there are these opposite to the nucleus, the rhoptries are attached to a repeating patterns in the life cycle of Theileria, it is submembrane specialization, although in T. parva, unlikely that the three processes evolved indepen- unlike other Apicomplexa (e.g. , see dently. What would appear more likely is that the Nichols and Chiappino, 1987), the merozoites and developmental pattern of cellularization has been sporozoites do not possess a clearly demarcated apical evolved once, and that the same process is used complex. Because of these structural similarities be- repeatedly by the parasite throughout its life cycle. The tween the different developmental stages in T. parva, it most logical way of repeatedly undergoing the same seems reasonable to suspect that the mechanisms of developmental process is for the organism to use the cellularization from the respective syncytia will be same cassette of genes with only the replacement of a similar to the process of merogony as described above. few genes each time to allow for minor differences in, Although the information on sporogony and piroplasm for example, the composition of the surface coat. differentiation in the literature is less than complete, Furthermore, the sporozoites and merozoites of T. what is known is consistent with the notion that during parva enter and establish within their respective host sporogony the process whereby the rhoptry complex cells in a morphologically similar manner (see Shaw et initially associates with the spindle pole bodies and al. 1991; Shaw, unpublished observations), indicating subsequently with a sub-plasmalemmal specialization that the parasite probably uses an identical or a very (see Fig. 26, Fawcett et al. 1985) is identical to what we similar method of entry into the different host cells. A have found in merogony. Similarly, in both sporogony logical conclusion from these observations would be and merogony the or microspheres appear that T. parva and by extension many, if not all, to be formed either just before or immediately after the members of the Apicomplexa which undergo similar if completion of zoite formation (Fawcett et al. 1985). not identical developmental processes a number of The only major difference between merogony and times during their life cycles, use a single cassette of sporogony in T. parva is the apparent absence in genes to perform each of these similar and repeated sporogony of a basket of tubules similar to the ones processes. Thus the parasite may be able to undergo a formed during merozoite budding. As we have been number of seemingly disparate developmental pro- unable to see this basket of tubules in schizonts cesses numerous times throughout its life cycle without undergoing merogony fixed in the same manner as the requirement of an extensive genome. Fawcett et al. (1982, 1985) it is likely that this is not a real difference in the two processes. Similarities between Theileria and related protozoan parasites, and how by knowing their comparative cell How the life cycle of Theileria may have evolved and biology one can progress more rapidly in what controls it understanding any one of the Up until now investigators have concentrated their The protozoan , Apicomplexa, includes a large attention on specific stages in the life cycle of a single number of parasitic organisms of both medical and parasitic protozoan. There are several reasons for this, veterinary importance. The processes of sporogony, not the least of which is the problem of obtaining merogony and gametogenesis are a common feature of infected material from more than one or two stages of the life cycle and all involve the formation of individual the life cycle that invariably involves two separate cells from a multinucleate syncytial body. Furthermore, hosts. For example, T. parva is a tick-borne protozoan each newly formed cell contains a single nucleus and a parasite of cattle and in each host different cells are precise complement of organelles. Therefore, it is not infected at different times (i.e. in the tick intestinal too surprising that the processes of sporogony, mero- epithelial cells and then selective cells in the salivary gony and gametogenesis appear to proceed in a similar gland, in the cow a subpopulation of lymphocytes and manner. From the various descriptions of the individual then red blood cells). Thus to compare what is processes in a wide range of Apicomplexa (e.g. happening in different hosts and in different host cells Vargliese, 1977; Pacherco and Fayer, 1977; Schein et al. requires many animals (both ticks and cattle) that have 1978; Wong and Desser, 1978; Dubremetz and Eisner, been infected at different times. In fact some of these 1979; Entzeroth, 1983; Fawcett et al. 1982, 1985; intermediate stages have not been well described, due Mehlhorn and Schein, 1984; Meis et al. 1985; Barta et 122 M. K. Shaw and L. G. Tilney al. 1987; Klein et al. 1988) sporogony, merogony and and McBrlde, J. S. (1989). 46-53 kilodalton from the gametogenesis all invariably proceed in a synchronized surface of falciparum merozoites. Mol. Biochem. Parasitol. 32, 15-24. manner and involve a co-ordinated sequence of events Conrad, P. A., Denham, D. and Brown, C. G. D. (1986). resulting in a large number of cells being formed Intraerythrocytic multiplication of Theileria parva in vitro: an simultaneously from a syncytium. Characteristically, ultrastructural study. Int. J. Parasitol. 16, 223-229. the co-ordinated sequence of events involves extensive Conrad, P. A., Kelly, B. G. and Brown, C. G. D. (1985). Intraerythrocytic schizogony of Theileria annulata. Parasitology 91, mitotic nuclear division followed by some degree of 67-82. condensation of the nuclear material, the formation, at Dubremetz, J. F. and Eisner, Y. Y. (1979). Ultrastructural study of an early stage, of a distinct extracellular coat (e.g. schizogony of bovis in cell cultures. J. Protozool. 26, 367- Fawcett et al. 1982, 1985; Glascodine et al. 1990; Clark 376. et al. 1989; Hamilton et al. 1988; Posthuma et al. 1988), Entzeroth, R. (1983). Electron microscope study of merogony preceding cyst formation of sp. in roe deer (Capreolus multiplication of the mitochondria, an increase in capreolus). Zeit. Parasitenk. 69, 447-456. endoplasmic reticulum and ribosomes, and the pro- Fawcett, D. W., Buscher, G. and Doxsey, S. (1982). Salivary gland of duction of various secretory organelles (e.g. rhoptries, the tick vector of East Coast fever. III. The ultrastructure of micronemes and microspheres). The formation of the sporogony in Theileria parva. Tissue and Cell 14, 183-206. individual cells from the syncytium occurs by a Fawcett, D. W., Conrad, P. A., Grootenhuls, J. G. and Morzaria, S. P. (1987). Ultrastructure of the intraerythrocytic stage of Theileria progressive budding process with the apical complex (a from cattle and waterbuck. Tissue and Cell 19, 643-655. characteristic feature of the Apicomplexa consisting, in Fawcett, D. W., Young, A. S. and Leltch, B. L. (1985). Sporogony in the majority of cases, of the polar ring and conoid Theileria (Apicomplexa: ). J. Submicrosc. Cytol. 17, complex) forming the leading pole of the developing 299-314. cell. In the merozoites and sporozoites of T. parva, the Fineran, B. A., Wild, D. J. C. and Ingerfeld, M. (1982). Initial wall formation in endosperm of wheat, Triticum aestivum: a conoid is absent, and the polar ring-inner membrane reevaluation. Can. J. Botany 60, 1776-1795. complex very much reduced as compared with the Foe, V. E. and Alberts, B. M. (1983). Studies of nuclear and zoites of other Apicomplexa. The budding of the cytoplasmic behaviour during the five mitotic cycles that precede individual cells is associated with, and presumably gastrulation in . /. Cell Sci. 61, 31-70. induced by, the development of an orderly array of Glascodine, J., Tetley, L., Tait, A., Brown, D. and Shiels, B. (1990). Developmental expression of a Theileria annulata merozoite tubules arising from the apical structure. These tubules surface . Mol. Biochem. Parasitol. 40, 105-112. form the subpellicular cytoskeleton of the developing Hamilton, A. J., Davies, C. S. and Sinden, R. E. (1988). Expression of cell. In some organisms such as T. parva, the mature circumsporozoite protein revealed in situ in the mosquito stages of merozoites have secondarily lost this cytoskeletal by the Lowicryl-immunogold technique. complex while in many others (e.g. Toxoplasma, Parasitology 96, 237-280. Klein, T. A., Akin, D. C, Young, D. G., Telford, S. R. and Butler, J. Eimeria) it is a characteristic feature of the free zoites F. (1988). Sporogony, development and ultrastructure of extrinsic and is involved in parasite motility and in the process of stages of Plasmodium mexicanum. Int. J. Parasitol. 18, 463-476. host cell invasion (e.g. Russell and Sinden, 1981; Lalor, P. A., Morrison, W. I., Goddeeris, B. M., Jack, R. J. and Russell, 1983). Thus, what is now apparent is that by Black, S. J. (1986). Monoclonal antibodies identify phenotypically being aware of how related protozoan parasites func- and functionally distinct cell types in the bovine lymphoid system. Vet. Immunol. Immunopathol. 13, 121-140. tion we can in many cases predict how a particular Mehlhorn, H. and Schein, E. (1984). The Piroplasms: Life cycle and parasite may function. sexual stages. Advances in Parasitology 23, 37-103. Meis, J. F. G. M., Verhave, J. P., Jap, P. H. K. and Meuwissen, J. H. We are grateful to Patrick Theuri and Daniel Ngugi for E. T. (1985). Fine structure of exoerythrocytic merozoite formation technical assistance, Francis Mwakima and colleagues in the of Plasmodium berghei in rat liver. J. Protozoology 32, 694- ILRAD Tick Unit for provision of sporozoites, Dr Tom 699. Dolan for providing the lymph node biopsy samples and other Miller, K. G., Field, C. M. and Alberts, B. M. (1989). 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