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Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis
Journal of Structural Biology xxx (2016) xxx–xxx
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Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis Juliana C. Vidal, Carolina de L. Alcantara, Wanderley de Souza, Narcisa L. Cunha-e-Silva ⇑ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil
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Article history: Received 21 April 2016 Received in revised form 20 July 2016 Accepted 27 July 2016 Available online xxxx Keywords: T. cruzi Metacyclogenesis Cytostome-cytopharynx Endocytosis
a b s t r a c t Trypanosoma cruzi epimastigotes uptake nutrients by endocytosis via the cytostome-cytopharynx complex – an anterior opening (cytostome) continuous with a funnel-shaped invagination (cytopharynx) that extends to the posterior of the cell, accompanied by microtubules. During metacyclogenesis – the transformation of epimastigotes into human-infective metacyclic trypomastigotes – the cytostomecytopharynx complex disappears, as trypomastigotes lose endocytic ability. To date, no studies have examined cytostome-cytopharynx complex disappearance in detail, or determined if endocytic activity persists during metacyclogenesis. Here, we produced 3D reconstructions of metacyclogenesis intermediates (Ia, Ib, Ic) using electron microscopy tomography and focused ion beam-scanning electron microscopy (FIB-SEM), concentrating on the cytostome-cytopharynx complex and adjacent structures, including the preoral ridge (POR). Parasite endocytic potential was examined by incubation of intermediate forms with the endocytic tracer transferrin (Tf)-Au. Ia, Ib and Ic cells were capable of internalizing Tf-Au, and had a shorter cytopharynx than that of epimastigotes, with the cytostome/POR progressively displaced towards the posterior, following the movement of the kinetoplast/flagellar pocket. While some Ic cells had a short cytopharynx with an enlarged proximal end (300 nm in diameter, larger than that of the cytostome), other Ic cells had no cytopharynx invagination, but retained the cytopharynx microtubules, which were also present in metacyclics. We conclude that cytostome-cytopharynx disappearance and loss of endocytic ability are late events in metacyclogenesis, during which the cytostome is displaced towards the posterior, probably due to a link to the kinetoplast/flagellar pocket. Retention of the cytopharynx microtubules by metacyclics may allow prompt cytostome-cytopharynx reassembly in amastigotes, upon host cell infection. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Trypanosoma cruzi – the causative agent of Chagas disease – has a complex life cycle involving mammalian and invertebrate hosts. Epimastigotes, the proliferating forms found in the insect vector gut, are capable of internalizing large amounts of transferrin, peroxidase, albumin and low-density lipoprotein from the extracellular milieu (Soares and de Souza, 1991). In this stage of the life cycle, the main site of endocytosis is the cytostome-cytopharynx complex (Porto-Carreiro et al., 2000), rather than the flagellar pocket, the exclusive site for endocytosis in other trypanosomatids (Webster and Russell, 1993). ⇑ Corresponding author at: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, bloco G subsolo, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 21941-902, Brazil. E-mail address: [email protected] (N.L. Cunha-e-Silva).
The cytostome consists of an opening at the epimastigote anterior region, continuous with a funnel-shaped invagination of the plasma membrane called the cytopharynx (Milder and Deane, 1969). Next to the cytostome, a specialized membrane domain called the preoral ridge (POR) separates the cytostome from the flagellar pocket (De Souza et al., 1978; Galbraith and McElrath, 1988; Pimenta et al., 1989; Vatarunakamura et al., 2005). The epimastigote preoral ridge has a thick electron-dense glycocalyx that makes it easily detectable by transmission electron microscopy (TEM)(Vatarunakamura et al., 2005). The participation of microtubules in the structure of the cytostome-cytopharynx complex is a hallmark of trypanosomatids (Chang et al., 1975; Sassa et al., 1975) and bodonids (Attias et al., 1996). The long and thin invagination of the cytopharynx is accompanied by two microtubule sets: a triplet that extends from underneath the cytostome membrane, and a quartet that originates close to the flagellar-pocket and continues underneath the
http://dx.doi.org/10.1016/j.jsb.2016.07.018 1047-8477/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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preoral ridge membrane domain, following the path of the cytopharynx towards the posterior of the cell (Alcantara et al., 2014). In T. cruzi, the cytostome-cytopharynx complex is present only in epimastigotes and amastigotes (Chang et al., 1975; Meyer and de Souza, 1973), but not in trypomastigotes. Thus, at the end of metacyclogenesis – the stage transition from epimastigotes to human-infective metacyclic trypomastigotes – the entire cytostome-cytopharynx complex must be disassembled, as trypomastigotes lose endocytic ability. Metacyclogenesis involves morphological, functional and protein expression changes, and can be reproduced in vitro, which allowed the description of several intermediate stages between epimastigotes and metacyclic trypomastigotes (Brener, 1973; Cortez et al., 2006; Ferreira et al., 2008; Perlowagora-Szumlewicz and Moreira, 1994; Schaub, 1988). In an ultrastructural study, Ferreira et al. (2008) reported the presence of the following types of T. cruzi intermediate forms between epimastigotes and trypomastigotes: intermediate Ia, with the kinetoplast anterior and very close to the nucleus (similarly to that observed in epimastigote forms); intermediate Ib, with the kinetoplast and nucleus side by side; and intermediate Ic, where the kinetoplast is posterior to the nucleus (as in trypomastigotes forms). A schematic drawing illustrating the intracellular distribution of organelles in T. cruzi intermediate forms (Ia, Ib and Ic) in comparison with epimastigote forms is shown in Fig. S1. In Ic forms both the nucleus and the kinetoplast exhibit signs of ultrastructural remodeling into the morphologies observed in metacyclic trypomastigotes, which have an elongated nucleus and a round kinetoplast at the posterior end of the cell. Although overall morphological descriptions of the intermediate forms have been reported, no studies have described in detail the restructuring/ disassembly of the cytostome-cytopharynx complex during metacyclogenesis. Also, the endocytic potential of metacyclogenesis intermediates has not been examined to date. In this work, we used advanced electron microscopy techniques – namely tomography and focused ion beam scanning electron microscopy (FIB) – to produce 3D reconstructions of the three metacyclogenesis intermediates (Ia, Ib, Ic, as described in (Ferreira et al., 2008) between epimastigotes and trypomastigotes. Aside from providing a detailed description of the ultrastructural changes involved in cytostome-cytopharynx disappearance during metacyclogenesis, we used transferrin as an endocytic tracer, followed by TEM observation, to evaluate the endocytic ability of the intermediate forms directly. 2. Materials and methods 2.1. Parasites Trypanosoma cruzi epimastigotes from the Dm28c clone were cultivated in liver infusion tryptose (LIT) medium (Camargo, 1964) supplemented with 10% (v/v) foetal calf serum (FCS) at 28 °C. Stationary-phase parasites from six-day-old cultures were used in all experiments. 2.2. In vitro metacyclogenesis Epimastigotes were induced to differentiate to metacyclic trypomastigotes as described previously (Contreras et al., 1985). Briefly, epimastigotes were collected by centrifugation at 3000g for 30 min, washed once in triatomine artificial urine (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM sodium phosphate buffer, pH 6.0) and adjusted to 5 108 cells/mL in the same medium. After 2 h at 28 °C, parasites were diluted 100-fold in TAU supplemented with 50 mM sodium glutamate,
10 mM L-proline, 2 mM sodium aspartate and 10 mM glucose (TAU3AAG) in cell culture flasks, and maintained at 28 °C, to induce metacyclogenesis. Metacyclogenesis induction was halted (at 24 or 48 h) by fixation for transmission electron microscopy. 2.3. Endocytosis assay Parasites undergoing metacyclogenesis were washed twice in phosphate-buffered saline (10 mM sodium phosphate buffer with 0.9%, NaCl, w/v, pH 7.2) and then incubated in RPMI medium containing transferrin coupled to 10 nm gold particles (Tf-Au) (Slot and Geuze, 1985) for 30 min at 28 °C. Parasites were fixed and processed for transmission electron microscopy observation, as detailed below. 2.4. Sample preparation for transmission electron microscopy (TEM) Parasites undergoing metacyclogenesis were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. Then, cells were washed twice in 0.1 M cacodylate buffer, pH 7.2, and post-fixed using an OTO (osmium-thiocarbohydrazide-osmium) protocol (Willingham and Rutherford, 1984). Briefly, cells were incubated in a post-fixative solution consisting of 1% (v/v) osmium tetroxide, 0.8% (v/v) potassium ferrocyanide, 5 mM calcium chloride, in 0.1 M cacodylate buffer (pH 7.2) for 40 min, washed twice in water and then incubated in a solution of 1% (w/v) thiocarbohydrazide (TCH – Sigma, St. Louis, USA) in water for 5 min. After three washes in water, cells were incubated again in the post-fixative osmium solution for 3 min. Following postfixation, samples were washed in water, dehydrated in an acetone series and embedded in Epoxy resin (EMbed-812/ Electron Microscopy Sciences). Images were recorded using a Helios 200 NanoLab dual-beam microscope (Eindhoven, NL) equipped with a gallium ion source for focused-ion beam milling, and a field emission gun with an in-lens secondary electron detector, for scanning electron microscopy imaging by SmartSEM software (V05.07 SP4, Carl Zeiss). Blocks were subjected to focused ion beam scanning electron microscopy or serial electron tomography (see below). 2.5. Serial electron tomography For tomography, 200-nm-thick serial sections were collected onto formvar-coated copper slot grids, stained with 5% (w/v) uranyl acetate and lead citrate (Reynolds, 1963) and incubated with 10-nm colloidal gold particles (Gold colloid, Sigma Aldrich) for 5 min. Then, sections were washed with distilled water and observed on a Tecnai-G2 transmission electron microscope operating at 200 kV (FEI Company, Eindhoven, Netherlands) equipped with a 4 k CCD camera. Single-axis tilt series (±60° with 1° increments) were produced. Alignments were applied using gold as fiducial markers and back projections with IMOD software package. 2.6. Focused ion beam-scanning electron microscopy (FIB-SEM) For observation by FIB-SEM, parasites undergoing metacyclogenesis for 24 h or 48 h were processed for TEM as described above. The tips of resin blocks containing the sample were then trimmed to a pyramidal shape and mounted on a support for FIB-SEM. Images were recorded using a Helios 200 NanoLab dual-beam microscope (Eindhoven, NL) equipped with a gallium ion source for focused-ion beam milling, and a field emission gun with an in-lens secondary electron detector, for scanning electron microscopy imaging by SmartSEM software, release number V05.07 SP4 (Carl Zeiss). ‘Slice-and-view’ image series were generated using a milling step size of 25 nm. After image capturing, the
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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contrast of backscattered electron images, recorded by Energy selective Backscattered (EsB) Detector, was inverted so that they resembled conventional TEM images. Sequential slices were automatically aligned using the XfAlign algorithm of IMOD (Kremer et al., 1996) and a fine alignment was then performed using MIDAS. All 3D models and measurements were performed with 3DMOD. 2.7. Negative staining TEM Cells were harvest by centrifugation at 1500g, for 30 min, resuspended in LIT medium (liver infusion tryptose) (Camargo, 1964) and allowed to adhere to glow-discharged and formvar-coated grids. Then, cells were extracted in 1% (v/v) NP-40 in PEME buffer (100 mM PIPES, 1 mM MgSO4, 0.1 mM EDTA and 2 mM EGTA, pH 6.9) for 5 min, and fixed in 2.5% (v/v) glutaraldehyde in PEME, for 10 min. Grids were negatively stained with 0.7% (v/v) aurothioglucose in water and observed in a Tecnai Spirit transmission electron microscope, operating at 120 kV. 3. Results 3.1. Metacyclogenesis intermediate forms are capable of endocytosis An important physiological hallmark of metacyclogenesis is a decrease in endocytic ability, as highly endocytic epimastigotes transform into infective metacyclic trypomastigotes; nevertheless, the dynamics of the main endocytic structure of epimastigotes – the cytostome-cytopharynx complex – during metacyclogenesis has not been described in detail, and little is known about the endocytic capacity of intermediate forms between epimastigotes and metacyclics.
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To study cytostome-cytopharynx dynamics during metacyclogenesis, we successfully recapitulated this process in vitro using a method described previously (Contreras et al., 1985), whereby incubation of epimastigotes in TAU3AAG medium triggered parasite adhesion to culture flasks and started morphological differentiation into trypomastigote forms. Cells were classified into one of the following T. cruzi developmental stages: epimastigotes, metacyclic trypomastigotes or metacyclogenesis intermediate forms, Ia, Ib and Ic (Ferreira et al., 2008), based on nucleus and kinetoplast/flagellum position. To investigate the effect of metacyclogenesis induction on endocytic activity, parasites were harvested after 24 h and 48 h of metacyclogenesis, subjected to an endocytic assay using transferrin coupled to 10 nm gold particles (Tf-Au) as cargo, fixed and observed by TEM (Fig. 1). Tf-Au cargo was found inside reservosomes – the late endosomes of T. cruzi (Soares and de Souza, 1991; Soares et al., 1992) – in intermediate Ia (Fig. 1A and D, white arrows), Ib (Fig. 1B and E, white arrows) cells, and also in reservosomes and vesicle in intermediate Ic (Fig. 1C and F, white arrows). Also, intermediate Ib had a POR membrane domain with its characteristic dense coat (black arrows in Fig. 1B). These findings indicated that inducing metacyclogenesis did not block parasite endocytic activity. Morphologically, intermediate Ia was often difficult to distinguish unambiguously from epimastigotes; thus, we focused our morphological analysis on intermediates Ib and Ic. 3.2. The cytostome-cytopharynx complex is displaced towards the posterior during metacyclogenesis At 48 h of metacyclogenesis numerous parasites were at stages Ib and Ic, which are clearly distinguishable from epimastigotes (Ferreira et al., 2008); thus, this time-point was chosen for the
Fig. 1. Endocytic activity in metacyclogenesis intermediates. After 24 h of metacyclogenesis induction, cells were incubated with transferrin coupled to 10-nm gold particles (used as an endocytic tracer), and then processed for transmission electron microscopy (TEM). Both intermediate Ia (with kinetoplast – K – immediately anterior to the nucleus – N; in A and D), intermediate Ib (with the kinetoplast adjacent to the nucleus, in B and E) and intermediate Ic (with kinetoplast posterior to the nucleus, in C and F) cells had late endosomal structures (reservosomes, R) and vesicles containing transferrin-gold particles (white arrows), indicative of endocytosis. D, E and F show higher magnification images of the areas indicated by the rectangles in A (D), B (E) and C (F). In intermediate Ic cells (note the kinetoplast at the posterior, near a reservosome), a vesicle loaded with gold particles is located immediately anterior to the kinetoplast. Intermediate b cells had a clear preoral ridge membrane domain (POR, black arrows, in B). Gc, Golgi complex; F, flagellum. Scale bars: 1 lm (A, B and C); 200 nm (D, E, F).
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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analysis of mid/late metacyclogenesis intermediates by electron microscopy. The cytostome-cytopharynx is a long and curved invagination of the plasma membrane that cannot be visualized in its entirety by conventional ultrathin-section TEM. Therefore, we used FIB-SEM to produce 3D reconstructions of the entire cytostome-cytopharynx complex of metacyclogenesis intermediates at an ultrastructural level, and combined these data with ‘targeted’ serial electron tomography or ultrathin-section TEM of specific morphological aspects that required high-resolution structural analysis. Samples were processed using thiocarbohydrazide, which enhanced the contrast of the cytopharynx membrane domain, facilitating its recognition in TEM images. We analyzed the cytostome-cytopharynx area (or the expected area of the cytostome-cytopharynx) from 51 mid (Ib)/late (Ic) metacyclogenesis intermediates. Among the 12 cells observed in FIB-SEM series, 3 representative cells were selected for a detailed analysis by 3D reconstruction. We reconstructed 4 intermediate Ib cells (with the kinetoplast adjacent to nucleus) and all had a clear preoral ridge (POR) and a curved cytopharynx invagination that reached the posterior of the cell (Fig. 2A–G). We have considered the hypothesis that, during metacyclogenesis, the cytostome-cytopharynx complex may accompany the kinetoplast/flagellar pocket complex in its migration to the posterior region of the cell. To examine this possibility, we compared FIB-SEM-based 3D reconstructions of epimastigotes and Ib cells that were in a similar orientation relative to the plane of viewing (and had been imaged at the same magnification; Fig. 2H). Positioning of the two models showed that the distance between the POR and nucleus decreased gradually with the progression metacyclogenesis, from epimastigotes to Ib cells (Fig. 2). Epimastigote forms had a spherical nucleus with a disk-shaped kinetoplast (K, in green), and a flagellum (F, in yellow) emerging from the anterior portion of the cell (Fig. 2H). Note that, as
differentiation proceeded, both the kinetoplast and the point of flagellum emergence from the cell surface moved towards the middle of the cell. As a consequence of this migration, the nucleus became sinuous and the POR (in purple) of intermediate Ib cells was localized immediately adjacent to the nucleus (Fig. 2D–G). 3.3. The cytostome disappears late during the Ic stage of metacyclogenesis Intermediate Ic differs from metacyclic trypomastigotes by having rod shaped kinetoplast, although both forms have a posterior kinetoplast. During the Ic stage, the Golgi complex migrates from the anterior of the cell to the posterior; thus, Ic cells with an anterior Golgi are early in the Ic stage of metacyclogenesis, although Golgi migration to the posterior has already started. We observed the region of the cytostome-cytopharynx (partly, or in its entirety) from a total of 27 intermediate Ic cells by thin-section TEM and 8 by FIB-SEM, or serial electron tomography (7). To show the ultrastrutural changes in the Ic stage in detail, a representative cell from this stage was selected from a serial electron tomography series produced by joining tomograms of seven adjacent 200 nm-thick sections, resulting in 660 virtual slices (Fig. 4). Also, for 3D reconstructions of 8 cells were produced based on FIB-SEM data (Fig. 5). When viewed by thin-section TEM, some intermediate Ic cells (aproxim. 27 cells observed by this technique) had a clearly identifiable cytopharynx microtubule quartet underlying the preoral ridge (Fig. 3A and B) adjacent to nucleus, or had all seven cytopharynx microtubules – a quartet and a triplet (indicated by blue and green arrows, respectively, in Fig. 3C and D) – running alongside the cytopharynx, in a typical ‘gutter’ configuration, at the posterior of the cell, near the kinetoplast (Fig. 3C and D). This microtubule arrangement is characteristic of the cytopharynx portion just below the cytostome opening (Alcantara et al., 2014); thus, it is
Fig. 2. 3D model of the cytostome-cytopharynx area of metacyclogenesis intermediate Ib cell by FIB-SEM. Images of individual FIB-SEM Z-slices (A–C) and corresponding 3D model (G) of the cytostome-cytopharynx area. To the right of each Z-slice, the slice position and orientation within the 3D model is indicated by a gray rectangle (D–F). Nucleus (N), blue; Kinetoplast (K), green; Flagellum (F), yellow; Flagellar pocket (FP), white; Golgi complex (Gc), brown; Preoral ridge (POR), purple; Cytopharynx (Cy), pink; Reservosomes (R), red. Note the kinetoplast next to the nucleus – a hallmark of Ib cells. The z-slices in A–C follow the path of the cytostome-cytopharynx complex from the anterior region (z-slice #62), near the POR, to the posterior (z-slice #172). The 3D model (G) shows that the cytopharynx reaches the posterior region of the cell. A comparison with a 3D model of an epimastigote cell (H) indicates that the POR in Ib cells is displaced towards the posterior, nearer the nucleus in the same direction. Scale bars, 200 nm.
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 3. Localization of the preoral ridge microtubules quartet and proximal region of the cytopharynx in intermediate Ic cells. A) Image of an intermediate Ic cell (as identified by the presence a rod-shaped kinetoplast – K – posterior to the nucleus – N), showing that the microtubule quartet that runs below the preoral ridge (and then follows the path of the cytopharynx) is located at the posterior of the cell. B) Higher magnification of the area indicated by the square in A, with detailed view of the microtubule quartet (blue arrows). C) Image of a different intermediate Ic cell (note the kinetoplast at the posterior) showing a transversal view of cytopharynx (pink asterisk). D) Higher magnification of the area indicated by the square in C, showing that the cytopharynx (pink asterisk) was accompanied by seven microtubules (a triplet and a quartet, indicated by green and blue arrows, respectively), as observed for the proximal region of the cytopharynx of epimastigotes (Alcantara et al., 2014).
clear that, in intermediate Ic cells that have a cytopharynx, the opening of the cytostome is located in the posterior of the cell, between the nucleus and the kinetoplast. The reconstructed 3D architecture of intermediate Ic cells (7) by serial electron tomography and all these cells showed alterations in the ultrastructure of the cytopharynx (Fig. 4B and C, Z Slice #100), compared with that observed in epimastigotes and in earlier metacyclogenesis intermediates. These alterations included an enlargement in the proximal portion of the cytopharynx, close to the cytostome, where the diameter of the cytopharynx itself (300 nm) was larger than the largest diameter of the
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epimastigote cytostome-cytopharynx complex previously reported (90 nm at the cytostome opening; (Vatarunakamura et al., 2005). At one side of this dilation, we could clearly observe five microtubules that had one end underneath the flagellar pocket membrane and followed the path of the cytopharynx (Fig. 4C), while accompanied by vesicles (Fig. 4E; Z Slice #184/orange arrows). In samples that had been subjected to an endocytic assay with Tf-Au, serial sections of intermediate Ic cells with the cytopharynx dilation showed endocytic tracers bound to the cytostome opening (Fig. 4F–I), an indication that the dilated section of the cytostome-cytopharynx complex was capable of endocytosis. In intermediates Ic cells with a dilated proximal portion of the cytopharynx, the Golgi complex was positioned further towards the posterior than in epimastigote cells, but had not yet reached the posterior region of the cell, past the nucleus (Gc; Fig. 4B/ brown). Importantly, our dataset of Ic cells also included 10 cells (imaged by FIB-SEM or thin-section TEM) where the cytopharynx was clearly absent (Fig. 5). In one of these cells (the thin-section series; Fig. 5A and B), the Golgi complex was positioned clearly between the nucleus and the kinetoplast, indicating that this was a late intermediate Ic cell; Golgi positioning could not be established with certainty for the remaining Ic cells without a cytopharynx, imaged by FIB-SEM (Fig. 5C–I). FIB-SEM-based 3D reconstructions of 8 late intermediate Ic cells (Fig. 5C–I) showed that these cells retained a preoral ridge (albeit smaller than that of earlier intermediates), despite the absence of a cytostome aperture (Fig. 5D, video 1 in supplementary material). The quartet of microtubules likely corresponding to that of the cytopharynx (from its positioning) also remained, although the cytopharynx itself was absent. The 3D reconstructions showed that this microtubule quartet had one end near the flagellar pocket, while the other end reached the posterior region of the cell (Fig. 5I). Note that the kinetoplast is localized posterior to a reservosome, indicating an advanced stage of kinetoplast repositioning towards the posterior of the cell, as observed in metacyclic trypomastigotes. The localization of microtubules was also included in the model. Our observations of intermediate Ic cells suggested that, although the cytostome and the cytopharynx invagination disappeared late in metacyclogenesis, part of the complex – the quartet of microtubules – remained. Thus, we observed negatively stained cytoskeletons (i.e., detergent-extracted cells) of metacyclic trypomastigotes by TEM, to verify if these cells contained structures that could correspond to the cytopharynx microtubules. In cytoskeletons of metacyclic trypomastigotes, we could clearly observe a cytoskeletal structure never previously noticed in these cells, representing a short microtubule quartet that extended from the flagellar pocket region towards the nucleus (Fig. 6). As opposed to that expected for the flagellum attachment zone (FAZ) microtubule quartet (MtQ), this short microtubule quartet did not follow the path of the FAZ (Fig. 6B), and is likely to represent a remnant of the microtubule cytoskeleton that follows the path of the cytopharynx, in epimastigotes and early metacyclogenesis intermediates. 4. Discussion Metacyclogenesis – the transformation of non-infective epimastigote into infective metacyclic trypomastigotes – is a key process in the T. cruzi developmental cycle, and comprises a progressive morphological transformation, including transitional forms described as ‘intermediates’. During metacyclogenesis, the cytostome-cytopharynx complex and the reservosomes (i.e., late endosomes) disappear, as cells lose endocytic ability (Soares and de Souza, 1991). Despite the importance of the cytostomecytopharynx complex as the main site of endocytosis in epimastigotes,
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 4. Morphology and endocytic potential of the enlarged cytopharynx observed late in metacyclogenesis. Tomographic reconstruction of the proximal portion of the cytopharynx of a metacyclogenesis intermediate Ic cell (as identified by the presence a rod-shaped kinetoplast – K – posterior to the nucleus – N). A, D and E show virtual slices of the tomogram used to produce the 3D model displayed in B and C. Nucleus (N), blue; Kinetoplast (K), green; Flagellum (F), yellow; Golgi (Gc), brown; Preoral ridge (POR), purple; Cytopharynx (Cy), pink; Vesicles, orange. The model shows a cytopharynx with an enlarged proximal portion (z-slice#100), but still associated with vesicles (orange arrows) and with the two microtubules sets (white arrows) (z-slice#184), as seen in epimastigotes. F–J Serial thin-sections of an intermediate Ic cell with an enlarged cytopharynx (at the proximal portion), showing endocytic tracer (transferrin coupled to 10-nm gold/black arrows) bound to the cytostome entrance. Scale bars: 500 nm (A, B); 200 nm (C, E and F–I) and 100 nm (J).
the morphological alterations on the path to cytostomecytopharynx disappearance during metacyclogenesis and the endocytic potential of intermediate forms had not been examined previously. In this study, we used FIB-SEM combined with high-resolution electron tomography to explore the cytostome-cytopharynx ultrastructural changes in the three described morphotypes of metacyclogenesis intermediates (Ia, Ib, Ic), and we examined the endocytic ability of these cells. We observed that all intermediate
forms (Ia, Ib and Ic) were capable of internalizing the endocytic tracer Tf-Au (Figs. 1 and 4), indicating that all of these forms retain endocytic activity. This is in agreement with ultrastructural data showing that intermediates Ia, Ib and a proportion of intermediated Ic cells have a clear cytostome-cytopharynx complex and a preoral ridge with the typical dense membrane coat (Figs. 1–4). The cytopharynx of intermediate Ib forms appears shorter than that of epimastigotes, with the preoral ridge displaced towards the posterior, and the cytostome aperture placed at or near the midline
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
J.C. Vidal et al. / Journal of Structural Biology xxx (2016) xxx–xxx
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Fig. 5. Ultrastructutral analysis of Ic metacyclogenesis intermediates lacking the cytopharynx. A and B) Transmission electron microscopy images showing an intermediate Ic cell (with a rod-shaped kinetoplast posterior to the nucleus). Note the location of the Golgi complex (Gc), between nucleus (N) and kinetoplast (K), as in metacyclic trypomastigotes. B) High magnification view of the area indicated by the rectangle in A, showing that the cytopharynx itself is absent, but microtubules likely corresponding to those that accompany the cytopharynx in earlier intermediate forms are still present. C–I) FIB-SEM images (C–E) and 3D reconstruction (I) of an intermediate Ic cell, focusing on the posterior of the cell. In F–H, the position and orientation of each Z-slice is indicated by a gray rectangle. A discrete preoral ridge (POR; black arrow in E) was present in this cell, but the cytostome-cytopharynx complex was absent. A quartet of microtubules (blue brackets in E and I) likely corresponding to that of the cytopharynx (due to its position) ran towards the posterior of the cell, and was clearly distinct from the MtQ (orange brackets in E, inset) that follows the path of the flagellum.
of the cell body (Fig. 3). In epimastigotes the cytopharynx microtubule quartet follows the path of the preoral ridge and then bends towards the center of the cell (Alcantara et al., 2014). These microtubules remain closely associated with each other along the entire path to the posterior of the cell (Alcantara et al., 2014). The physical link between the cytopharynx microtubules and the base of the flagellum (Okuda et al., 1999) was maintained during metacyclogenesis, and flagellar pocket migration appeared to ‘drag’ the cytostome-cytopharynx complex towards the posterior (see scheme in Fig. 7). Special and strong connections are likely to exist between the microtubules of the cytostome-cytopharynx quartet, because they were repositioned as a unit towards the posterior, during metacyclogenesis.
Interestingly, some intermediate Ic cells – those with the Golgi anterior to the kinetoplast – still had a structured cytostomecytopharynx complex accompanied by the characteristic seven microtubules. Also, endocytic assay data indicate that the cytostome-cytopharynx complex of Ic cells is functional, since Ic cells were able to uptake gold-labeled transferrin (Fig. 1C and F), whose internalization is mostly (85%) via the cytostomecytopharynx, in epimastigotes (Porto-Carreiro et al., 2000). Some parasites in the Ic phase (Fig. 4) had a short and enlarged cytopharynx (300 nm of diameter, the same measure of the cytostome aperture) accompanied by microtubules. In the cell shown in Fig. 4, the Golgi complex was still localized anterior to the nucleus. Although we could not determine Golgi positioning
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 6. Negative staining of a detergent-extracted metacyclic trypomastigote showing the flagellum (F) as well as remnants of the kinetoplast (K) and the nucleus (N). A) The set of four microtubules (square) that extends from the flagellar pocket area towards the nuclear remnant. B) High magnification of square in A showing the set of four microtubules likely corresponds to cytopharynx microtubule quartet, and does not follow the path of the FAZ (as expected for the FAZ microtubule quartet, or MtQ). Scale bars: 200 nm.
for all cells in our FIB-SEM dataset, our combined FIB-SEM and thin-section EM data suggest that Golgi repositioning between nucleus and kinetoplast may coincide with the start of cytopharynx disassembly. Besides the enlargement in width, we could see vesicles aligned with the ‘naked’ side of the cytopharynx (i.e., the side devoid of microtubules) (Fig. 4). The presence of vesicles near cytopharynx, in the epimastigote form, had already been shown in conventional TEM ultrathin sections (Milder and Deane, 1969; Salzman et al., 1985). In our previous work, vesicles with or without the endocytic tracer Tf–Au were observed clearly budding from or fusing with the cytopharynx membrane, in epimastigotes (Alcantara et al., 2014). The presence of vesicles aligned with the cytopharynx, in intermediate Ic cells, is a strong indication that, at least some Ic parasites still have the normal mechanism of vesicle budding and fusing and, consequently, efficient endocytic activity via the cytopharynx. In a subset of intermediate Ic, the cytopharynx invagination was absent, but a group of microtubules likely corresponding to those of the cytopharynx remained. The most drastic ultrastructural modification observed in the cytostome-cytopharynx complex were parasites where a microtubule quartet likely corresponding to that of the cytopharynx was present, but the membrane invagination of the cytopharynx was absent (Fig. 5A–I). The representative cell (Fig. 5A and B) appeared to be a late intermediate of metacyclogenesis (late Ic stage) because the kinetoplast was posterior to nucleus, but was still rod-shaped and the Golgi complex localized between nucleus and kinetoplast, as seen in trypomastigotes form. In a different slice and view series of an intermediates Ic cell where the kinetoplast was posterior to a reservosome, indicating a very late stage in metacyclogenesis (late Ic, but still different from metacyclic trypomastigotes, because the kinetoplast was rod-shaped), the cytostome and the cytopharynx were absent, but the preoral ridge was present (albeit smaller than in epimastigotes), and we could also identify the four microtubules of the cytopharynx quartet (Fig. 5F–I). In this late intermediate, cytopharynx disassembly probably occurred by vesiculation or endocytic budding without membrane replacement. The reorganization of the Golgi is a crucial phenomenon in the polarization and migration of many cell types (Coppens et al., 1987). The Golgi apparatus is a station for the sorting and transport of lipids and proteins that are essential for secretory pathways (Amersi et al., 2006); thus, Golgi migration may be very important in supplying membrane components for rearrangements in cellular architecture during metacyclogenesis. In T. cruzi epimastigotes, the
Golgi apparatus is localized near the flagellar pocket and the cytostome-cytopharynx complex, in the anterior region of parasite (Alcantara et al., 2014), and this proximity may be essential to deliver content from the Golgi to these two active membrane domains. Previously we suggested that this proximity allows Golgi vesicles to fuse quickly with the cytopharynx invagination, delivering essentials molecules and membrane for the maintenance this membrane domain (Alcantara et al., 2014). Early in metacyclogenesis, the cytostome-cytopharynx migration (to the posterior region) starts. Our observations indicate that the Golgi complex is maintained in the anterior region during the Ib stage, and its migration to the posterior of the cell starts in the Ic stage, but may only be concluded late in the Ic stage, after cytostomecytopharynx posterior-ward migration. Late Golgi migration to the posterior is expected to increase the spatial separation between this organelle and cytostome-cytopharynx, and might decrease membrane replacement to this organelle. This effect, coupled with the maintenance of endocytic activity – with budding of cytopharynx membrane into vesicles – may underlie the ultrastrutural changes observed in the cytostome-cytopharynx complex, culminating in cytopharynx shortening and disappearance, in Ic intermediates. In line with this hypothesis, Johnson et al. (2016) showed that, in a mammalian cell line, peripheral lysosomes pH were less acid than internal ones and had decreased proteolytic activity. This effect was explained by the increased distance between the Golgi and peripheral lysosomes, which had, therefore, decreased access to Golgiderived enzymatic and acidifying content (Johnson et al., 2016). Interestingly, negatively-stained cytoskeletons of metacyclic trypomastigotes display a previously unnoticed structure: a microtubule quartet that describes a path from the flagellar pocket area towards the nucleus, as expected for the cytopharynx microtubule quartet (but not for the FAZ MtQ) (Fig. 6). These data represent clear evidence that the microtubule quartet of the cytopharynx is maintained in metacyclic trypomastigotes, which may enable the fast reassembly of the cytostome-cytopharynx in amastigotes, after trypomastigote entry into host cells. Fig. 7 illustrates a sequential description of these events that includes kinetoplast/ flagellum displacement, shortening of the cytopharynx, Golgi displacement, and finally, cytopharynx disappearance, with maintenance of the microtubule quartet that accompanies the cytopharynx in other life cycle forms. The work presented herein provided evidence that metacyclogenesis involves active cellular reorganization with maintenance of endocytic activity.
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 7. Model showing the displacement, shortening and disappearance of the cytopharynx during metacyclogenesis. A) Epimastigote with the kinetoplast anterior to nucleus, Golgi complex and flagellar pocket at the anterior region of the cell; B) Intermediate Ia, with the kinetoplast and flagellum repositioned closer to the nucleus, but still located in the anterior region of the cell, similarity to that observed in the epimastigote; C) Intermediate Ib, showing kinetoplast/flagellum migration to a position adjacent to the nucleus, which may ‘drag’ cytostome aperture repositioning. D) Intermediate Ic, with the kinetoplast posterior to the nucleus, while the Golgi remained in the anterior region. Note the cytostome aperture in the posterior of the cell, between the kinetoplast and the nucleus, and the short cytopharynx invagination; E) Intermediate Ic, with a posterior Golgi complex. In these cells, the cytopharynx invagination itself is absent, and only the microtubule quartet (blue) of cytopharynx remains. F) Metacyclic has globular kinetoplast localized at the posterior end of the cell and an elongate nucleus and Golgi complex between these organelles. The microtubule quartet of cytopharynx remains.
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and shortening, and cytopharynx disappearance – clarifies important aspects of the intricate progression of morphological events during the formation T. cruzi infective forms. Acknowledgements The authors would like to thank Luis Sergio Júnior (Inmetro, Rio de Janeiro, Brazil) and Thiago Luis de Barros Moreira (Centro Nacional de Bioimagem, Rio de Janeiro, Brazil) for their technical assistance. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number 472262/2012-2], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) scholarships to C.L.A and J.C.V., Financiadora de Estudos e Projetos (FINEP), Programas Núcleos de Excelência (PRONEX) [grant number E-26/110.576/2010] and Cientista do Nosso Estado [grant number E-26/102.850/2012] from the Fundação Carlos Chagas Filho de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2016.07.018. References
Our study of the 3D architecture of the cytostome-cytopharynx complex during metacyclogenesis – with the description of morphological milestones such as preoral ridge and cytostome posterior-ward displacement, cytopharynx proximal end enlargement
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Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis Juliana C. Vidal, Carolina de L. Alcantara, Wanderley de Souza, Narcisa L. Cunha-e-Silva ⇑ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil
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Article history: Received 21 April 2016 Received in revised form 20 July 2016 Accepted 27 July 2016 Available online xxxx Keywords: T. cruzi Metacyclogenesis Cytostome-cytopharynx Endocytosis
a b s t r a c t Trypanosoma cruzi epimastigotes uptake nutrients by endocytosis via the cytostome-cytopharynx complex – an anterior opening (cytostome) continuous with a funnel-shaped invagination (cytopharynx) that extends to the posterior of the cell, accompanied by microtubules. During metacyclogenesis – the transformation of epimastigotes into human-infective metacyclic trypomastigotes – the cytostomecytopharynx complex disappears, as trypomastigotes lose endocytic ability. To date, no studies have examined cytostome-cytopharynx complex disappearance in detail, or determined if endocytic activity persists during metacyclogenesis. Here, we produced 3D reconstructions of metacyclogenesis intermediates (Ia, Ib, Ic) using electron microscopy tomography and focused ion beam-scanning electron microscopy (FIB-SEM), concentrating on the cytostome-cytopharynx complex and adjacent structures, including the preoral ridge (POR). Parasite endocytic potential was examined by incubation of intermediate forms with the endocytic tracer transferrin (Tf)-Au. Ia, Ib and Ic cells were capable of internalizing Tf-Au, and had a shorter cytopharynx than that of epimastigotes, with the cytostome/POR progressively displaced towards the posterior, following the movement of the kinetoplast/flagellar pocket. While some Ic cells had a short cytopharynx with an enlarged proximal end (300 nm in diameter, larger than that of the cytostome), other Ic cells had no cytopharynx invagination, but retained the cytopharynx microtubules, which were also present in metacyclics. We conclude that cytostome-cytopharynx disappearance and loss of endocytic ability are late events in metacyclogenesis, during which the cytostome is displaced towards the posterior, probably due to a link to the kinetoplast/flagellar pocket. Retention of the cytopharynx microtubules by metacyclics may allow prompt cytostome-cytopharynx reassembly in amastigotes, upon host cell infection. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Trypanosoma cruzi – the causative agent of Chagas disease – has a complex life cycle involving mammalian and invertebrate hosts. Epimastigotes, the proliferating forms found in the insect vector gut, are capable of internalizing large amounts of transferrin, peroxidase, albumin and low-density lipoprotein from the extracellular milieu (Soares and de Souza, 1991). In this stage of the life cycle, the main site of endocytosis is the cytostome-cytopharynx complex (Porto-Carreiro et al., 2000), rather than the flagellar pocket, the exclusive site for endocytosis in other trypanosomatids (Webster and Russell, 1993). ⇑ Corresponding author at: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, bloco G subsolo, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 21941-902, Brazil. E-mail address: [email protected] (N.L. Cunha-e-Silva).
The cytostome consists of an opening at the epimastigote anterior region, continuous with a funnel-shaped invagination of the plasma membrane called the cytopharynx (Milder and Deane, 1969). Next to the cytostome, a specialized membrane domain called the preoral ridge (POR) separates the cytostome from the flagellar pocket (De Souza et al., 1978; Galbraith and McElrath, 1988; Pimenta et al., 1989; Vatarunakamura et al., 2005). The epimastigote preoral ridge has a thick electron-dense glycocalyx that makes it easily detectable by transmission electron microscopy (TEM)(Vatarunakamura et al., 2005). The participation of microtubules in the structure of the cytostome-cytopharynx complex is a hallmark of trypanosomatids (Chang et al., 1975; Sassa et al., 1975) and bodonids (Attias et al., 1996). The long and thin invagination of the cytopharynx is accompanied by two microtubule sets: a triplet that extends from underneath the cytostome membrane, and a quartet that originates close to the flagellar-pocket and continues underneath the
http://dx.doi.org/10.1016/j.jsb.2016.07.018 1047-8477/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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preoral ridge membrane domain, following the path of the cytopharynx towards the posterior of the cell (Alcantara et al., 2014). In T. cruzi, the cytostome-cytopharynx complex is present only in epimastigotes and amastigotes (Chang et al., 1975; Meyer and de Souza, 1973), but not in trypomastigotes. Thus, at the end of metacyclogenesis – the stage transition from epimastigotes to human-infective metacyclic trypomastigotes – the entire cytostome-cytopharynx complex must be disassembled, as trypomastigotes lose endocytic ability. Metacyclogenesis involves morphological, functional and protein expression changes, and can be reproduced in vitro, which allowed the description of several intermediate stages between epimastigotes and metacyclic trypomastigotes (Brener, 1973; Cortez et al., 2006; Ferreira et al., 2008; Perlowagora-Szumlewicz and Moreira, 1994; Schaub, 1988). In an ultrastructural study, Ferreira et al. (2008) reported the presence of the following types of T. cruzi intermediate forms between epimastigotes and trypomastigotes: intermediate Ia, with the kinetoplast anterior and very close to the nucleus (similarly to that observed in epimastigote forms); intermediate Ib, with the kinetoplast and nucleus side by side; and intermediate Ic, where the kinetoplast is posterior to the nucleus (as in trypomastigotes forms). A schematic drawing illustrating the intracellular distribution of organelles in T. cruzi intermediate forms (Ia, Ib and Ic) in comparison with epimastigote forms is shown in Fig. S1. In Ic forms both the nucleus and the kinetoplast exhibit signs of ultrastructural remodeling into the morphologies observed in metacyclic trypomastigotes, which have an elongated nucleus and a round kinetoplast at the posterior end of the cell. Although overall morphological descriptions of the intermediate forms have been reported, no studies have described in detail the restructuring/ disassembly of the cytostome-cytopharynx complex during metacyclogenesis. Also, the endocytic potential of metacyclogenesis intermediates has not been examined to date. In this work, we used advanced electron microscopy techniques – namely tomography and focused ion beam scanning electron microscopy (FIB) – to produce 3D reconstructions of the three metacyclogenesis intermediates (Ia, Ib, Ic, as described in (Ferreira et al., 2008) between epimastigotes and trypomastigotes. Aside from providing a detailed description of the ultrastructural changes involved in cytostome-cytopharynx disappearance during metacyclogenesis, we used transferrin as an endocytic tracer, followed by TEM observation, to evaluate the endocytic ability of the intermediate forms directly. 2. Materials and methods 2.1. Parasites Trypanosoma cruzi epimastigotes from the Dm28c clone were cultivated in liver infusion tryptose (LIT) medium (Camargo, 1964) supplemented with 10% (v/v) foetal calf serum (FCS) at 28 °C. Stationary-phase parasites from six-day-old cultures were used in all experiments. 2.2. In vitro metacyclogenesis Epimastigotes were induced to differentiate to metacyclic trypomastigotes as described previously (Contreras et al., 1985). Briefly, epimastigotes were collected by centrifugation at 3000g for 30 min, washed once in triatomine artificial urine (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM sodium phosphate buffer, pH 6.0) and adjusted to 5 108 cells/mL in the same medium. After 2 h at 28 °C, parasites were diluted 100-fold in TAU supplemented with 50 mM sodium glutamate,
10 mM L-proline, 2 mM sodium aspartate and 10 mM glucose (TAU3AAG) in cell culture flasks, and maintained at 28 °C, to induce metacyclogenesis. Metacyclogenesis induction was halted (at 24 or 48 h) by fixation for transmission electron microscopy. 2.3. Endocytosis assay Parasites undergoing metacyclogenesis were washed twice in phosphate-buffered saline (10 mM sodium phosphate buffer with 0.9%, NaCl, w/v, pH 7.2) and then incubated in RPMI medium containing transferrin coupled to 10 nm gold particles (Tf-Au) (Slot and Geuze, 1985) for 30 min at 28 °C. Parasites were fixed and processed for transmission electron microscopy observation, as detailed below. 2.4. Sample preparation for transmission electron microscopy (TEM) Parasites undergoing metacyclogenesis were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. Then, cells were washed twice in 0.1 M cacodylate buffer, pH 7.2, and post-fixed using an OTO (osmium-thiocarbohydrazide-osmium) protocol (Willingham and Rutherford, 1984). Briefly, cells were incubated in a post-fixative solution consisting of 1% (v/v) osmium tetroxide, 0.8% (v/v) potassium ferrocyanide, 5 mM calcium chloride, in 0.1 M cacodylate buffer (pH 7.2) for 40 min, washed twice in water and then incubated in a solution of 1% (w/v) thiocarbohydrazide (TCH – Sigma, St. Louis, USA) in water for 5 min. After three washes in water, cells were incubated again in the post-fixative osmium solution for 3 min. Following postfixation, samples were washed in water, dehydrated in an acetone series and embedded in Epoxy resin (EMbed-812/ Electron Microscopy Sciences). Images were recorded using a Helios 200 NanoLab dual-beam microscope (Eindhoven, NL) equipped with a gallium ion source for focused-ion beam milling, and a field emission gun with an in-lens secondary electron detector, for scanning electron microscopy imaging by SmartSEM software (V05.07 SP4, Carl Zeiss). Blocks were subjected to focused ion beam scanning electron microscopy or serial electron tomography (see below). 2.5. Serial electron tomography For tomography, 200-nm-thick serial sections were collected onto formvar-coated copper slot grids, stained with 5% (w/v) uranyl acetate and lead citrate (Reynolds, 1963) and incubated with 10-nm colloidal gold particles (Gold colloid, Sigma Aldrich) for 5 min. Then, sections were washed with distilled water and observed on a Tecnai-G2 transmission electron microscope operating at 200 kV (FEI Company, Eindhoven, Netherlands) equipped with a 4 k CCD camera. Single-axis tilt series (±60° with 1° increments) were produced. Alignments were applied using gold as fiducial markers and back projections with IMOD software package. 2.6. Focused ion beam-scanning electron microscopy (FIB-SEM) For observation by FIB-SEM, parasites undergoing metacyclogenesis for 24 h or 48 h were processed for TEM as described above. The tips of resin blocks containing the sample were then trimmed to a pyramidal shape and mounted on a support for FIB-SEM. Images were recorded using a Helios 200 NanoLab dual-beam microscope (Eindhoven, NL) equipped with a gallium ion source for focused-ion beam milling, and a field emission gun with an in-lens secondary electron detector, for scanning electron microscopy imaging by SmartSEM software, release number V05.07 SP4 (Carl Zeiss). ‘Slice-and-view’ image series were generated using a milling step size of 25 nm. After image capturing, the
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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contrast of backscattered electron images, recorded by Energy selective Backscattered (EsB) Detector, was inverted so that they resembled conventional TEM images. Sequential slices were automatically aligned using the XfAlign algorithm of IMOD (Kremer et al., 1996) and a fine alignment was then performed using MIDAS. All 3D models and measurements were performed with 3DMOD. 2.7. Negative staining TEM Cells were harvest by centrifugation at 1500g, for 30 min, resuspended in LIT medium (liver infusion tryptose) (Camargo, 1964) and allowed to adhere to glow-discharged and formvar-coated grids. Then, cells were extracted in 1% (v/v) NP-40 in PEME buffer (100 mM PIPES, 1 mM MgSO4, 0.1 mM EDTA and 2 mM EGTA, pH 6.9) for 5 min, and fixed in 2.5% (v/v) glutaraldehyde in PEME, for 10 min. Grids were negatively stained with 0.7% (v/v) aurothioglucose in water and observed in a Tecnai Spirit transmission electron microscope, operating at 120 kV. 3. Results 3.1. Metacyclogenesis intermediate forms are capable of endocytosis An important physiological hallmark of metacyclogenesis is a decrease in endocytic ability, as highly endocytic epimastigotes transform into infective metacyclic trypomastigotes; nevertheless, the dynamics of the main endocytic structure of epimastigotes – the cytostome-cytopharynx complex – during metacyclogenesis has not been described in detail, and little is known about the endocytic capacity of intermediate forms between epimastigotes and metacyclics.
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To study cytostome-cytopharynx dynamics during metacyclogenesis, we successfully recapitulated this process in vitro using a method described previously (Contreras et al., 1985), whereby incubation of epimastigotes in TAU3AAG medium triggered parasite adhesion to culture flasks and started morphological differentiation into trypomastigote forms. Cells were classified into one of the following T. cruzi developmental stages: epimastigotes, metacyclic trypomastigotes or metacyclogenesis intermediate forms, Ia, Ib and Ic (Ferreira et al., 2008), based on nucleus and kinetoplast/flagellum position. To investigate the effect of metacyclogenesis induction on endocytic activity, parasites were harvested after 24 h and 48 h of metacyclogenesis, subjected to an endocytic assay using transferrin coupled to 10 nm gold particles (Tf-Au) as cargo, fixed and observed by TEM (Fig. 1). Tf-Au cargo was found inside reservosomes – the late endosomes of T. cruzi (Soares and de Souza, 1991; Soares et al., 1992) – in intermediate Ia (Fig. 1A and D, white arrows), Ib (Fig. 1B and E, white arrows) cells, and also in reservosomes and vesicle in intermediate Ic (Fig. 1C and F, white arrows). Also, intermediate Ib had a POR membrane domain with its characteristic dense coat (black arrows in Fig. 1B). These findings indicated that inducing metacyclogenesis did not block parasite endocytic activity. Morphologically, intermediate Ia was often difficult to distinguish unambiguously from epimastigotes; thus, we focused our morphological analysis on intermediates Ib and Ic. 3.2. The cytostome-cytopharynx complex is displaced towards the posterior during metacyclogenesis At 48 h of metacyclogenesis numerous parasites were at stages Ib and Ic, which are clearly distinguishable from epimastigotes (Ferreira et al., 2008); thus, this time-point was chosen for the
Fig. 1. Endocytic activity in metacyclogenesis intermediates. After 24 h of metacyclogenesis induction, cells were incubated with transferrin coupled to 10-nm gold particles (used as an endocytic tracer), and then processed for transmission electron microscopy (TEM). Both intermediate Ia (with kinetoplast – K – immediately anterior to the nucleus – N; in A and D), intermediate Ib (with the kinetoplast adjacent to the nucleus, in B and E) and intermediate Ic (with kinetoplast posterior to the nucleus, in C and F) cells had late endosomal structures (reservosomes, R) and vesicles containing transferrin-gold particles (white arrows), indicative of endocytosis. D, E and F show higher magnification images of the areas indicated by the rectangles in A (D), B (E) and C (F). In intermediate Ic cells (note the kinetoplast at the posterior, near a reservosome), a vesicle loaded with gold particles is located immediately anterior to the kinetoplast. Intermediate b cells had a clear preoral ridge membrane domain (POR, black arrows, in B). Gc, Golgi complex; F, flagellum. Scale bars: 1 lm (A, B and C); 200 nm (D, E, F).
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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analysis of mid/late metacyclogenesis intermediates by electron microscopy. The cytostome-cytopharynx is a long and curved invagination of the plasma membrane that cannot be visualized in its entirety by conventional ultrathin-section TEM. Therefore, we used FIB-SEM to produce 3D reconstructions of the entire cytostome-cytopharynx complex of metacyclogenesis intermediates at an ultrastructural level, and combined these data with ‘targeted’ serial electron tomography or ultrathin-section TEM of specific morphological aspects that required high-resolution structural analysis. Samples were processed using thiocarbohydrazide, which enhanced the contrast of the cytopharynx membrane domain, facilitating its recognition in TEM images. We analyzed the cytostome-cytopharynx area (or the expected area of the cytostome-cytopharynx) from 51 mid (Ib)/late (Ic) metacyclogenesis intermediates. Among the 12 cells observed in FIB-SEM series, 3 representative cells were selected for a detailed analysis by 3D reconstruction. We reconstructed 4 intermediate Ib cells (with the kinetoplast adjacent to nucleus) and all had a clear preoral ridge (POR) and a curved cytopharynx invagination that reached the posterior of the cell (Fig. 2A–G). We have considered the hypothesis that, during metacyclogenesis, the cytostome-cytopharynx complex may accompany the kinetoplast/flagellar pocket complex in its migration to the posterior region of the cell. To examine this possibility, we compared FIB-SEM-based 3D reconstructions of epimastigotes and Ib cells that were in a similar orientation relative to the plane of viewing (and had been imaged at the same magnification; Fig. 2H). Positioning of the two models showed that the distance between the POR and nucleus decreased gradually with the progression metacyclogenesis, from epimastigotes to Ib cells (Fig. 2). Epimastigote forms had a spherical nucleus with a disk-shaped kinetoplast (K, in green), and a flagellum (F, in yellow) emerging from the anterior portion of the cell (Fig. 2H). Note that, as
differentiation proceeded, both the kinetoplast and the point of flagellum emergence from the cell surface moved towards the middle of the cell. As a consequence of this migration, the nucleus became sinuous and the POR (in purple) of intermediate Ib cells was localized immediately adjacent to the nucleus (Fig. 2D–G). 3.3. The cytostome disappears late during the Ic stage of metacyclogenesis Intermediate Ic differs from metacyclic trypomastigotes by having rod shaped kinetoplast, although both forms have a posterior kinetoplast. During the Ic stage, the Golgi complex migrates from the anterior of the cell to the posterior; thus, Ic cells with an anterior Golgi are early in the Ic stage of metacyclogenesis, although Golgi migration to the posterior has already started. We observed the region of the cytostome-cytopharynx (partly, or in its entirety) from a total of 27 intermediate Ic cells by thin-section TEM and 8 by FIB-SEM, or serial electron tomography (7). To show the ultrastrutural changes in the Ic stage in detail, a representative cell from this stage was selected from a serial electron tomography series produced by joining tomograms of seven adjacent 200 nm-thick sections, resulting in 660 virtual slices (Fig. 4). Also, for 3D reconstructions of 8 cells were produced based on FIB-SEM data (Fig. 5). When viewed by thin-section TEM, some intermediate Ic cells (aproxim. 27 cells observed by this technique) had a clearly identifiable cytopharynx microtubule quartet underlying the preoral ridge (Fig. 3A and B) adjacent to nucleus, or had all seven cytopharynx microtubules – a quartet and a triplet (indicated by blue and green arrows, respectively, in Fig. 3C and D) – running alongside the cytopharynx, in a typical ‘gutter’ configuration, at the posterior of the cell, near the kinetoplast (Fig. 3C and D). This microtubule arrangement is characteristic of the cytopharynx portion just below the cytostome opening (Alcantara et al., 2014); thus, it is
Fig. 2. 3D model of the cytostome-cytopharynx area of metacyclogenesis intermediate Ib cell by FIB-SEM. Images of individual FIB-SEM Z-slices (A–C) and corresponding 3D model (G) of the cytostome-cytopharynx area. To the right of each Z-slice, the slice position and orientation within the 3D model is indicated by a gray rectangle (D–F). Nucleus (N), blue; Kinetoplast (K), green; Flagellum (F), yellow; Flagellar pocket (FP), white; Golgi complex (Gc), brown; Preoral ridge (POR), purple; Cytopharynx (Cy), pink; Reservosomes (R), red. Note the kinetoplast next to the nucleus – a hallmark of Ib cells. The z-slices in A–C follow the path of the cytostome-cytopharynx complex from the anterior region (z-slice #62), near the POR, to the posterior (z-slice #172). The 3D model (G) shows that the cytopharynx reaches the posterior region of the cell. A comparison with a 3D model of an epimastigote cell (H) indicates that the POR in Ib cells is displaced towards the posterior, nearer the nucleus in the same direction. Scale bars, 200 nm.
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 3. Localization of the preoral ridge microtubules quartet and proximal region of the cytopharynx in intermediate Ic cells. A) Image of an intermediate Ic cell (as identified by the presence a rod-shaped kinetoplast – K – posterior to the nucleus – N), showing that the microtubule quartet that runs below the preoral ridge (and then follows the path of the cytopharynx) is located at the posterior of the cell. B) Higher magnification of the area indicated by the square in A, with detailed view of the microtubule quartet (blue arrows). C) Image of a different intermediate Ic cell (note the kinetoplast at the posterior) showing a transversal view of cytopharynx (pink asterisk). D) Higher magnification of the area indicated by the square in C, showing that the cytopharynx (pink asterisk) was accompanied by seven microtubules (a triplet and a quartet, indicated by green and blue arrows, respectively), as observed for the proximal region of the cytopharynx of epimastigotes (Alcantara et al., 2014).
clear that, in intermediate Ic cells that have a cytopharynx, the opening of the cytostome is located in the posterior of the cell, between the nucleus and the kinetoplast. The reconstructed 3D architecture of intermediate Ic cells (7) by serial electron tomography and all these cells showed alterations in the ultrastructure of the cytopharynx (Fig. 4B and C, Z Slice #100), compared with that observed in epimastigotes and in earlier metacyclogenesis intermediates. These alterations included an enlargement in the proximal portion of the cytopharynx, close to the cytostome, where the diameter of the cytopharynx itself (300 nm) was larger than the largest diameter of the
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epimastigote cytostome-cytopharynx complex previously reported (90 nm at the cytostome opening; (Vatarunakamura et al., 2005). At one side of this dilation, we could clearly observe five microtubules that had one end underneath the flagellar pocket membrane and followed the path of the cytopharynx (Fig. 4C), while accompanied by vesicles (Fig. 4E; Z Slice #184/orange arrows). In samples that had been subjected to an endocytic assay with Tf-Au, serial sections of intermediate Ic cells with the cytopharynx dilation showed endocytic tracers bound to the cytostome opening (Fig. 4F–I), an indication that the dilated section of the cytostome-cytopharynx complex was capable of endocytosis. In intermediates Ic cells with a dilated proximal portion of the cytopharynx, the Golgi complex was positioned further towards the posterior than in epimastigote cells, but had not yet reached the posterior region of the cell, past the nucleus (Gc; Fig. 4B/ brown). Importantly, our dataset of Ic cells also included 10 cells (imaged by FIB-SEM or thin-section TEM) where the cytopharynx was clearly absent (Fig. 5). In one of these cells (the thin-section series; Fig. 5A and B), the Golgi complex was positioned clearly between the nucleus and the kinetoplast, indicating that this was a late intermediate Ic cell; Golgi positioning could not be established with certainty for the remaining Ic cells without a cytopharynx, imaged by FIB-SEM (Fig. 5C–I). FIB-SEM-based 3D reconstructions of 8 late intermediate Ic cells (Fig. 5C–I) showed that these cells retained a preoral ridge (albeit smaller than that of earlier intermediates), despite the absence of a cytostome aperture (Fig. 5D, video 1 in supplementary material). The quartet of microtubules likely corresponding to that of the cytopharynx (from its positioning) also remained, although the cytopharynx itself was absent. The 3D reconstructions showed that this microtubule quartet had one end near the flagellar pocket, while the other end reached the posterior region of the cell (Fig. 5I). Note that the kinetoplast is localized posterior to a reservosome, indicating an advanced stage of kinetoplast repositioning towards the posterior of the cell, as observed in metacyclic trypomastigotes. The localization of microtubules was also included in the model. Our observations of intermediate Ic cells suggested that, although the cytostome and the cytopharynx invagination disappeared late in metacyclogenesis, part of the complex – the quartet of microtubules – remained. Thus, we observed negatively stained cytoskeletons (i.e., detergent-extracted cells) of metacyclic trypomastigotes by TEM, to verify if these cells contained structures that could correspond to the cytopharynx microtubules. In cytoskeletons of metacyclic trypomastigotes, we could clearly observe a cytoskeletal structure never previously noticed in these cells, representing a short microtubule quartet that extended from the flagellar pocket region towards the nucleus (Fig. 6). As opposed to that expected for the flagellum attachment zone (FAZ) microtubule quartet (MtQ), this short microtubule quartet did not follow the path of the FAZ (Fig. 6B), and is likely to represent a remnant of the microtubule cytoskeleton that follows the path of the cytopharynx, in epimastigotes and early metacyclogenesis intermediates. 4. Discussion Metacyclogenesis – the transformation of non-infective epimastigote into infective metacyclic trypomastigotes – is a key process in the T. cruzi developmental cycle, and comprises a progressive morphological transformation, including transitional forms described as ‘intermediates’. During metacyclogenesis, the cytostome-cytopharynx complex and the reservosomes (i.e., late endosomes) disappear, as cells lose endocytic ability (Soares and de Souza, 1991). Despite the importance of the cytostomecytopharynx complex as the main site of endocytosis in epimastigotes,
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 4. Morphology and endocytic potential of the enlarged cytopharynx observed late in metacyclogenesis. Tomographic reconstruction of the proximal portion of the cytopharynx of a metacyclogenesis intermediate Ic cell (as identified by the presence a rod-shaped kinetoplast – K – posterior to the nucleus – N). A, D and E show virtual slices of the tomogram used to produce the 3D model displayed in B and C. Nucleus (N), blue; Kinetoplast (K), green; Flagellum (F), yellow; Golgi (Gc), brown; Preoral ridge (POR), purple; Cytopharynx (Cy), pink; Vesicles, orange. The model shows a cytopharynx with an enlarged proximal portion (z-slice#100), but still associated with vesicles (orange arrows) and with the two microtubules sets (white arrows) (z-slice#184), as seen in epimastigotes. F–J Serial thin-sections of an intermediate Ic cell with an enlarged cytopharynx (at the proximal portion), showing endocytic tracer (transferrin coupled to 10-nm gold/black arrows) bound to the cytostome entrance. Scale bars: 500 nm (A, B); 200 nm (C, E and F–I) and 100 nm (J).
the morphological alterations on the path to cytostomecytopharynx disappearance during metacyclogenesis and the endocytic potential of intermediate forms had not been examined previously. In this study, we used FIB-SEM combined with high-resolution electron tomography to explore the cytostome-cytopharynx ultrastructural changes in the three described morphotypes of metacyclogenesis intermediates (Ia, Ib, Ic), and we examined the endocytic ability of these cells. We observed that all intermediate
forms (Ia, Ib and Ic) were capable of internalizing the endocytic tracer Tf-Au (Figs. 1 and 4), indicating that all of these forms retain endocytic activity. This is in agreement with ultrastructural data showing that intermediates Ia, Ib and a proportion of intermediated Ic cells have a clear cytostome-cytopharynx complex and a preoral ridge with the typical dense membrane coat (Figs. 1–4). The cytopharynx of intermediate Ib forms appears shorter than that of epimastigotes, with the preoral ridge displaced towards the posterior, and the cytostome aperture placed at or near the midline
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
J.C. Vidal et al. / Journal of Structural Biology xxx (2016) xxx–xxx
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Fig. 5. Ultrastructutral analysis of Ic metacyclogenesis intermediates lacking the cytopharynx. A and B) Transmission electron microscopy images showing an intermediate Ic cell (with a rod-shaped kinetoplast posterior to the nucleus). Note the location of the Golgi complex (Gc), between nucleus (N) and kinetoplast (K), as in metacyclic trypomastigotes. B) High magnification view of the area indicated by the rectangle in A, showing that the cytopharynx itself is absent, but microtubules likely corresponding to those that accompany the cytopharynx in earlier intermediate forms are still present. C–I) FIB-SEM images (C–E) and 3D reconstruction (I) of an intermediate Ic cell, focusing on the posterior of the cell. In F–H, the position and orientation of each Z-slice is indicated by a gray rectangle. A discrete preoral ridge (POR; black arrow in E) was present in this cell, but the cytostome-cytopharynx complex was absent. A quartet of microtubules (blue brackets in E and I) likely corresponding to that of the cytopharynx (due to its position) ran towards the posterior of the cell, and was clearly distinct from the MtQ (orange brackets in E, inset) that follows the path of the flagellum.
of the cell body (Fig. 3). In epimastigotes the cytopharynx microtubule quartet follows the path of the preoral ridge and then bends towards the center of the cell (Alcantara et al., 2014). These microtubules remain closely associated with each other along the entire path to the posterior of the cell (Alcantara et al., 2014). The physical link between the cytopharynx microtubules and the base of the flagellum (Okuda et al., 1999) was maintained during metacyclogenesis, and flagellar pocket migration appeared to ‘drag’ the cytostome-cytopharynx complex towards the posterior (see scheme in Fig. 7). Special and strong connections are likely to exist between the microtubules of the cytostome-cytopharynx quartet, because they were repositioned as a unit towards the posterior, during metacyclogenesis.
Interestingly, some intermediate Ic cells – those with the Golgi anterior to the kinetoplast – still had a structured cytostomecytopharynx complex accompanied by the characteristic seven microtubules. Also, endocytic assay data indicate that the cytostome-cytopharynx complex of Ic cells is functional, since Ic cells were able to uptake gold-labeled transferrin (Fig. 1C and F), whose internalization is mostly (85%) via the cytostomecytopharynx, in epimastigotes (Porto-Carreiro et al., 2000). Some parasites in the Ic phase (Fig. 4) had a short and enlarged cytopharynx (300 nm of diameter, the same measure of the cytostome aperture) accompanied by microtubules. In the cell shown in Fig. 4, the Golgi complex was still localized anterior to the nucleus. Although we could not determine Golgi positioning
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
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Fig. 6. Negative staining of a detergent-extracted metacyclic trypomastigote showing the flagellum (F) as well as remnants of the kinetoplast (K) and the nucleus (N). A) The set of four microtubules (square) that extends from the flagellar pocket area towards the nuclear remnant. B) High magnification of square in A showing the set of four microtubules likely corresponds to cytopharynx microtubule quartet, and does not follow the path of the FAZ (as expected for the FAZ microtubule quartet, or MtQ). Scale bars: 200 nm.
for all cells in our FIB-SEM dataset, our combined FIB-SEM and thin-section EM data suggest that Golgi repositioning between nucleus and kinetoplast may coincide with the start of cytopharynx disassembly. Besides the enlargement in width, we could see vesicles aligned with the ‘naked’ side of the cytopharynx (i.e., the side devoid of microtubules) (Fig. 4). The presence of vesicles near cytopharynx, in the epimastigote form, had already been shown in conventional TEM ultrathin sections (Milder and Deane, 1969; Salzman et al., 1985). In our previous work, vesicles with or without the endocytic tracer Tf–Au were observed clearly budding from or fusing with the cytopharynx membrane, in epimastigotes (Alcantara et al., 2014). The presence of vesicles aligned with the cytopharynx, in intermediate Ic cells, is a strong indication that, at least some Ic parasites still have the normal mechanism of vesicle budding and fusing and, consequently, efficient endocytic activity via the cytopharynx. In a subset of intermediate Ic, the cytopharynx invagination was absent, but a group of microtubules likely corresponding to those of the cytopharynx remained. The most drastic ultrastructural modification observed in the cytostome-cytopharynx complex were parasites where a microtubule quartet likely corresponding to that of the cytopharynx was present, but the membrane invagination of the cytopharynx was absent (Fig. 5A–I). The representative cell (Fig. 5A and B) appeared to be a late intermediate of metacyclogenesis (late Ic stage) because the kinetoplast was posterior to nucleus, but was still rod-shaped and the Golgi complex localized between nucleus and kinetoplast, as seen in trypomastigotes form. In a different slice and view series of an intermediates Ic cell where the kinetoplast was posterior to a reservosome, indicating a very late stage in metacyclogenesis (late Ic, but still different from metacyclic trypomastigotes, because the kinetoplast was rod-shaped), the cytostome and the cytopharynx were absent, but the preoral ridge was present (albeit smaller than in epimastigotes), and we could also identify the four microtubules of the cytopharynx quartet (Fig. 5F–I). In this late intermediate, cytopharynx disassembly probably occurred by vesiculation or endocytic budding without membrane replacement. The reorganization of the Golgi is a crucial phenomenon in the polarization and migration of many cell types (Coppens et al., 1987). The Golgi apparatus is a station for the sorting and transport of lipids and proteins that are essential for secretory pathways (Amersi et al., 2006); thus, Golgi migration may be very important in supplying membrane components for rearrangements in cellular architecture during metacyclogenesis. In T. cruzi epimastigotes, the
Golgi apparatus is localized near the flagellar pocket and the cytostome-cytopharynx complex, in the anterior region of parasite (Alcantara et al., 2014), and this proximity may be essential to deliver content from the Golgi to these two active membrane domains. Previously we suggested that this proximity allows Golgi vesicles to fuse quickly with the cytopharynx invagination, delivering essentials molecules and membrane for the maintenance this membrane domain (Alcantara et al., 2014). Early in metacyclogenesis, the cytostome-cytopharynx migration (to the posterior region) starts. Our observations indicate that the Golgi complex is maintained in the anterior region during the Ib stage, and its migration to the posterior of the cell starts in the Ic stage, but may only be concluded late in the Ic stage, after cytostomecytopharynx posterior-ward migration. Late Golgi migration to the posterior is expected to increase the spatial separation between this organelle and cytostome-cytopharynx, and might decrease membrane replacement to this organelle. This effect, coupled with the maintenance of endocytic activity – with budding of cytopharynx membrane into vesicles – may underlie the ultrastrutural changes observed in the cytostome-cytopharynx complex, culminating in cytopharynx shortening and disappearance, in Ic intermediates. In line with this hypothesis, Johnson et al. (2016) showed that, in a mammalian cell line, peripheral lysosomes pH were less acid than internal ones and had decreased proteolytic activity. This effect was explained by the increased distance between the Golgi and peripheral lysosomes, which had, therefore, decreased access to Golgiderived enzymatic and acidifying content (Johnson et al., 2016). Interestingly, negatively-stained cytoskeletons of metacyclic trypomastigotes display a previously unnoticed structure: a microtubule quartet that describes a path from the flagellar pocket area towards the nucleus, as expected for the cytopharynx microtubule quartet (but not for the FAZ MtQ) (Fig. 6). These data represent clear evidence that the microtubule quartet of the cytopharynx is maintained in metacyclic trypomastigotes, which may enable the fast reassembly of the cytostome-cytopharynx in amastigotes, after trypomastigote entry into host cells. Fig. 7 illustrates a sequential description of these events that includes kinetoplast/ flagellum displacement, shortening of the cytopharynx, Golgi displacement, and finally, cytopharynx disappearance, with maintenance of the microtubule quartet that accompanies the cytopharynx in other life cycle forms. The work presented herein provided evidence that metacyclogenesis involves active cellular reorganization with maintenance of endocytic activity.
Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018
J.C. Vidal et al. / Journal of Structural Biology xxx (2016) xxx–xxx
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Fig. 7. Model showing the displacement, shortening and disappearance of the cytopharynx during metacyclogenesis. A) Epimastigote with the kinetoplast anterior to nucleus, Golgi complex and flagellar pocket at the anterior region of the cell; B) Intermediate Ia, with the kinetoplast and flagellum repositioned closer to the nucleus, but still located in the anterior region of the cell, similarity to that observed in the epimastigote; C) Intermediate Ib, showing kinetoplast/flagellum migration to a position adjacent to the nucleus, which may ‘drag’ cytostome aperture repositioning. D) Intermediate Ic, with the kinetoplast posterior to the nucleus, while the Golgi remained in the anterior region. Note the cytostome aperture in the posterior of the cell, between the kinetoplast and the nucleus, and the short cytopharynx invagination; E) Intermediate Ic, with a posterior Golgi complex. In these cells, the cytopharynx invagination itself is absent, and only the microtubule quartet (blue) of cytopharynx remains. F) Metacyclic has globular kinetoplast localized at the posterior end of the cell and an elongate nucleus and Golgi complex between these organelles. The microtubule quartet of cytopharynx remains.
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and shortening, and cytopharynx disappearance – clarifies important aspects of the intricate progression of morphological events during the formation T. cruzi infective forms. Acknowledgements The authors would like to thank Luis Sergio Júnior (Inmetro, Rio de Janeiro, Brazil) and Thiago Luis de Barros Moreira (Centro Nacional de Bioimagem, Rio de Janeiro, Brazil) for their technical assistance. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number 472262/2012-2], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) scholarships to C.L.A and J.C.V., Financiadora de Estudos e Projetos (FINEP), Programas Núcleos de Excelência (PRONEX) [grant number E-26/110.576/2010] and Cientista do Nosso Estado [grant number E-26/102.850/2012] from the Fundação Carlos Chagas Filho de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2016.07.018. References
Our study of the 3D architecture of the cytostome-cytopharynx complex during metacyclogenesis – with the description of morphological milestones such as preoral ridge and cytostome posterior-ward displacement, cytopharynx proximal end enlargement
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Please cite this article in press as: Vidal, J.C., et al. Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.07.018