Dynamics and 3D organization of secretory organelles of Toxoplasma gondii

Journal of Structural Biology 177 (2012) 420–430

Contents lists available at SciVerse ScienceDirect

Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi

Dynamics and 3D organization of secretory organelles of Toxoplasma gondii Tatiana Christina Paredes-Santos a,b, Wanderley de Souza a,b,c, Márcia Attias a,b,⇑ a

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Brazil c Instituto Nacional de Metrologia, Normalização e Qualidade Industrial-Inmetro, Brazil b

a r t i c l e

i n f o

Article history: Received 25 August 2011 Received in revised form 21 November 2011 Accepted 28 November 2011 Available online 4 December 2011 Keywords: Toxoplasma gondii Secretory organelles FIB-serial sections 3D reconstruction Stereology Apical complex

a b s t r a c t Micronemes, rhoptries and dense granules are secretory organelles of Toxoplasma gondii crucial for host cell invasion and formation of the parasitophorous vacuole (PV). We examined whether their relative volumes change during the intracellular cycle. Stereological analysis of random ultrathin sections taken at 5 min of interaction, 7 and 24 h post-infection demonstrated that the relative volume of each type of organelle decreases just after the respective peak of secretion. Micronemes are radially arranged below the polar ring, while rhoptries converge to but only a few reach the inside of the conoid. In contrast to the apical and polarized organelles, dense granules were found scattered throughout the cytoplasm, with no preferential location in the parasite cell body. Extensive observation of random sections indicated that each organelle probably secretes in a different region. Micronemes secrete just below the posterior ring and probably require that the conoid is extruded. The rhoptries passing through the conoid secrete at a porosome-like point at the most apical region. Dense granules secrete laterally, probably at fenestrations in the inner membrane complex. Immunocytochemistry showed that there are no subpopulations of rhoptries or dense granules, as a single organelle can contain more than one kind of its specific proteins. The vacuolar-like profiles observed at the apical portion of parasites just after invasion were confirmed to be empty rhoptries, as they were positively labeled for rhoptry proteins. These findings contribute for a better understanding of the essential behavior of secretory organelles. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction The phylum Apicomplexa comprises numerous species of protozoan parasites, some of which are human and animal pathogens. The phylum includes Plasmodium sp., Eimeria sp., Cryptosporidium sp. and Toxoplasma gondii (Dubey et al., 1998) and is characterized by the presence of the apical complex, a structure formed by elements of the cytoskeleton and specialized secretory organelles essential for invasion of the host cell (Morisaki et al., 1995). T. gondii is an obligate intracellular parasite capable of invading the nucleated cells of all warm-blooded animals, and successful invasion is a major determinant of the establishment of infection (Jones et al., 1972). The tachyzoite is a rapidly dividing form characteristic of the acute stage of the infection. It has three types of secretory organelle: micronemes, rhoptries and dense granules. These organelles secrete their contents sequentially (Carruthers and Sibley, 1997). The first organelle to secrete are micronemes, which are small, ⇑ Corresponding author at: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, Ilha do Fundão, 21941-902 Rio de Janeiro-RJ, Brazil. Fax: +55 21 22602364. E-mail address: [email protected] (M. Attias). 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.11.028

numerous, rod-shaped, membrane-bound structures located at the apical portion of the cell (Carruthers and Sibley, 1997). Microneme secretion is triggered by an increase in intracellular calcium (Carruthers and Sibley, 1999), which results in the release of a mixture of proteins called MICs (Zhou et al., 2005). Some MICs possess adhesive domains that are responsible for adhesion to the host cell and parasite gliding. MICs allow the tachyzoites to move, to adhere and to recognize new host cells to invade (Keeley and Soldati-Frave, 2004). The second type of organelles to secrete are the rhoptries. Larger and less abundant (6–14 per cell) than micronemes, rhoptries are club-shaped (Scholtyseck and Melhorn, 1970) and acidic (Shaw et al., 1998). There are two recognized compartments in rhoptries: a bulb with a honeycombed appearance and a tapered neck oriented towards the conoidal channel. As observed by freezefracture replicas, the rhoptries present a linear distribution of inner-membrane particles along the peduncle (Lemgruber et al., 2010). The contents of the rhoptries are both lipidic, giving the organelle its shape (Besteiro et al., 2008), and proteic: RONs and ROPs. Those are derived from necks and bulbs, respectively (reviewed by Boothroyd and Dubremetz, 2008). The RON proteins are responsible for the assembly of the moving junction, a transitory structure between the parasite and the host cell membrane that is believed to be essential for active invasion and parasitophorous

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

vacuole formation (Alexander et al., 2005), while ROP proteins are responsible for the establishment of infection, acting on several targets inside the host cell (Bradley and Sibley, 2007). The dense granules are the third type of secreting organelle (Sibley et al., 1995). Dense granules constitutively secrete their contents inside the parasitophorous vacuole throughout the intracellular cycle; however, there is a calcium-independent peak of secretion that occurs 20 min after invasion (Chaturvedi et al., 1999). The secretory products are used to build the intravacuolar network, a membranous net of tubules that helps to support the parasitophorous vacuole (PV) (Magno et al., 2005) and the increase of the PV membrane. These products include the GRA proteins, nucleoside triphosphate hydrolases (NTPases) and other enzymes (Cesbron-Delauw, 1994; Mercier et al., 2005). All of the above properties of these secretory organelles have been elucidated in previous studies; however, no previous study considers its distribution and behavior throughout the intracellular cycle or the location and mode of secretion of these organelles. Furthermore, detailed ultrastructural descriptions of the secretory organelles were published in the 70s, 80s and 90s (Scholtyseck and Melhorn, 1970; Nichols et al., 1983; Saffer et al., 1992), when some of the techniques employed here were not available. In this paper, we focused on formulating an updated description of the ultrastructure of secretory organelles throughout the T. gondii intracellular cycle and investigated as-yet-unanswered questions about the site of secretion of each organelle. Our methods included classic techniques such as chemical fixation and morphometric analysis of random ultrathin sections from samples fixed at different times after interaction with culture cells; we also utilized some more recent on ‘‘state of the art’’ technical resources, such as 3D reconstruction from serial sections using a Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), a tool that allows the reconstruction of whole cells from sections much thinner than can be obtained via serial sectioning with an ultramicrotome (Bennett et al., 2009). This method yields better resolution and alignment of the models. To visualize smaller volumes at an even higher resolution, electron tomography was employed. Tachyzoites fixed by freeze-substitution also contributed with data confirming the position and shape of these organelles. 2. Materials and methods 2.1. Parasites Tachyzoites of the virulent RH strain of T. gondii were maintained by intraperitoneal passage in CF1 mice and were harvested in Hank’s solution 48 h after infection. The ascites fluid suspension obtained from infected mice was centrifuged at 1000g for 10 min to remove cells and debris. The pellet obtained was re-suspended to a density of 107 parasites/ml in RPMI without fetal bovine serum (FBS) and immediately allowed to interact with the host cells. 2.2. Host cells LLCMK2 cells (Rhesus monkey kidney epithelial cells) were maintained in RPMI medium supplemented with 10% FBS and 2 mg/ml garamicin. Serial passages were conducted by trypsinization when the cell density approached confluence in a monolayer. One day before the experiments began, LLCMK2 cells were inoculated into 25-cm2 flasks and maintained at 37 °C in 5% CO2. 2.3. Initial invasion assay For the initial invasion assay, a parasite-to-cell ratio of 50:1 cell was used. To synchronize the invasions, after 15 min at 4 °C, the

421

temperature was raised to 37 °C. Each interaction was allowed to continue for only 5 min. 2.4. Long term infections Parasites suspended in RPMI were challenged for 45 min with LLCMK2 cells using a 10:1 parasite-to-host cell ratio. After challenge, the monolayers were washed twice with RPMI to remove extracellular parasites. The monolayers were then evenly divided in two groups, one incubated for 7 h and the other for 24 h, at 37 °C for 7 h and 24 h and processed for transmission electron microscopy (TEM). 2.5. Transmission electron microscopy LLCMK2 cells were inoculated into 25-cm2 flasks and allowed to interact with the parasites, as described above. After interaction, the cultures were washed with RPMI and fixed overnight at 4 °C with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. Then the cultures were washed three times in cacodylate buffer and post-fixed for 1 h in a solution containing 1% OsO4, 1.25% potassium ferrocyanide, and 5 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.2. After post-fixation, the cells were washed in cacodylate buffer to remove osmium residues and dehydrated in an ethanol gradient from 30% to 100% (v/v). After dehydration, the specimens were infiltrated and embedded with PolybedÒ resin (Polysciences). Ultrathin sections were cut and stained with 5% uranyl acetate and lead citrate, then observed in a Zeiss EM900 transmission electron microscope. Serial sections were obtained as previously described (Attias et al., 1996). 2.6. Scanning electron microscopy For scanning electron microscopy, the LLC-MK2 cells were grown over round coverslips in 24-well plates, challenged with the parasites, fixed, post-fixed, and dehydrated, as described above. After dehydration in ethanol, the coverslips were critical-point dried. Next, the monolayer was sputtered with a 3 nm thick gold coat in a Balzers apparatus and observed in a JEOL 6340F field emission scanning electron microscope at 5.0 kV at a working distance of 8 mm. 2.7. High-pressure freezing and freeze substitution (HPF-FS) The free tachyzoites were cryofixed by high-pressure freezing (HPM-010 Balzer’s) and freeze-substituted in a freeze substitution (FS) medium consisting of anhydrous acetone, 2% osmium tetroxide, 0.1% uranyl acetate and 1% water. The samples were kept at 80 °C for 72 h, then at 20 °C for 24 h. They were finally rinsed twice (30 min/rinse) in acetone on ice. After rinsing, the samples were gradually infiltrated with increasing concentrations of PolybedÒ:acetone solution (ratios of 1:2, 1:1 and 2:1, with pure PolybedÒ used in the final step). Samples were held in each infiltration step for at least 24 h. The final polymerization was performed at 60 °C for 72 h. 2.8. Immunocytochemistry For immuno-electron microscopy, samples of short (5-min) interactions of host cells and parasites were quickly rinsed in PBS (phosphate-buffered saline); fixed in 0.2% glutaraldehyde, 4% freshly prepared formaldehyde and 3% sucrose in 0.1 M cacodylate buffer, pH 7.3; dehydrated at 4 °C in ethanol, infiltrated in Unicryl resin (Pelco) at 20 °C and polymerized under UV radiation for 96 h. Ultrathin sections of Unicryl-embedded parasites were collected on nickel grids, incubated in 50 mM ammonium chloride in PBS for 30 min to quench free aldehyde groups and transferred

422

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

to blocking buffer for 1 h at room temperature. Thin sections were subsequently incubated with a 1:100 diluted primary antibody anti-ROP-2,4; anti-GRA-3; anti-RON-4 and anti-GRA-7 in blocking buffer (1.5% bovine serum albumin, 0.025% fish gelatin and 0.02% Tween-20 in PBS, pH 7.2) for 1 h, washed and incubated with 10nm gold-labeled goat anti-mouse IgG secondary antibody (BBI, England) and with 15-nm gold-labeled goat anti-rabbit IgG, diluted 1:100 (BBI, England). In control samples, incubation with the primary antibodies was omitted. The sections were successively washed in PBS and water, stained with uranyl acetate and lead citrate and observed in a JEOL 1200 EX transmission electron microscope. 2.9. Stereologic analysis The morphometric measurements were made with images acquired at 20,000 magnification in a ZEISS 900 transmission electron microscope. One hundred cell profiles in random sections were recorded from each sample (5 min, 7 and 24 h post-invasion). ITEM software (Olympus) was used for the measurements. The sum of the areas of the nucleus, rhoptries, micronemes and dense granules was compared with the total plasma membrane area, which was considered to represent 100% of the total area of the cell; these measurements followed the principle of Delesse’s theorem to estimate the relative volume of each measured organelle. 2.10. FIB-dual-beam electron microscopy (ion abrasion scanning electron microscopy) Resin blocks from standard flat-embedding molds (EMS, Hatfield, PA) were trimmed to a pyramidal shape using a razor blade, with block faces typically about 2 mm2 in area. The surface was smoothed by sectioning, using a conventional 45° diamond knife from Diatome (distributed by EMS, Hatfield, PA). The entire pyramidal block was removed and mounted with the wider base on a aluminum stub with silver paint (SPI Supplies, West Chester, PA) such that the ultramicrotome-prepared, flat surface of the resin block pointed upwards, perpendicular to the electron column. The images were recorded using a Helios 200 NanoLab dual-beam instrument (Eindhoven, NL) equipped with a gallium ion source for focused-ion beam milling and a field emission gun scanning electron microscope with an in-lens secondary electron detector for imaging. Prior to milling and SEM imaging, the entire sample surface was coated with a platinum/palladium layer (1 lm thick) using the gas injector system (GIS) in the main specimen chamber. The specimen stage was tilted to 52° and exposed to the focused ion beam such that the plane of the stage was parallel to the ion beam. A cross-sectional cut was introduced in two stages. First, a coarse cut was made at high beam currents (typically 7–20 nA) and at an accelerating voltage of 30 kV to create a trench that enabled viewing of the cross-section. Usually, 50-to-150-lm-wide trenches were cut into the specimen. In the second step, the ion beam was scanned using a current of 3–7 nA to polish and smooth the surface. Secondary electron SEM images were typically recorded at accelerating voltages of 3 kV and 10,000 magnification, with a beam current of 68–270 pA in the immersion lens mode. To create a slice-and-view image series, a step size of 25 nm was chosen for the removal of material from the specimen surface with the focused ion beam. All images are presented with inverted contrast in the figures and videos for ease of comparison to images obtained from transmission electron microscopy. 2.10.1. Modeling and analysis of serial sections data The sequential slices were aligned by MIDAS (IMOD package), and the data were rendered with 3D MOD from the IMOD package (Kremer et al., 1996).

2.11. Electron tomography (ET) In this study, we performed ET on semi-thin sections (200– 250 nm) of Polybed-embedded samples, applying additional postsectioning contrast with uranyl acetate and lead citrate without fiducial markers. The specimens were placed in a high-tilt specimen holder, and datasets were recorded at 200 kV (in a Tecnai 20 LaB6, (FEI Company), at 1-degree-increments and with an angular tilt range from 60 to +60 for single-axis tomography. Images (1024  1024-square pixels) were recorded using a charge-coupled-device (CCD) camera (Temcam F214, TVIPS GmbH). The sections were pre-irradiated to avoid shrinking effects during recording. Automated data acquisition in the tilt series was carried out using Xplore 3D (FEI Company). Tomograms were computed for each tilt axis using the R-weighted back-projection algorithm and combined into one double-tilt tomogram with the IMOD software package (Kremer et al., 1996). 2.11.1. Modeling and analysis of tomographic data Single-tilt tomograms were analyzed and modeled using the IMOD software package (Kremer et al., 1996). Features of interest were contoured manually in serial optical slices extracted from the tomogram. The image slicer window in IMOD was used to facilitate the recognition of membranous structures. 3D models were displayed and rotated to study its 3D geometry. For accurate interpretation of the data, especially regarding the distance between the membranes, the graphic window in IMOD was used. 3. Results and discussion Rhoptries, micronemes and dense granules are secretory organelles present in T. gondii, as well as in other members of the phylum Apicomplexa. Secretion by micronemes is essential for parasite gliding and adhesion to the host cell surface. Rhoptries, in contrast, secrete during active invasion, thus contributing to the formation of the nascent parasitophorous vacuole and the establishment of the dynamic moving junction between the plasma membranes of the parasite and the host cell. Once the parasite is internalized in the parasitophorous vacuole, dense granules begin to secrete their products. Secretion occurs very intensely at first and at a slower but constant rate as the endodyogenic cycles proceed (Cesbron-Delauw, 1994). As these organelles begin secretion in sequence and at specific points throughout the cycle of invasion and multiplication of T. gondii, an investigation into the distribution and behavior of these organisms was carried out and the results compiled in this manuscript. 3.1. Stereological aspects along the intracellular cell cycle To analyze the behavior of secretory organelles throughout the intracellular cycle, we examined random ultrathin profiles of tachyzoites under the following conditions: extracellular parasites adhered to the host cell (group 1) and intracellular parasites at 5 min (group 2), 7 (group 3) and 24 (group 4) h post-infection. The relative areas occupied by micronemes, rhoptries and dense granules were measured in relation to the total cell area in 100 profiles for each condition. All measurements were converted to volume percentages (v/v), as represented in graphs in Fig. 1. The stereologic data showed that micronemes occupied 0.21% v/v when the parasites were adhered; reached 0.27% v/v just after internalization; 0.21% v/v at 7 hpi, when the first division is about to end; and 0.1% v/v at 24 hpi, when huge parasite rosettes are observed and 46% of the parasites are dividing (Fig. 1A). Rhoptries (Fig. 1B) occupied 3.1% v/v in group 1, but this relative volume dropped significantly (to 1.7% v/v) immediately after

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

Fig.1. Stereologic data of the relative volume occupied by secretory organelles along the intracellular cycle. Measurements were made in extracellular parasites adhered to the host cell (group 1), recently internalized intracellular parasites (group 2), intracellular parasites after 7 (group 3) and 24 (group 4) h of infection. (A) Micronemes: reached the smallest relative volume in the extracellular stage (0.21%). After internalization the volume increases to 0.26%, decreasing again while the division cycles are taking place from 0.21% to 0.17% at 24 hpi. (B) Rhoptries occupied approximately 3.1% of total volume, decreasing just after internalization to 1.7%. In the group 4 (24 hpi) a drastic reduction of volume to 1.7% was observed. (C) Dense granules occupied 5.7% of the total volume decreasing to 4.7% after the internalization, 3.2% at 7 hpi and 1.8% at 24 hpi with the progress of infection.

internalization (group 2) and recovered to 4% v/v at 7 hpi (group 3). At 24 hpi, however, the volume dropped to 1.9% v/v. The third organelle evaluated, dense granules, occupied 5.7% v/v in adhered parasites, 4.9% v/v just after internalization and 3.4% v/v at 7 hpi; the lowest relative volume, 2.3% v/v, was observed at 24 hpi (Fig. 1C). Micronemes occupied less volume in comparison with rhoptries and dense granules, reaching their lowest volume while the parasites were still outside the host cells, when gliding and adhesion are intense and require secreted MICs (Zhou et al., 2005).

423

Microneme volume increased slightly after invasion (group 2), and very low values were reached again during division cycles (group 4). In random sections of extracellular parasites recovered from peritoneal exudate of infected mice, microneme profiles were seldom seen, although their presence was unequivocally detected in some favorable section planes. As Delesse’s theorem is based on a random distribution of structures, these observations are probably an artifact of the small size of micronemes, their rod-shaped appearance and their non-random distribution in the cells. When many of the cells were dividing (group 4), micronemes were even less frequently observed but this lack of data does not indicate their absence. Another artifact of stereologic measurements is the Holmes effect that is a consequence of the overprojection due to the positive thickness of a section. A nontransparent object embedded in a section is seen as large as or larger than it should be due to the thickness of a section greater than 0. However, we believe this effect did not influence the results since the sections analyzed from the different samples observed under equal conditions (microscope, magnification and voltage used). Besides it, the thin and elongated shape of both micronemes and rhoptries and its non random distribution and orientation in the cell, reduces the impact of the Holmes effect in our measurements. As would be expected, the highest decrease in the relative volume occupied by rhoptries occurred just after invasion, as RON-2, -4, -5 and -8, which are secreted by rhoptry necks, are essential for the formation of the moving junction (Alexander et al., 2005; Besteiro et al., 2009). Furthermore, ROP proteins are required for establishment of the parasitophorous vacuole (reviewed by Boothroyd and Dubremetz, 2008). At 24 h postinfection (group 4), however, there was another drastic reduction in rhoptry volume. Most probably, this reduction was a consequence of the fact that 46% of the parasites were dividing and that pre-rhoptries were not included in the measurements. The relative volume occupied by dense granules decreased at a constant rate as the intracellular cycles proceeded, in accordance with the constitutive secretion pattern observed during the development of the parasitophorous vacuole (Fig. 1C). As the parasitophorous vacuole develops, dense granule secretion products are used to build the intravacuolar network, which helps to support increases in vacuole volume and in the number of parasites (Magno et al., 2005; Mercier et al., 2005). In a relationship that would be expected but had not previously been described, the results of stereological analysis showed that, for each organelle, the relative volume decreased just after the respective peak of secretion, which was described by Carruthers and Sibley (1997). During the endodyogenic cycle, rhoptries and micronemes are being synthesized de novo being, probably for this reason, seldom detected. 3.2. 3D view of secretory organelles The stereologic data indicated that each organelle was reduced in relative volume after the peak of secretion in the intracellular cycle. On the other hand, it is clear from observation of random sections and from previous publications (Vivier and Petitprez, 1972; Monteiro et al., 2001; Lemgruber et al., 2010) that rhoptries and micronemes are not randomly distributed in the tachyzoite. To investigate further, we employed a new method, using a FIB-dual beam scanning electron microscope, to obtain serial sections for 3D reconstruction. This equipment, besides the electron beam and detectors for secondary and backscattered electrons as in a regular SEM, has a second beam that emits focused Gallium ions. As the Gallium ions scan the surface of the sample, they cause an abrasion few nanometers thick. Subsequently, each exposed surface is scanned with the electron beam, and the signal is captured

424

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

by the detector of backscattered electrons. All membranes and structures that were impregnated with OsO4 during post-fixation generate a signal that results in an inverted image, analogous to an ultrathin section observed by transmission electron microscopy. This ‘‘slice-and-view’’ tool allowed the acquisition of a series comprising several whole tachyzoites that could be examined along the whole length of each cell (Bennett et al., 2009). Two of those extracellular parasites were reconstructed (Figs. 2 and 3), and the Z-series of sections used is in the Supplemental material 1 (video file). The first model rendered (Fig. 2D) was from a whole parasite, transversally sliced (Fig. 2A–C). At this slicing orientation, it was clear that micronemes were radially disposed below the conoid, around the polar ring (Fig. 2A). In the section shown in Fig. 2B, dense granules were already visible. As the medial portion of the cell body is reached (Fig. 2C), the nucleus, more dense granules, elements of the Golgi complex, and profiles of the mitochondrion and endoplasmic reticulum were also seen, but micronemes and rhoptries were absent. The model, shown in Fig. 2D, displays the following features: dense granules scattered in the cell body, rhoptries concentrated in one side of the cell and micronemes forming a crown around the polar ring. Only one of the ten rhoptries of this cell extended inside the conoid (Fig. 2D). A video of this model is included in Supplemental material 2 (video file). The second model was rendered from a parasite sliced longitudinally. The section in Fig. 3A shown the inside of the conoid where several aligned vesicles are seen passing through. Profiles corresponding to rhoptry necks and micronemes were also seen, but could not be distinguished in this single plane. In contrast, in the

section shown in Fig. 3B, the wall of the conoid was tangentially included. In this same plane, there are about ten parallel profiles of regularly spaced micronemes, demonstrating that these organelles are set peripherally around and below the conoid. Several profiles typical of rhoptries are also present in this section. In the reconstructed model, the radial disposition of the micronemes around and below the conoid is very clear, as are the membrane-bound vesicles inside the conoidal channel that run parallel to the inner pair of microtubules (Fig. 3C). In Fig. 3D, fourteen rhoptries, each displayed in a different color, could be counted, but only four extended into the conoidal channel. In the Fig. 3E was observed the capacity of the conoid which supports four rhoptry necks. The FIB-SEM dual-beam system, used for serial sectioning of chemically fixed and epoxy-embedded samples, brought threedimensional reconstruction to a new level; this system made it possible to cover a volume much larger than could previously be covered using only serial sections obtained by ultramicrotomy. Furthermore, the slice-and-view procedures (Bennett et al., 2009) result in thinner sections, avoiding artifacts due to section compression or dilation. In addition, a large number of cells can be traced in a single volume (Supplemental material 2). A whole T. gondii tachyzoite (Melo et al., 2000) had already been reconstructed from serial sections, showing features such as the location of rhoptries and dense granules and the single mitochondrion. This technique, however, has some intrinsic and unavoidable artifacts and does not have the necessary resolution to reconstruct the fine-level organization of several structures, such as the micronemes and the conoid. On the other hand, electron tomography,

Fig.2. 3D reconstruction of an extracellular tachyzoite. (A–D) Transversal slices of a parasite used for reconstruction from the apical (A), (B) to the middle (C) and posterior portions of the cell body, by FIB-SEM dual beam. (A) Face view of the polar ring (arrow) with micronemes (arrowhead) around it and rhoptry necks inside. (B) Between the polar ring and the nucleus, a rhoptry (r) and a dense granule (dg) can be identified. (C) At the upper level of the nucleus (N) elements from the Golgi complex (GC) and mitochondrial profiles (m) are seen. (D) Rendered model. The plasma membrane is transparent white. Inside the parasite several dense granules (blue) are scattered in the cytoplasm, 10 rhoptries (green) and many micronemes (red) are seen around the conoid (arrow). The neck of one rhoptry is seen inside the conoid (white arrow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

425

Fig.3. 3D reconstruction of the apical complex of T. gondii. (A and B) Two slices of the parasite used for the reconstruction shown in C and D, by FIB-SEM dual beam. (A) Section showing the inside of the conoid where vesicles aligned (small arrows) and a pore-like structure (arrowhead) at the apex of the cell are shown. (B) Conoidal fibers (arrow) and below them the radial profile of the micronemes (arrowheads). Several rhoptries (R) are also seen in this section. (C and D) Models rendered. (C) The five spherical vesicles from A are depicted in yellow, running parallel to the two microtubules (green) inside the conoid. Micronemes (red) encircle the basis of the conoid (gray). (D) The 14 rhoptries of this cell are represented, each in a different color. (E) Higher magnification of the model showing that only four rhoptries (light and dark blue, magenta and purple), reach the inside of the conoid, practically filling its entire internal diameter.

despite its power of resolution, is limited to relatively small volumes, making it incapable of encompassing entire parasites. In conclusion, the slice-and-view approach with the dual-beam SEM sheds new light on issues such as the number of rhoptries that reach the inside of the conoid and the organization of micronemes and dense granules. For Toxoplasma, the FIB serial sectioning followed by 3D reconstruction demonstrated that up to and no more than four rhoptry necks can fit inside the conoid (Fig. 3E). In addition, this technique gave further evidence of the radial disposition of the micronemes, which are found just below the posterior ring and surrounding the conoid. Micronemes have been mistakenly identified as pre-rhoptries by Vivier and Petitprez (1972). Furthermore, Lemgruber et al. (2010) showed, in freeze-fracture replicas, the presence of bridges between the micronemes. Those bridges may help to maintain this radial organization. The 3D model also shows the scattered distribution of dense granules, which were already seen in random sections. To confirm the distribution of secretory organelles throughout the intracellular cycle, classical ultrathin serial sectioning was performed. The 3D models rendered from serial sections had a much lower resolutions than when FIB-SEM slice and view was employed. Nevertheless, those series consistently correlated with the information from morphometric analyses of random sections at the same times of development (45 min, 7 and 24 h post infection) (Supplemental materials 4 and 5).

3.3. Ultrastructural aspects of secretion along the intracellular cell cycle Some of the ultrastructural features observed in random sections were observed frequently enough to be considered significantly related to the adhesion and invasion of the parasites to the host cell and secretion processes, particularly in short-term interactions. Fig. 4A shows a tachyzoite with the apical portion adhered to a host cell membrane, issuing from which are numerous filopodia that surround and touch the parasite. The conoid of this parasite is not extruded, and the micronemes display the same radial and even distribution that has already been described in the 3D models and seen in higher detail in Fig. 4B. Fig. 4B shows that micronemes are oriented, not towards the conoidal channel, but around it. Randomly oriented micronemes are also seen, suggesting that, as the aligned micronemes are exocytosed, new micronemes will dock at the site of secretion. The microneme-docking site is probably located in the plasma membrane at the level of the posterior polar ring and would be available only when the conoid is in the upper position, when a double membrane covers the area. To further confirm the position of the micronemes, we fixed the cells by high-pressure freezing in association with freeze-substitution to ensure the most reliable preservation of the shape and arrangement of these organelles. The pattern obtained was equal to what we observed after chemical fixation (Fig. 4C). These

426

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

Fig.4. Positioning of micronemes. (A) In a thicker section, the parasite is seen adhered to the host cell (HC) by the conoid and surrounded by filopodia (arrow), (R) Rhoptry, (DG) dense granule. (B) High magnification of the apical portion of a parasite with the porosome-like structure on the membrane over the conoid (arrowhead) apparent. Micronemes (arrow) are radially disposed just below the base of the conoid (c). Microneme profiles randomly distributed (white arrow) are also observed. (C) Radial disposition of micronemes (arrows) below the conoid in a tachyzoites fixed by HPF-FS. Non oriented microneme profiles are also seen (arrowheads). (D) Scanning electron microscopy of a parasite with vesicles (arrowhead) at the base of the extruded conoid.

observations are reinforced by the results generated by Field Emission Scanning Electron Microscopy (FE-SEM): parasites are seen with the conoid extruded and show evidence of secretion (Fig. 4D). After extensive observation of random sections, we propose that microneme secretion always take place at the level of the posterior polar ring. We consider this proposition reasonable, as microneme secretion and conoid extrusion are both dependent on calcium and can be induced by calcium ionophores (Carruthers and Sibley, 1999; Lovett and Sibley, 2003; Mondragon and Frixione, 1996; González Del Carmen et al., 2009). Thus, micronemes would require that the conoid be extruded in order to dock and fuse with a specific domain at the plasma membrane. Furthermore, fusion at this region would be favored because it lacks the two inner membranes that constitute the inner pellicle. This hypothesis is an alternative to the proposal of Carruthers and Sibley (1999), who proposed that micronemes migrate to the inside of the conoid and secrete at the same place as the rhoptries. No micronemes were observed inside the conoid, although we obtained and observed several series of sections that covered the entire volume of the conoid (Fig. 4A–C). Indeed, although extensive observations have shown micronemes positioned radially around the conoid and below the posterior polar ring, no micronemes were ever seen in the process of secretion. This assembly may be maintained by the thin filaments that connect adjacent micronemes that were observed by Lemgruber et al. (2010) in replicas of freeze fractured-deep etched tachyzoites.

As seen in the 3D models, not all rhoptries reach the inside of the conoid (Fig. 3A–E). Among those few, some appeared to be docking to a specific region at the apical portion of the tachyzoite, which was clearly visible in ultrathin sections as a circular structure approximately 50 nm in diameter, positioned at the tip of the membrane covering the conoid (Figs. 4B and 5A and B). The presence and location of this structure was further confirmed by electron tomography of a 200-nm section of an extracellular parasite (Fig. 6A and B and Supplemental material 3, video file). The tomogram showed membrane regions over the conoid that had a different texture at the probable point of rhoptry docking. Similar structures have already been observed in acinar cells from mammals and are called porosomes (Jena et al., 2003). This porosome-like structure was also observed by FE-SEM, as shown in Fig. 5C and D, as a slight depression at the center of the conoid. This structure was also present in samples that were high-pressure-frozen and freeze-substituted (Fig. 5A). Evidence that this structure may be a docking point for rhoptries is shown in Fig. 5B, which depicts a chemically fixed parasite with a semi-empty rhoptry just below it. The exact position of and trigger for rhoptry secretion are still unknown; however, Porchet and Torpier (1977) had already demonstrated the existence of a rosette of particles at the tip of the conoid that could correspond to these porosome-like structures. Porosomes are preferential sites for vesicle fusion; they measure 125–185 nm in diameter and have been described in specialized secretory cells (Jena et al., 2003; Jena, 2009). Electron tomography

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

427

Fig.5. (A) A parasite fixed by HPF-FS showing four vesicles aligned along the central pair of microtubules (small arrows), a rhoptry (R) neck runs inside the conoid and micronemes (small arrowheads) are positioned below it. The arrowhead on the right points to a bump at the plasma membrane just at a point where the inner pellicle is absent. (B) Apical complex of adhered parasite chemically fixed with a rhoptry probably secreting (arrowhead) docked to a porosome-like structure (arrow). (C) Scanning electron microscopy of extracellular parasites pointing to the probable site of rhoptry secretion (arrow). Inset: face view of the apical portion of T. gondii. The arrow points to the porosome-like structure.

Fig.6. Electrontomography A and B two Z sections of the apical complex of an extracellular tachyzoites. The porosome-like structure is pointed by the arrow.

revealed a structure just above a rhoptry neck that measures 50 nm in diameter. The difference between the size of the porosomes described in acinar cells and the structure observed in T. gondii can be explained by the size of the secretory granules and of the rhoptry necks. Plattner and Kissmehl (2003) have described dense core vesicles in Paramecium that secrete at specialized sites in the plasma membrane that are comparable with porosomes. Thus, we can hypothesize that the same phenomenon would occur at different scales in large cells like Paramecium and small cells like T. gondii. Although each parasite has a large number of rhoptries (Scholtyseck and Melhorn, 1970), at most one empty rhoptry per cell was observed, reinforcing the hypothesis that each rhoptry has to dock at the porosome-like to expel its contents.

As shown in the FIB-SEM series and in the model in Fig. 3A–C and E, along with the pair of microtubules and the rhoptry necks, we often observed membrane-bound vesicles measuring an average of 50 nm of diameter inside the conoid (Figs. 5A and 7A). These vesicles have previously been mentioned by several authors (Vivier and Petitprez, 1972; Porchet-Hennere and Nicolas, 1983; Lemgruber et al., 2010), but their origin and role are still unknown. Parasites captured during or immediately after internalization often show a structure at the conoidal inner channel that contains several membrane-bound vesicles called empty rhoptries (Fig. 7A and B). This structure differs from other vacuoles because it is elongated and passes through the conoidal channel. The structure coexists with intact rhoptries. Immunocytochemistry using antibodies against rhoptry proteins ROP-2,4 and RON-4 positively labeled

428

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

Fig.7. (A) High magnification of an invading parasite. An empty rhoptry with the neck (arrowhead) passing through the conoidal channel and five vesicles aligned parallel to it (arrow) A sixth vesicle (asterisk is seen outside the parasite. The conoid of this parasite is nor in the extruded position (thick arrows). (B–D) Immunocytochemistry. (B) Empty rhoptry in an intracellular parasite labeled for ROP-2,4, confirming its nature. (C) Double labeling for rhoptry neck protein RON-4 (15 nm Au) (arrow) and ROP-2,4 (10 nm Au) (double arrow) in a parasite adhered to a host cell, the bulb presents an homogeneous pattern of labeling. Fifteen nanometers gold particles are seen only in the upper portion (neck) of the rhoptry. (D) Dense granules in a tachyzoite double labeled for GRA-3 (10 nm Au) and GRA-7 (15 nmAu) (arrowhead), showing that there are no subpopulations of dense granules.

these large vesicles, confirming that they are rhoptries that have partially secreted their contents (Fig. 7B). We also tested the proposition of Sibley et al. (1995) that, given that one organelle secretes at a time, there may be subpopulations of organelles in different states of maturation. To this end, we employed antibodies against various proteins associated with rhoptries and dense granules. Ultimately, this hypothesis was not supported by immunolabeling for two different rhoptry proteins (ROP/RON) and two distinct dense granule proteins (GRA). For each organelle, we tested two antibodies: anti-GRA-3 and anti-GRA-7 for dense granules (Fig. 7D) and anti-RON-4 (a rhoptry-neck protein) and anti-ROP-2,4 (a rhoptry-bulb protein) for rhoptries (Fig. 7C). This method revealed no subpopulations of dense granules or rhoptries (Fig. 7C and D). As the infection proceeds and the parasitophorous vacuole grows, the secretion of dense granules becomes more intense; this event, however, is rarely observed, probably because occurs very quickly. Fig. 8A and B depict two of these moments. In Fig. 8A the secretion seems to be accumulating between the inner pellicle and the plasma membrane, while in Fig. 8B the granule seems to be fusing with the plasma membrane in a classical manner. Dubremetz et al. (1993) have shown that dense granule secretion takes place preferentially in the lateral, apical portion of the parasite and have demonstrated that the accumulation of secretion seen in Fig. 8A was an effect of the plane of section; in another plane, the contents of the granule would be seen sorting to the intravacuolar space. This kind of fusion and the ‘‘bumps’’ corresponding to the accumulation of dense-granule contents had also

previously been reported by Monteiro et al. (2001), and would be a consequence of the inner pellicle structure, as described by Porchet and Torpier (1977). The inner pellicle fenestrations, if too small, wouls impair granule fusion. A similar phenomenon has been described in the secretion of microvsicles by immune cells (Théry et al., 2009). 4. Conclusions Taken together, the data resulting from stereological analysis demonstrate that microneme secretion is very intense during recognition and adhesion by the T. gondii tachyzoites. Rhoptry secretion, in contrast, occurs upon entry, and dense granules are constantly exocytosed after entry, causing their numbers to decrease; however, none of these organelles are eliminated. Observation of random sections from samples processed by routine chemical fixation or high-pressure freezing and freeze-substitution strongly suggests that micronemes dock and fuse with the plasma membrane at a point below the posterior polar ring, while the conoid is in its upward position. Rhoptries, in turn, expel their contents after passing through the conoidal channel and docking, one at a time, to a porosome-like structure in the plasma membrane above. Up to four rhoptries at a time can reach the inside of the conoid, although more than 10 could be counted in a single cell. Finally, dense granules constitutively fuse and are secreted from the very first moments of invasion of the host cell. For clarity, we have created a model of the secretory process in T. gondii tachyzoites in which rhoptries secrete through the porosome in the

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

429

middle of the conoid tip, the micronemes secrete just below the posterior polar ring, and the dense granules probably secrete at sites of interruption in the inner-membrane complex (Fig. 9). These results also raise and leave unanswered a number of other questions: why are there so many rhoptries if a few at most are used in invasion? What is the nature of the 50-nm vesicles that were so frequently observed inside the conoid; are they secretory or endocytic in nature? Is the calcium trigger for microneme secretion independent of the trigger for conoid extrusion, or does conoid extrusion free the site for microneme docking, fusion and secretion? Acknowledgments The authors are grateful to FEI-Company Nanoport Eidhoven, David Wall for the FIB-SEM Dual Beam series, and to Felix de Haas for the tomogram. To Renata Travassos and Nathalia Muller for technical assistance in high pressure freezing experiments. To J.F. Dubremetz and John Boothroyd, for the primary antibodies, and to Dirceu Esdras do Nascimento for the Toxoplasma model. The present work was supported by CNPq, FAPERJ and PRONEX. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jsb.2011.11.028. References

Fig.8. Dense granule secretion in intracellular parasites. (A) The content of a dense granule is seen accumulated between the plasma membrane and the inner membrane complex forming a bubble (arrowhead). (B) A dense granule is seen fusing with the plasma membrane (arrow).

Fig.9. Model compiling the results presented: secretion of rhoptries (green) takes place one at a time, inside the conoid through docking to the porosome. Micronemes (red) dock to the plasma membrane just below the posterior polar ring requiring that the conoid is extruded; and dense granules (dark blue) fuse and release its contents through the fenestrated inner membrane complex. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Alexander, D.L., Mital, J., Ward, G.E., Bradley, P., Boothroyd, J.C., 2005. Identification of the Moving Junction Complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog. 1, 137–149. Attias, M., Vommaro, R.C., DeSouza, W., 1996. Computer aided three-dimensional reconstruction of the free-living protozoan Bodo sp. (Kinetoplastida Bodonida). Cell Struct. Funct. 21, 297–306. Bennett, A.E., Narayan, K., Shi, D., Hartnell, L.M., Gousset, K., et al., 2009. Ionabrasion scanning electron microscopy reveals surface-connected tubular conduits in HIV-infected macrophages. PLoS Pathog. 5, 1–10. Besteiro, S., Bertrand-Michel, J., Lebrun, M., Vial, H., Dubremetz, J.F., 2008. Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol. J. Biochem. 415, 87–96. Besteiro, S., Michelin, A., Poncet, J., Dubremetz, J.F., Lebrun, M., 2009. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5, 1–14. Boothroyd, J.C., Dubremetz, J.F., 2008. Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat. Rev. Microbiol. 6, 79–88. Bradley, P.J., Sibley, L.D., 2007. Rhoptries: an arsenal of secreted virulence factors. Curr. Opin. Microbiol. 10, 582–587. Carruthers, V.B., Sibley, L.D., 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur. J. Cell Biol. 73, 114–123. Carruthers, V.B., Sibley, L.D., 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol. Microbiol. 31, 421–428. Cesbron-Delauw, M.F., 1994. Dense-granule organelles of Toxoplasma gondii: their role in the host–parasite relationship. Parasitol. Today 10, 293–296. Chaturvedi, S., Qi, H., Coleman, D., Rodriguez, A., Hanson, P.I., et al., 1999. Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab Machinery. J. Biol. Chem. 274, 2424– 2431. Dubey, J.P., Lindsay, D.S., Speer, C.A., 1998. Structures of Toxoplasma gondii Tachyzoites, Bradyzoites, and Sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267–299. Dubremetz, J.F., Achbarou, A., Bermudes, D., Joiner, K.A., 1993. Kinetics and pattern of organelle exocytosis during Toxoplasma gondii host–cell interaction. Parasitol. Res. 79, 402–408. González Del Carmen, M., Mondragón, M., González, S., Mondragón, R., 2009. Induction and regulation of conoid extrusion in Toxoplasma gondii. Cell. Microbiol. 11, 967–982. Jena, B.P., 2009. Porosome: the secretory portal in cells. Biochemistry 48, 4009– 4018. Jena, B.P., Cho, S.J., Jeremic, A., Stromer, M.H., Abu-Hamdah, R., 2003. Structure and composition of the fusion pore. Biophys. J. 84, 1337–1343. Jones, T.C., Yeh, S., Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells: mechanism of entry and intracellular fate of the parasite. J. Exp. Med. 136, 1557–1572. Keeley, A., Soldati, D., 2004. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol. 14, 528–532.

430

T.C. Paredes-Santos et al. / Journal of Structural Biology 177 (2012) 420–430

Kremer, J.R., Mastronarde, D.N., Mcintosh, J.R., 1996. Computer visualization of three dimensional image data using IMOD. J. Struct. Biol. 116, 71–76. Lemgruber, L., Lupetti, De., Souza, W., Vommaro, R.C., 2010. New details on the fine structure of the rhoptry of Toxoplasma gondii. Microsc. Res. Tech. 2, 1–8. Lovett, J.L., Sibley, L.D., 2003. Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells. J. Cell Sci. 116, 3009–30016. Magno, R.C., Lemgruber, L., Vommaro, R.C., Souza, W., Attias, M., 2005. Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole. Microsc. Res. Tech. 67, 45–52. Melo, E.J.L., Attias, M., De Souza, W., 2000. The single mitochondrion of tachyzoites of Toxoplasma gondii. J. Struct. Biol. 130, 27–33. Mercier, C., Adjogbleb, K.D.Z., Daubenerb, W., Cesbron-Delauw, M.F., 2005. Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? Int. J. Parasitol. 35, 829–849. Mondragon, R., Frixione, E., 1996. Ca(2+)-dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J. Eukarytic. Microbiol. 43, 120–127. Monteiro, V.G., de Melo, E.J.T., Attias, M., de Souza, W., 2001. Morphological changes during conoid extrusion in Toxoplasma gondii tachyzoites treated with calcium ionophore. J. Struct. Biol. 136, 181–189. Morisaki, J.H., Heuser, J.E., Sibley, L.D., 1995. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci. 108, 2457–2464. Nichols, B.A., Chiappino, M.L., O’Connor, G.R., 1983. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J. Ultrastruct. Res. 83, 85–98. Plattner, H., Kissmehl, R., 2003. Dense-core secretory vesicle docking and exocytotic membrane fusion in Paramecium cells. Biochim. Biophys. Acta 164, 183–193.

Porchet, E., Torpier, G., 1977. Freeze fracture study of Toxoplasma and Sarcocystis infective stages. Z. Parasitenk. 54, 101–124. Porchet-Hennere, E., Nicolas, G., 1983. Are rhoptries of Coccidia really extrusomes? J. Ultrastruct. Res. 84, 194–203. Saffer, L.D., Mercereau-Puijalon, O., Dubremetz, J.F., Schwartzman, J.D., 1992. Localization of a Toxoplasma gondii rhoptry protein by immunoelectron during and after host cell penetration. J. Protozool. 39, 526–530. Scholtyseck, E., Melhorn, H., 1970. Ultrastructural study of characteristic organelles Paired organelles, micronemes, micropores of Sporozoa and related organisms. Z. Parasitenk. 34, 97–127. Shaw, M.K., Ross, D.S., Tilney, L.G., 1998. Acidic compartments and rhoptry formation in Toxoplasma gondii. Parasitology 117, 435–443. Sibley, D.L., Niesman, I.R., Parmley, S.F., Cesbron-Delauw, M.F., 1995. Regulated secretion of multi-lamelar vesicles leads to formation of a tubule-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell Sci. 108, 1669–1677. Théry, C., Ostrowski, M., Segura, E., 2009. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol., 581–593. Vivier, E., Petitprez, A., 1972. Données ultrastructurales complémentaires, morphologiques et cytochimiques, sur. Toxoplasma gondii. Protistologica 7, 110–123. Zhou, X.W., Kafsack, B.F.C., Cole, R.N., Beckett, P., Shen, R.F., et al., 2005. The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival. J. Biol. Chem. 280, 34233–34244.