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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Pathogen–host interactions are modulated at multiple levels by both the pathogen and the host cell. Modulation of host cell functions is particularly intriguing in the case of the intracellular Theileria parasite, which resides as a multinucleated schizont free in the cytosol of the host cell. Direct contact between the schizont plasma membrane and the cytoplasm enables the parasite to affect the function of host cell proteins through direct interaction or through the secretion of regulators. Structure and dynamics of the schizont plasma membrane are poorly understood and whether schizont membrane dynamics contribute to parasite propagation is not known. Here we show that the intracellular Theileria schizont can dynamically change its shape by actively extending filamentous membrane protrusions. We found that isolated schizonts bound monomeric tubulin and in vitro polymerized microtubules, and monomeric tubulin polymerized into dense assemblies at the parasite surface. However, we established that isolated Theileria schizonts free of host cell microtubules maintained a lobular morphology and extended filamentous protrusions, demonstrating that host microtubules are dispensable both forthe maintenance of lobular schizont morphology and for the generation of membrane protrusions. These protrusions resemble nanotubes and extend in an actin polymerization-dependent manner; using cryo-electron tomography, we detected thin actin filaments beneath these protrusions, indicating that their extension is driven by schizont actin polymerization. Thus the membrane of the schizont and its underlying actin cytoskeleton possess intrinsic activity for shape control and likely function as a peri-organelle to interact with and manipulate host cell components.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

To understand the intricate relationship of intracellular pathogens with their host cells requires many different angles of observation. These include pathogen-dependent gene and protein expression profiling, biochemical pathway analysis and the description of immune reactions to intracellular parasitism. To complement these quantitative approaches, we examined surface structure and membrane dynamics of the apicomplexan parasite Theileria annulata by different microscopy methods inside its host cell and isolated from it.

Theileria are important intracellular parasites of ruminants in tropical and subtropical regions. Two species – T. parva and T. annulata – induce the lympho- and myeloproliferative disorders East Coast Fever and tropical Theileriosis in cattle, and thereby constitute the two most pathogenic variants (Mehlhorn et al., 1994). Pathogenesis is caused by massive, parasite-dependent amplification of infected lymphoid and myeloid cells and of their concomitant spread inside the infected host animal. Inside the cell, the parasite resides free as a syncytium (schizont) within the host cell cytoplasm, not surrounded by a parasitophorous vacuole like some other better-known apicomplexan parasites, such as Toxoplasma or Plasmodium (Mehlhorn et al., 1994; Dobbelaere and Baumgartner, 2009) but similar to Babesia (Lobo et al., 2012). Theileria parasites transform their host cells. This transformation results in continuous proliferation of the infected cells and in protection against the induction of apoptosis. The amplification of the parasite population depends on partition of syncytia onto the two daughter cells. This process is ensured by the physical association of the schizont with the host cell mitotic spindle apparatus and the co-ordination of host cell and schizont cell cycles. It is dependent on the regulated association of host cell polo-like kinase 1 (PLK1) and PLK1 activity, which position the schizont on the equatorial plane of the dividing mother cell (von Schubert et al., 2010). In infected animals, Theileria-transformed cells form new foci of proliferating cells at sites distant to the original inoculation by the tick vector. This requires migration of infected cells across tissue barriers, and such host colonization behaviour has been referred to as metastatic, in analogy to invasive dissemination of human cancer cells (Adamson and Hall, 1996; Forsyth et al., 1999; Lizundia et al., 2006). Parasite spread results from the acquired motility of infected cells, which requires co-ordinate, parasite-dependent modifications in the host cell actin cytoskeleton (Baumgartner, 2011a,b). These acquired changes in host cell behaviour depend on poorly understood interactions of Theileria schizonts with host cell components that trigger and mediate the underlying molecular modifications.

A key group of host cell molecules with activities that change after parasite elimination are protein kinases. Theileria-dependent changes in host cell kinase activities have been observed under different contexts, including anti-apoptosis, proliferation and cell migration (Galley et al., 1997; Chaussepied et al., 1998; Baumgartner et al., 2000; 2003; Heussler et al., 2002; Baumgartner, 2011b). However, parasite-dependent mechanisms of kinase activation and the substrates and effectors of these kinases remain largely unknown. Binding of several host cell proteins to the schizont surface including the I kappaB kinase (IKK) (Heussler et al., 2002), PLK1 (von Schubert et al., 2010) and tubulin (Seitzer et al., 2010) were reported in recent years. These findings suggest that the plasma membrane of the schizont could function as an interface for protein and information exchange between host cell and schizont cytoplasms. Indeed, compared with a flat membrane surface, the lobular structure of intracellular Theileria schizonts provides the parasite with a comparatively large membrane surface. The functional significance of this particular shape and the parasite intrinsic mechanism to maintain it has not yet been addressed. Due to the relative lack of information on structural and functional properties of the schizont surface inside its host cell, we investigated schizont morphology and membrane dynamics. We combined fix-cell immune-fluorescence light microscopy with high-resolution scanning electron microscopy, cryo-electron tomography and live cell imaging. The combination of these approaches has allowed us to describe surface characteristics, structural specificities and membrane dynamics of Theileria schizonts at unprecedented spatiotemporal resolution and revealed for the first time apicomplexan actin filaments in situ.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Parasites extend filamentous membrane protrusions

Intracellular Theileria schizonts reside as lobular structures inside the cytoplasm of their host cells (Fig. 1). Host cell microtubule binding to the parasite surface has been suspected to maintain lobular shape and no active schizont process for shape control has been described so far. In routine IF analysis using antibodies against T. annulata surface protein (TaSP) we detected filamentous membrane protrusions visible under standard cell culture conditions in approximately 20% of cells (Fig. 1). These membranous protrusions extend either towards the cell periphery (Fig. 1A a), the nucleus (Fig. 1A b) or – in migrating cells – towards the leading edge of the host cell (Fig. 1A c). Intracellular Theileria schizonts are closely associated with the host cell microtubule network and the microtubule-organizing centre (MTOC) (von Schubert et al., 2010), which is located adjacent to the host cell nucleus. Consistently, the confocal microscopy image of a surface-labelled parasite shows schizont localization near the host cell nucleus (Fig. 1B). The schizont shown extends two protrusions: a shorter one that appears to extend towards a position close to the host cell nucleus, possibly the MTOC (arrow), and a thin, long second protrusion (arrowheads). Latter is of considerable length (5 μm) and appears to circle the host cell nucleus (Fig. 1B). Differential interference contrast (DIC) imaging allows visualizing the intracellular parasite in intact cells (Fig. 1C). Using DIC live-cell imaging we were able to monitor movements of the schizont inside the host cell and to visualize dynamic alterations in the schizont plasma membrane. Using this technique we monitored a membranous protrusion during extension towards the host cell plasma membrane (Fig. 1C, Movie S1). Although the protrusion did not seem to attach firmly to any cellular structure, it remained extended for more than 30 min. Taken together, we found that Theileria parasites are able to generate membranous protrusions inside their host cells that extend either towards the cell periphery or towards the regions close to the host cell nucleus. The mechanical and structural properties of these protrusions and their functional significance are unknown.

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Figure 1. Intracellular parasite can extend small protrusions inside host cell.

A. Visualization of intracellular T. annulata (anti-TaSP, green), host cell actin cytoskeleton (phalloidin Tx-red) and DNA (Hoechst, blue). In 1A c, T. annulata-infected macrophages were seeded onto fibronectin, which leads to front-back polarity. Boxed areas are magnified fourfold and anti-TaSP staining is shown in inverted grey scale.

B. Confocal microscopy analysis of a parasite protrusion in the host cell cytoplasm. Arrow indicates region where schizont is in close proximity with host nucleus. Arrowheads indicate a long and thin membranous protrusion.

C. Still images of Movie S1 showing the extension of membranous parasite protrusion inside host cell. Contours of protrusions are outlined in green. Localization of intracellular parasite relative to host cell nucleus is shown schematically. Boxed areas are magnified fourfold and shown to the right.

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Tubulin polymerization on the surface of schizonts

The interaction of host cell tubulin with the membrane of T. annulata schizonts was recently investigated and punctuated alpha tubulin staining described on the schizont surface (Seitzer et al., 2010). We wondered whether host cell microtubule binding is functionally linked to the membranous protrusions on the schizont membrane. We first confirmed tubulin binding to the surface of isolated schizonts by IFM. Using an antibody directed against mammalian tubulin, we detected punctate tubulin staining on the schizont and – on some occasions – tubulin enrichment in the grooves between the lobes (Fig. 2A). Schizont isolation requires nocodazole treatment for microtubule depolymerization; consistently, no bound microtubules were detected on isolated schizonts (Fig. 2A and data not shown). We next tested whether the schizont surface interacts de novo only with polymerized host microtubules or also with tubulin in order to trigger microtubule polymerization. To this end, we incubated isolated schizonts adhering to glass slides with rhodamine-tubulin (rhod-tub) and performed a tubulin polymerization assay in the presence of schizonts. By detecting rhodamine fluorescence by IFM, we could then visualize polymerized tubulin as dense aggregates on the schizont surface (Fig. 2B). To generate a meshwork of microtubules, we performed tubulin polymerization assays on schizonts in test tubes and in the presence of taxol, which stabilizes microtubules. Microtubules did not bind to uncoated glass slides. To recover the microtubule meshwork, we seeded isolated schizonts after in vitro tubulin polymerization on poly-l-lysine-coated glass slides (Fig. 2C c). As controls we used untreated isolated schizonts (Fig. 2C a) or schizonts where tubulin polymerization was performed in the absence of taxol (Fig. 2C b). Before processing for IFM, we incubated the schizonts in medium for 2 h to trigger the extension of membrane protrusions. Control schizonts without rhod-tubulin and incubated with anti-tubulin antibodies showed that host cell tubulin remained bound to the surface of isolated schizonts throughout the procedure and demonstrated that host cell tubulin could bind tightly to the surface of the schizont (Fig. 2C a). The addition of rhod-tubulin to isolated schizonts without the microtubule-stabilizing drug taxol resulted in punctate staining on the schizont surface (Fig. 2C b, arrowheads). In the presence of taxol, tubulin polymerized to a mesh of microtubules (Fig. 2C c). The isolated schizonts were trapped in this microtubule mesh, with microtubules tightly surrounding the individual schizonts (Fig. 2C c, arrow). Combined, these data demonstrate that: (i) host cell tubulin interacts robustly with the surface of the schizont, (ii) de novo polymerization of tubulin can occur on the surface of the schizont, and (iii) the schizont can sequester both tubulin monomers and microtubules on its surface. Thus, the outer surface of the Theileria schizont plasma membrane serves both as a docking structure for host cell protein kinases such as Iκ_B kinase (Heussler et al., 2002) and PLK1 (von Schubert et al., 2010) and as a catalyst surface to facilitate microtubule binding and polymerization.

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Figure 2. Tubulin polymerizes on the surface of isolated schizonts.

A. Non-permeabilized, isolated T. annulata schizonts were labelled with anti-tubulin antibody (red) to reveal host cell tubulin bound to the parasite membrane after isolation. The schizont membrane was visualized with anti-TaSP antibody (green). DNA is stained in blue. Boxed areas are magnified fourfold, TaSP and tubulin staining are shown in inverted grey scale.

B. Recombinant, rhodamin-labelled tubulin (red) was incubated with isolated schizonts bound to un-coated glass slides and allowed to polymerize in the presence of taxol for 30 min on the surface of the parasite. The surface of the schizont was visualized with anti-TaSP antibody (green). DNA is stained in blue. Boxed areas are magnified fourfold, TaSP and rhod-tub staining are shown in inverted grey scale.

C. Isolated schizonts were incubated in test tubes in the absence (a) or presence of rhod-tubulin (b and c) and without (b) or with taxol (c) for 30 min. Parasites were then seeded onto poly-l-lysine-coated glass slides and incubated with medium for another 2 h before processing for IFM. Host-cell tubulin is in green, rhod-tubulin in red and DNA in blue. Arrows indicate polymerized microtubules interacting with parasite and arrowheads punctuate rhod-tubulin on parasite surface. Inverted grey-scale of rhod-tubulin fluorescence is shown for better visualization of tubulin and microtubule binding.

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The surface of T. annulata schizonts at high resolution

Descriptions of the lobular structure of Theileria schizonts, with grooves separating the individual lobes, have been reported in the past (Dobbelaere and Kuenzi, 2004; Seitzer et al., 2010; von Schubert et al., 2010) but the mechanisms maintaining the lobes are not known. A recent report has established that the interaction of the schizont with host cell microtubules is necessary for partition of the schizont onto the two daughter cells (von Schubert et al., 2010). This study visualized the deformability of the schizont and the dynamic acquisition of shapes of great diversity, depending on constrictions imposed by the host cell. Although some of these variations can be attributed to microtubule binding, others cannot because nocodazole, a drug that disrupts host cell microtubules, does not affect the lobular structure of the schizont (von Schubert et al., 2010). This observation indicates that the peculiar lobular shape of Theileria schizonts does not depend on bound host cell microtubules but on a parasite-intrinsic scaffold. Parasite cytoskeleton components that provide the necessary rigidity and shape have not yet been described; additionally, the surface topology of the schizont membrane side that faces the host cell cytoplasm is not known. To learn more about morphological characteristics of the schizont surface, we visualized its membrane by confocal and scanning electron microscopy. Confocal microscopy confirmed that the lobular structure consisting of multiple, membrane-enclosed but connected cavities, is maintained also in isolated schizonts (Fig. 3A) that are devoid of bound microtubules (Fig. 3B and C). Scanning electron micrographs of the surface of isolated schizonts (Fig. 3B and C) show that the schizont surface is relatively smooth but humpy, with grooves between the individual lobes (Fig. 3B). On platinum-sputtered specimens, allowing higher resolution (Fig. 3C), we observed knob-like structures decorating the schizont surface evenly. No traces of host cell cytoskeleton structures bound to isolated schizonts were detected with this method. Combined, these images suggest that an intrinsic structure and not host cell cytoskeleton components shapes the surface of isolated schizont, generating plasma membrane modifications with specific and well-circumscribed structures. The presence of membrane knobs indicates the possibility of clustered protein accumulation on the surface of the schizont.

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Figure 3. Lobular with knobs: the surface of isolated T. annulata.

A. Isolated schizonts were investigated by confocal IFM after surface staining with anti-TaSP antibody. The left panel shows an intensity projection of multiple individual confocal sections as the one shown in the right panel.

B and C. Images of the schizont surface generated by scanning electron microscopy (SEM). In (B) a gold-sputtered specimen and in (C) a platinum-sputtered specimen is shown. Boxed areas are magnified fourfold and shown to the right.

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Protrusions depend on parasite actin polymerization

We next investigated morphology and dynamics of the membranous protrusions extending from the schizont surface in order to explore possible structural components. Towards this end, we visualized protrusions on isolated parasites by four different imaging approaches: by IFM, by scanning electron microscopy (SEM), by live-cell imaging and by cryo-electron tomography. The protrusions measured up to 10 μm in length with a minimum width of 10 nm (Fig. 4A). Marked protrusions extended from the schizont surface within 2 h after isolation; the extension of the protrusions is a dynamic, fluctuating process (Movie S2) and appeared to involve the passage of plasma membrane material from the base of the protrusion outward towards its tip in a wave-like movement (Movie S3). The protrusion ended in a bulge, which persisted throughout the whole extension process (Fig. 4A and Movie S4). The bulge does not interact with tubulin and we observed no binding to microtubules. To determine whether protrusion formation depends on actin or tubulin polymerization, we treated isolated schizonts either with nocodazole or with cytochalasin D (CytoD), to inhibit tubulin or actin polymerization respectively. CytoD but not nocodazole blocked protrusion formation, which suggested that their extension is an actin polymerization-driven process (Fig. 4B). At lower CytoD concentrations (0.4 μM), the schizonts still extended protrusions (Fig. 4C). However, their morphology markedly differed from untreated schizonts in that bulges along the stem of the protrusions formed (serial ‘pearls on a string’) and the protrusions started to branch off near the tip (Fig. 4D b and 4E a + b). The distance between bulges was approximately 0.75 μm (Fig. 4D a and E). We could also observe the pearls on a string phenotype and branching off by live-video microscopy (Movie S5). After CytoD treatment, the branches developed perpendicular to the stem and the whole protrusion including the branches appeared to rigidify (Fig. 4D and E and Movie S5). Theileria actin filaments were not detectable using phalloidin (not shown) and since no antibody against Theileria actin is available, we used an antibody directed against Toxoplasma actin, which does not cross-react with bovine actin. Theileria actin shares 94% homology with Toxoplasma actin and 93% with Plasmodium actin (Fig. S1) and does not present the mutations G200S and K270M that were shown to decrease stability of actin filaments in Toxoplasma and Plasmodium (Skillman et al., 2011). This antibody revealed punctuated staining inside the schizont near the membrane and also inside protrusions (Fig. S2, arrowheads), which indicated that actin could indeed be involved in maintaining the lobular shape and in extending membranous protrusions.

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Figure 4. Membranous parasite protrusions are sensitive to inhibition of actin polymerization.

A. SEM images of membranous protrusions from schizont. Note bulge-like expansion at end of protrusion (arrow).

B. Inhibitors of actin (Cytochalasin D, CytoD) and microtubule (nocodazole, Noc) polymerization were used to test whether protrusions require actin or tubulin polymerization for extension respectively. IF images of isolated, anti-TaSP-labelled schizonts and quantification of lengths of protrusions are shown.

C. Comparison of isolated, anti-TaSP-labelled untreated and CytoD-treated schizonts. Note the stumpy, branched morphology of CytoD-treated parasites.

D. Inhibition of actin polymerization by 2 μM CytoD after protrusion extension had started results in the formation of multiple bulges that are either aligned in series or branched (see also Movie S5). Distance between bulges is approximately 0.75 μm.

E. SEM images of membranous parasite protrusions after CytoD treatment. Serial or branched appearances of multiple bulges are magnified. Boxed areas are magnified fourfold.

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Filamentous actin assemblies and an inner membrane inside membranous protrusions

The inhibition of protrusion formation by CytoD pointed towards F-actin polymerization as the driving force for their extension. We expected that the resulting actin filaments – if present – be detectable by high-resolution imaging approaches. One of these approaches is cryo-electron tomography, which has repeatedly visualized individual actin filaments in vertebrate cells (Medalia et al., 2002; Cyrklaff et al., 2007; 2011), although it failed to visualize actin filaments in Plasmodium sporozoites (Kudryashev et al., 2010). We employed cryo-electron tomography of isolated Theileria schizonts to investigate the membranous protrusions and the closely associated parts of the parasite cytoplasm. We observed thin (Fig. S3) and thicker protrusions (Fig. S4) and obtained tomographic reconstructions. On different sections of a reconstructed tomogram, we observed a bulge-like structure presenting the plasma membrane and beneath it an inner membrane, which ran along the entire protrusion (Fig. 5A and B). At higher magnifications of the thin membranous protrusion we observed filaments with average width of 6 nm and length of 40 nm, arranged parallel to the protrusion and suggestive of actin filaments (Fig. 5C and D). Reconstructed tomograms of thicker protrusions showed membranous structures underneath (Fig. S5), which appeared to be bi-layered and twisted. Also, long filaments with a width of 6 nm were observed beneath the plasma membrane (Fig. S5C). This protrusion also presented membranous structures underneath (Fig. 6A) and showed several actin-like filaments on different sections of the tomograms (Figs 6B and C and S6). The observed filaments appeared randomly oriented reaching up to 200 nm in length.

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Figure 5. Cryo-electron tomography analysis of a thin membranous protrusion from an isolated Theileria schizont.

A. A slice through a reconstructed tomogram of a thin membranous protrusion. At the bulge end (arrowhead) of the protrusion it is possible to observe an outer membrane (OM) and an inner membrane (IM).

B. A slice through the same tomogram at a different Z-projection showing more clearly the thin membranous protrusion (arrow).

C and D. Higher magnifications of the thin protrusion at different Z-projections showing the filamentous structures (arrows) beneath the protrusion membrane. Graphs show the electron density profile of these different slices, denoting the outer membrane (OM), the inner membrane (IM) and the filamentous structures (FS).

Bars: A and B = 200 nm; C and D = 100 nm.

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Figure 6. Cryo-electron tomography of filamentous structures within a Theileria protrusion.

A. A slice through a reconstructed tomogram of a thick protrusion (white arrows) with some membranous structures (arrowheads).

B. A different slice of the same tomogram showing some actin-like filaments (arrows) inside the protrusion at random orientations. Inbox shows a higher magnification of these filamentous structures.

C. At a different projection, more randomly oriented filamentous structures (arrows) inside the protrusion are observed. Inbox shows a higher magnification of these filamentous structures.

Bars = 200 nm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study we describe surface topology and morphological features of the plasma membrane of the intracellular parasite T. annulata at high resolution. Using isolated schizonts, we show here that both de-polymerized host cell tubulin and in vitro polymerized microtubules interact with the parasite membrane. We demonstrate that the lobular shape of the schizont is maintained by a mechanism intrinsic to the parasite and likely dependent on parasite actin polymerization. We found no evidence of host microtubule binding on isolated schizonts to maintain the lobular shape. Furthermore, we describe a novel structure in Theileria schizonts, a filamentous nanotube-like membrane protrusion, which depends on parasite actin dynamics. Finally, we visualized for the first time native actin filaments of an apicomplexan parasite. Combined, our data suggest that the schizont membrane could function as peri-organelle to dynamically interact with host cell components, possibly to catalyse molecular events for host cell manipulation.

One hallmark of Theileria schizonts is their lobular surface topology with numerous bulging expansions and grooves separating them. The present work is the first to address this phenomenon by applying high-resolution microscopy analysis combined with time-lapse video microscopy. The lobular shape of Theileria schizonts is maintained after isolation of the schizonts from the host cell despite drug-mediated depolymerization of bound host cell microtubules. Thus, host cell microtubules are dispensable for the maintenance of the lobular shape and they do not function as an exoskeleton for maintaining schizont shape. Nevertheless, besides their crucial function for parasite segregation during host cell division (von Schubert et al., 2010), microtubule binding to the intracellular schizont might be necessary to spatially arrange the parasite inside the host cell, which likely is relevant in interphase cells during cell migration. Indeed, in polarized macrophages infected with T. annulata, the schizont is caged by microtubules and maintained between the host cell nucleus and the trailing edge (Baumgartner, 2011a). Host cell microtubules polymerize around intracellular Theileria immediately after invasion (Fawcett et al., 1984) and remain tightly associated during schizont stage (Seitzer et al., 2010; von Schubert et al., 2010). Using recombinant tubulin, we now show in vitro that tubulin and microtubule binding to the schizont membrane does not require supplementing the polymerization reaction with microtubule-associated proteins, suggesting that the parasite surface provides abundant binding sites for tubulin and microtubules. However, we cannot fully exclude that host cell proteins associated with the schizont were co-purified and mediate in vitro microtubule interaction.

Our data indicate that the lobular shape of Theileria schizonts is maintained by an internal scaffolding structure that provides rigidity and curvature. This internal, parasite-regulated scaffolding structure likely allows the active extension of membranous protrusions from the schizont surface, both in intracellular and in isolated schizonts. We also observed membranous protrusions in migrating T. annulata-infected cells, which display a polarized morphology (Fig. 1A c); host cell polarization is hallmarked by pronounced F-actin assemblies at the leading edge and the formation of podosome type adhesions (Baumgartner, 2011b). Cell polarization along the axis of migration disrupts the structural remnants of cell division. Therefore, we do consider it unlikely that membranous protrusions observed in intracellular schizonts are the result of improper partition of the schizont after cell division. Furthermore, using DIC live-cell imaging, we could visualize a membranous protrusion extending from the intracellular schizont towards the cell periphery (Movie S1). The movements of this intracellular protrusion resemble the movements of membranous protrusions observed on isolated schizonts in vitro (Movie S2). This indicates that the mechanism driving membranous protrusions on intracellular and on isolated schizonts is identical. We observed membranous protrusions in only approximately 20% of cells in culture, whereas close to 100% of the schizonts displayed protrusions after isolation. One possible mechanism of regulation is the abundance of bound tubulin and/or microtubules, which in abundance could restrict protrusions extension. Consistently, none of the schizonts trapped in the microtubule mesh in vitro extended membranous protrusions.

Some of the membranous protrusions observed on isolated Theileria schizonts are of similar appearance to the recently described nanotube-like protrusions of Plasmodium gametocytes (Rupp et al., 2011). Using phalloidin the authors suggest that the protrusions are actin driven, but no actin filaments could be visualized (Rupp et al., 2011). Actin filaments are notoriously elusive in apicomplexan parasites, despite their essential role in parasite motility and invasion (Baum et al., 2008; Kudryashev et al., 2010; Sattler et al., 2011). Bundles of filaments have been shown after treatment of parasites with the actin depolymerization inhibiting drug jasplakinolide (Shaw and Tilney, 1999), by knocking out the Toxoplasma actin sequestering protein ADF (Mehta and Sibley, 2011) or when expressing a mutated form of Toxoplasma actin (Skillman et al., 2011). The latter report showed that most apicomplexans have a glycine at position 200 and a lysine or arginine at position 270, while most higher eukaryotes show a serine at position 200 and a methionine at position 270. Curiously, Theileria along with Babesia show a serine at position 200 and a methionine at position 270. When these mutations were introduced into Toxoplasma gondii and Plasmodium actin, these actins polymerized faster in vitro than their respective wild-type actins (Skillman et al., 2011). When mutated actin was expressed in Toxoplasma tachyzoites, these parasites formed more readily filamentous fibres when jasplakinolide was added (Skillman et al., 2011). Cytochalasin, a mycotoxin that blocks actin polymerization and leads to F-actin filament depolymerization, disrupts Theileria protrusions (Fig. 4) and filamentous actin structures could be revealed by cryo electron tomography (Figs 5 and 6). This provides evidence that Theileria actin is capable of polymerization into filaments to drive long extensions, whatever their function. Consistently, an antibody raised against the closely related actin of Toxoplasma, detects parasite actin in a dotted staining pattern throughout the parasite, underneath the plasma membrane and along the protrusions (Fig. S2). Curiously, Theileria can invade host cells without relying on its own actin (Shaw, 1999). The fact that we could readily image short actin filaments in thin protrusions and long filaments at their base further confirms the idea that G200/K270 is key for keeping actin of other apicomplexans at a short length (Skillman et al., 2011). The additional stability possibly conferred to Theileria actin by retaining S200/M270 might allow it to use actin for different purposes than host cell invasion. It might for example play a role in the maintenance of fairly stable lobes in isolated parasite. It might also explain the wave-like expansion of filamentous protrusions. Active protrusion extension alternates with flabby states (Movies S1–S3), where the flaccidity towards the tip of the protrusion could indicate local collapse or remodelling of short actin filaments.

The functional significance of the filamentous protrusions is not clear yet. Exact positioning of the parasite with respect to the host cell nucleus during interphase (Seitzer et al., 2010) and to the central spindle during host cell mitosis (von Schubert et al., 2010) is essential for parasite propagation. The filamentous protrusions inside cells tend to target positions near the host cell nucleus. Therefore, it is possible that the protrusion facilitates positioning of the parasite by a search and capture mechanisms in order to align and orient the parasite properly.

Combined, our data revealed that membrane curvature and dynamics are controlled by parasite intrinsic mechanisms and not by the host cell cytoskeleton. Furthermore, our data demonstrate the plasticity of the parasite surface and reveal its potential to bear organelle functions serving the orientation of the parasite relative to the host cell nucleus, the exchange of molecules between the parasite and the host cell cytosol and the modification of host cell functions.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Reagents

Schizonts were isolate from T. annulata-infected macrophage lines TaC12 (gift from Dirk Dobbelaere), from ‘Thei’ (Moreau et al., 1999) cells (gift from Gordon Langsley) or TaH12810 (Jensen et al., 2008) cells (gift from Elizabeth Glass). Antibodies used were mouse mc anti-tyrosine anti-tubulin (clone A2, SIGMA), mouse mc anti-phospho Tyrosine (p-Tyr-100, 9411 Cell Signaling), rabbit pc anti-Toxoplasma actin (generous gift from Dominique Soldati) and rabbit pc anti-TaSP (Schnittger et al., 2002) (generous gift from Jabbar Ahmed); Texas-red phalloidin (Molecular Probes).

Tubulin polymerization assay

In vitro microtubule polymerization was performed according to the manufacturer's (Cytoskeleton) instructions. In brief: 5 μg of rhodamine-tubulin in general tubulin buffer containing 1 mM GTP and 12% glycerol was allowed to polymerize with Taxol in the presence of isolated parasites bound to glass coverslips for 20 min at 37°C. Alternatively, tubulin was pre-polymerized with parasite in test tubes without or with Taxol for 20 min at 37°C. The schizont-microtubule mixture was then seeded either on uncoated or on poly-l-lysine-coated glass slides.

Schizont isolation and fixation on glass coverslips

Theileria annulata schizonts were isolated essentially according to the protocol described previously (Baumgartner et al., 1999). In brief: T. annulata-infected macrophages were treated with nocodazole to depolymerize microtubules for 2 h. Cells were then incubated with trypsin-activated aerolysin on ice. Excess aerolysin was removed and temperature was risen to 37°C to stimulate toxin-mediated permeabilization of the host cell plasma membrane. Permeabilization was stopped when majority of cells tested trypanblue positive. Schizonts were separated from host cell debris using Percoll gradient centrifugation. Isolated schizonts were allowed to adhere for 20 min at room temperature to either untreated or poly-l-lysine-coated glass coverslips. Schizont adhesion to untreated glass coverslips was only slightly less efficient.

IF microscopy

Isolated parasites were fixed in 4% paraformaldehyde, where indicated permeabilized in 0.5% Tx-100, and non-specific epitopes were blocked with 10% FCS in PBS. Primary and secondary antibodies were diluted in 10% FCS in PBS. If not otherwise stated, images were acquired in wide-field mode either on a Nikon 80i or on an inverted Nikon Eclipse TE2000-U microscope by using Openlab software. Confocal IF images were acquired on a laser-scanning microscope (Leica, SP2) using Leica software. Image procession was performed using Imaris and Adobe Photoshop software.

Time-lapse imaging

Time-lapse imaging using video microscopy was performed with isolated parasites adhering to glass bottom culture dishes (Willco Wells, the Netherlands) using a Nikon Eclipse TE2000-U inverted microscope equipped with a climate-controlled chamber. Data acquisition and image processing were performed using NIS software of Nikon Instruments. DIC and fluorescence images were acquired and assembled in AVI movies using NIS software.

Cryo-electron tomography

Isolated T. annulata were put onto holey carbon-coated grids, excess liquid blotted, and rapidly plunged frozen into liquid ethane and stored in liquid nitrogen. Grids were mounted in a Gatan cryo-holder and observed in a FEI Polara microscope (operating at 300 kV) equipped with a field emission gun. Tilt series with two to three degrees increments were recorded on a 2048 × 2048 pixel CCD camera, at a final magnification of 18 000 and an objective lens defocus of −12 μm. Images were aligned with MIDAS, and Etomo was used to reconstruct the tomograms (all programs are part of IMOD software package – Boulder Laboratory for 3D electron microscopy).

Scanning electron microscopy

Coverslips were sputter-coated with gold or platinum and treated with poly-l-lysine (Sigma, Buchs, Switzerland) to promote cell adhesion. Isolated parasites were allowed to adhere to the coverslips for 30 min. Thereafter, samples were fixed in 2.5% glutaraldehyde (Merck, Darmstadt, Germany), dehydrated through an ascending alcohol series and dried by evaporation of hexamethyldisilazane (Sigma, Buchs, Switzerland) (Ting-Beall et al., 1995a,b; Braet et al., 1997). Coverslips were then mounted on metal stubs by use of conductive carbon adhesives (Provac AG, Balzers, Liechtenstein). Samples were sputtered with 4 nm of platinum carbon by electron beam evaporation in a BalTec MED20 (Leica Microsystems, Heerbrugg, Switzerland) and examined in a field emission scanning electron microscope DSM 982 Gemini (Zeiss, Oberkochen, Germany) at an accelerating voltage of 5 kV, at a working distance of 6–8 mm.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Ulrike Seitzer and Jabbar Ahmed for providing anti-TaSP and Dominique Soldati for providing anti-T. gondii actin sera. We are grateful to Kirsty Jensen, Elizabeth Glass, Dirk Dobbelaere and Gordon Langsley for providing cell lines. We also thank Dirk Dobbelaere for stimulating discussions and Gordon Langsley and Marie Chaussepied for sharing unpublished data. We thank the EMBL for access to their EM facility. This study was supported by grants of the Swiss National Science Foundation (Grant SNF_31003A_127025/1) and the University of Bern to M.B., a postdoctoral fellowship from the University of Heidelberg Excellence Cluster CellNetwork to L.L., the Chica and Heinz Schaller Foundation to F.F. F.F. is a member of the European Union Network of Excellence EVIMalaR.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cmi12006-sup-0001-movie1.avi285K

Movie S1. Live-video microscopy of T. annulata-infected TaC12 cells. 640× magnification (cropped), 60 s intervals, 60 min recording, 600× acceleration.

cmi12006-sup-0002-movie2.m4v1527K

Movie S2. Live-video microscopy of isolated T. annulata schizonts. 640× magnification (cropped), 30 s intervals, 60 min recording, 300× acceleration.

cmi12006-sup-0003-movie 3.m4v1173K

Movie S3. Live-video microscopy of isolated T. annulata schizonts. 640× magnification (cropped), 30 s intervals, 60 min recording, 300× acceleration.

cmi12006-sup-0004-movie4.m4v2284K

Movie S4. Live-video microscopy of isolated T. annulata schizonts. 640× magnification (cropped), 30 s intervals, 120 min recording, 300× acceleration.

cmi12006-sup-0005-movie5.m4v879K

Movie S5. Isolated T. annulata schizonts were allowed to start protrusion formation for 45 min. Cytochalasin D at a concentration of 2 μM was the added and live-video microscopy was performed. 64× magnification (cropped), 30 s intervals, 45 min recording, 300× acceleration.

cmi12006-sup-0006-fS1.tif2127K

Fig. S1. Sequence alignment of actins from Toxoplasma gondii (TgACTI), Plasmodium falciparum (PfACTI) and Theileria annulata (TaACT1). Residues that were demonstrated to give instability to actin filaments (G200S and K270M) are indicated by an asterisk.

cmi12006-sup-0007-fS2.tif3215K

Fig. S2. Isolated parasites were incubated with antibody raised against Toxoplasma actin and anti-TaSP antibody. Isolated parasites were fixed immediately after purification (A) or after 1 h in medium (B) to induce membranous protrusions. Arrowheads indicate dotted anti-actin immunoreactivity in membranous protrusions. Arrowheads highlight actin dots inside membranous protrusions.

cmi12006-sup-0008-fS3.tif1247K

Fig. S3. Cryo-electron micrographs of isolated Theileria showing thin membranous protrusions (arrows) that end in a bulge-like structure (arrowhead). Some slices through the reconstructed tomograms are shown in Fig. 5. Bars = 400 nm.

cmi12006-sup-0009-fS4.tif1306K

Fig. S4. Cryo-electron micrographs of isolated Theileria showing thick membranous protrusions (arrows), presenting membranous structures inside them (arrowhead). Some slices through the reconstructed tomograms are shown in Fig. 6. Bars = 400 nm.

cmi12006-sup-0010-fS5.tif1530K

Fig. S5. Cryo-electron tomography analysis of a thick filamentous protrusion of Theileria.

A. A slice through a tomogram of a thick membranous protrusion (white arrows) of an isolated Theileria. Arrowhead indicates a membranous structure of twisted shape inside the protrusion.

B. Higher magnification of the membranous structure (arrowhead) inside the protrusion.

C. Slice through the tomogram in a different Z-projection showing some filamentous structures (black arrows).

Bars: A = 200 nm; B and C = 100 nm.

cmi12006-sup-0011-fS6.tif1696K

Fig. S6. Theileria protrusion shown in Fig. 8 after 3D modelling and rendering. The plasma membrane is in green, membranous structures in yellow and magenta, and the actin filaments in blue.

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