Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa

ARTICLE IN PRESS PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY Progress in Histochemistry and Cytochemistry 44 (2009) 67–124 www.elsevier.de/proghi

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa Wanderley de Souzaa,b,, Celso Sant’Annaa,b, Narcisa L. Cunha-e-Silvaa a Laborato´rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, bloco G subsolo, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro 21941-902, Brazil b Diretoria de Programas, Instituto Nacional de Metrologia, Normalizac¸a˜o e Qualidade Industrial-INMETRO, Av. Nossa Senhora das Grac¸as, 50, Xere´m, Duque de Caxias – Rio de Janeiro 25250-020, Brazil

Abstract Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania. r 2009 Elsevier GmbH. All rights reserved. Keywords: Protozoa; Electron microscopy cytochemistry; Trypanosomatids; Apicomplexa; Entamoeba; Trichomonas; Giardia; Endocytosis; Reservosome; Lysosome

Corresponding author at: Laborato´rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, bloco G subsolo, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro 21941-902, Brazil. Tel.: +55 21 2562 6581; fax: +55 21 22602364. E-mail address: [email protected] (W. de Souza).

0079-6336/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.proghi.2009.01.001

ARTICLE IN PRESS 68

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

1. Introduction During the evolution of prokaryotic to eukaryotic organisms, some properties were lost while others were acquired. Among the latter is the ability of eukaryotic cells to incorporate macromolecules, macromolecular complexes and even other cells through a process that involves the formation of endocytic vesicles and vacuoles. There are also examples of eukaryotic cells that secrete proteases into the extracellular space to degrade proteins into amino acids, which are then transported across the plasma membrane; this process is predominant in prokaryotic cells. For most eukaryotic cells, including pathogenic protozoa, endocytosis is the basic mechanism for ingesting macromolecules; these internalized molecules are later degraded in the endosomal–lysosomal system and provide important precursors for several key metabolic pathways. The extent of endocytic activity varies across different protozoa and even across various developmental stages of some protozoa. In most protozoa, endocytic activity is present throughout the cell surface. Some protozoa, however, are polarized, so that the endocytic activity is restricted to welldefined regions of the cell surface. The best examples of these specific protozoa are trypanosomatids, in which the endocytic activity is restricted to the flagellar pocket region. Before describing in some detail the endocytic pathway of pathogenic protozoa, especially of Trypanosoma cruzi, the causative agent of Chagas disease, we will briefly review the information that is available for mammalian cells, where this pathway has been intensely investigated using an arsenal of contemporary techniques. We chose to cite only recent reviews in this section as mammal or yeast cells endocytic pathway is not the scope of the present review but is summarized here as a guide to the present knowledge in this field. Further information on this subject can be found in excellent reviews that have been recently published (Mayor and Pagano, 2007; Cullen, 2008; van Meel and Klumperman, 2008; Sandvig et al., 2008).

2. Endocytosis in mammal cells The process of ingestion of macromolecules, macromolecular aggregates and larger particles, varying from small viruses to large protozoa, is fundamental for both cell nutrition and the elimination of dead cells from the intercellular space. In addition, viruses, bacteria and protozoa use this process to penetrate into host cells, where they can survive and multiply. Perhaps as a reflection of the multiple roles that the endocytic process has, many entry pathways have been identified. Based on gross morphological information, we can identify at least three endocytic processes: phagocytosis, macropinocytosis and pinocytosis. In the first two processes, large vacuoles are formed by plasma membrane extensions that are propelled by actin filament polymerization. In pinocytosis, small vesicles are formed by plasma membrane invagination. Pinocytic processes can be subdivided according

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

69

Fig. 1. Schematic representation of the endocytic pathway of mammal cells. The several modes of pinocytosis are represented at left: clathrin-dependent, caveolin-dependent and one type of clathrin- and caveolin-independent endocytosis use dynamin to promote vesicle budding; fluid-phase pinocytosis, especially from GPI-anchored protein-rich domains does not use dynamin for budding. The resulting vesicles initially keep similarity with their origins that are subsequently lost as they undergo homotypic fusion, forming the caveosome and GEEC. Fusion with the sorting endosome delivers molecules to an acidic environment that allows recycling of some molecules to the plasma membrane, directly, a Rab4-mediated event, or via recycling endosome, mediated by Rabs 8 and 11. The remaining molecules are addressed to degradation and transported to late endosome or multivesicular body (MVB) that exhibits many intralumenal vesicles, formed by inward movement of the organelle membrane. Degradation work begins in late endosome, performed by hydrolytic enzymes come from Golgi complex. Degradation is completed in lysosome that receives cargo and enzymes from late endosome. The right side of the scheme depicts internalization of big particles by phagocytosis or big fluid volumes by macropinocytosis, both by emission of membrane projections sustained by actin filaments. The vacuoles thus formed join endocytic pathway by successive fusion with endosomes and lysosome. All endocytic compartments receive H+ ions actively pumped from cytosol and possess identified molecular markers, Rab proteins being the most representative.

to the participation of the cytoplasmic proteins dynamin, clathrin and caveolin. Fig. 1 schematically presents the basic features of the various endocytic processes. Phagocytosis involves the assembly of membrane projections that surround the particles to be ingested; these particles were previously attached to the cell surface

ARTICLE IN PRESS 70

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

due to recognition of molecules exposed on the particle’s surface (specific ligands, charge or hydrophobic forces) by receptors on the cell surface. The assembly of actin filaments is an important step in the initial process, as is the involvement of PI3 kinase in the final step. The ingested particle is localized within a vacuole known as the phagosome. This phagosome, in most cases, sequentially fuses with early and late endosomes, as well as with lysosomes to form phagolysosomes. Phagocytosis also takes place in some pathogenic protozoa, especially in Entamoeba histolytica and Trichomonas vaginalis, as will be discussed later. Macropinocytosis, a process used to ingest large amounts of liquid, is characterized by the ruffling of membrane that is rich in actin filaments; the front portion of these ruffles can bend and fuse with the plasma membrane to form a large endocytic vacuole. This process can be a mere consequence of migration in adherent cells but may also be induced or intensified by growth factors and PI3 kinase. An interesting variation of macropinocytosis has been recently described (Orth et al., 2006); this process involves PI3 kinase and dynamin and consists of the assembly of actin-dependent circular dorsal ruffles that are able to rapidly internalize 50% of epidermal growth factor receptor. The formation of small endocytic vesicles, which is classically known as pinocytosis, is more complex since it varies according to the participation of different cytoplasmic molecules. The first, and one of the best characterized processes, depends on the assembly of a clathrin coat on the cytoplasmic side of the forming vesicle. This process also involves the participation of dynamin, which is fundamental for vesicle scission. In most cases, clathrin-dependent endocytosis also involves the previous recognition of the ligand to be ingested by receptors on the cell surface. From the time of mobilization of receptor–ligand complexes until vesicle budding, at least 15 molecules are known to participate, as identified in yeast by genetic techniques and live cell imaging (Kaksonen et al., 2005). Four sequentially assembled modules include coat formation, membrane invagination, local actin nucleation and vesicle budding. Recent immunocytochemical studies (Idrissi et al., 2008) have implicated actin and myosin I in yeast endocytic vesicle fission. The second variation of pinocytosis involves the participation of both caveolin and dynamin, with the formation of caveosomes prior to early endosomes (reviewed by Parton and Simons, 2007). Caveolae are 50–80 nm flask-shaped invaginations of the plasma membrane; they are enriched in sphingolipids and cholesterol, signalling proteins and GPI-anchored proteins. The third pinocytic process is dynamin-dependent but clathrin- and caveolinindependent and involves the formation of uncoated vesicles. This process is used to internalize the b-chain of the interleukin-2 receptor among other proteins. It involves the participation of the signalling pathway of Rac 1 and p21 activated kinases (Paks), which activates cortactin (Grassart et al., 2008). The fourth process does not depend on clathrin, caveolin or dynamin and also involves the formation of uncoated vesicles with the co-participation of flotillin-1 and flotillin-2 (Frick et al., 2007). Clusters of GPI-anchored proteins follow a similar route of internalization, involving the participation of actin nanomachinery controlled by Cdc42/Rac and cholesterol, and are subsequently found in GEEC

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

71

(GPI-anchored proteins enriched endosomal compartment). This pathway seems to be the most intensely used mechanism in the so-called fluid-phase pinocytosis (Chadda et al., 2007). The fifth process also does not depend on clathrin, caveolin or dynamin and involves the formation of uncoated vesicles that are delivered to early tubular recycling compartments. This process depends on the participation of the GTPase ARF6, flotillin-1, Rab11, Rab22 and PI3 kinase. Proteins such as b1 integrin and E-cadherin are internalized by this process (Balzac et al., 2005). In all of these processes, after the vesicles lose their coats, they fuse with early endosomes; this event is regulated by Rab5 and the early endosomal antigen 1 (EEA-1). Early endosomes display a variable morphology; they are characterized by a slightly acidic pH, around 6.5, which is established by the activity of vacuolar-type (V-type) proton ATPase. Endosomal compartments are progressively acidic; this acid environment is essential for key events, such as receptor–ligand uncoupling and lysosomal hydrolases activation (Lafourcade et al., 2008). The early endosome is the site of a distribution of internalized particles, which can be degraded, recycled back to the plasma membrane or secreted via a different plasma membrane domain (transcytosis). The transferrin receptor (TfR) has been the model for recycling studies, as it carries apotransferrin back to plasma membrane after iron delivery inside early endosomes. Due to their molecular selecting activity, early endosomes have been called ‘‘sorting’’ endosomes. Molecular sorting can be determined by the cytoplasmic tail, where signal sequences are recognized by a cytoplasmic coat through adaptor molecules (Bonifacino and Traub, 2003). Recently, they have been proposed that the peculiar early endosome morphology of tubules emanating from a round compartment functions as a sorting mechanism; in this model, different sorting nexins (SNXs) would form sorting complexes with actin nucleating complexes (N-WASP) or microtubule motor proteins (kinesins and dynein) to drive recycling to the plasma membrane (in a short route, mediated by Rab4, or a long, juxtanuclear route, mediated by Rab11) or to the trans-Golgi network (the retromer) (Cullen, 2008). Each early endosome domain has a molecular marker (Rabs 4, 5A, 5C, 8, 11, EEA1, TfR). It is not yet clear whether these domains, which have functions that are so essential that they have acquired different names, such as sorting endosome and recycling endosome, in addition to the early endosome itself, are one continuous branched compartment, similar to the endoplasmic reticulum, or a collection of vesicles and tubules with the common marking character of distributing internalized cargo. Internalized molecules that are destined for degradation are concentrated in the non-tubule region of the early endosome, aided by clathrin. Some of these are included during the formation of intraluminal vesicles that will constitute the next endosomal compartment, the late endosome or multivesicular body. Late endosomes are characterized by an acidic pH (around 6.0), the presence of Rabs 7 and 9 and a multivesicular morphology. The assembly of a special molecular machinery, ESCRT (endosomal sorting complex required for transport) seems to drive late endosomal limiting membrane inward vesiculation; this process depends on the orchestrated

ARTICLE IN PRESS 72

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

action of several proteins, including class E Vps (vacuolar protein sorting) and monoubiquitinating and deubiquitinating enzymes (Saksena et al., 2007). The degradation of membrane proteins follows this pathway. As multivesicular bodies mature, they acquire the capacity for fusion with lysosomes, the final station of the endocytic pathway. Lysosomes are characterized by a very low pH (around 5.0) and their electron-dense content. The presence of several active hydrolases, mainly proteases and acid phosphatases, is considered a lysosomal marker in mammal cells, in addition to the presence of heavily glycosylated membrane proteins (LAMPs, lysosome associated membrane proteins that are also lysosome markers) and the accumulation of most of the ingested protein which needs to be degraded (recently reviewed by De Matteis and Luini, 2008; van Meel and Klumperman, 2008). Cargo transfer between late endosomes and lysosomes is controversial. An elegant work that correlates live cell imaging and electron microscopy (Bright et al., 2005) showed that the two compartments fuse temporarily, forming a hybrid organelle, and then subsequently re-form the lysosomes. Similar observations have since been made in the majority of models where it has been investigated. The hybrid organelle appears to be the site where bulk digestion takes place (Luzio et al., 2007). The most important contribution of these studies is the characterization of lysosomes as fusogenic organelles, rather than endpoints of the endocytic pathway; as fusogenic organelles, they can play other roles, such as fusing with phagosomes and autophagosomes. This point of view has opened the way for new lysosome functions, such as membrane repair by secretory lysosomes (Andrews, 2002).

3. Endocytosis in anaerobic protozoa A significant number of protozoa live in anaerobic or microaerobic environments, usually in the lumen of the digestive and genito-urinary tract. The most important protozoa belong to (a) the genera Trichomonas, which includes T. vaginalis and Trichomonas foetus, agents of human and cattle trichomoniasis, respectively; (b) the genus Entamoeba, including E. histolytica and Entamoeba dispar, which are responsible for human amoebiasis; and (C) Giardia lamblia, the causative agent of giardiasis, which has a high prevalence throughout the world. 3.1. Endocytosis in Trichomonas The first observations of trichomonads by transmission electron microscopy highlighted the presence of a significant number of cytoplasmic vacuoles, some of which contained material in their matrix (Brugerolle, 1971; Francioli et al., 1983; Scholtyseck et al., 1985; Benchimol et al., 1986; Juliano et al., 1991; McGrory et al., 1994). Quantitative comparative analysis of different species has been carried out and the data obtained show clearly that some strains of T. vaginalis have more intense endocytic activity. For instance, in phagocytosis experiments it was shown that parasites from the T016 strain ingest twice the number of yeast cells as those of

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

73

the much less virulent Jt strain (Pereira-Neves and Benchimol, 2007). Experiments using proteins, such as horseradish peroxidase and ferritin, as well as gold particles coated with different ligands, such as transferrin, lactoferrin and LDL, showed that trichomonads ingest these labels through a typical endocytic process involving the formation of small vesicles, traditionally known as pinocytic vesicles, which subsequently become other larger compartments (Affonso et al., 1994). It is

Fig. 2. Endocytosis in trichomonads: (a) using transmission electron microscopy, cationized ferritin-coated 80 nm latex beads can be observed inside small vesicles (curved arrow), as well as bound to the parasite membrane (arrows); (b) the fluorescent tracer Lucifer yellow inside endocytic compartments (arrowheads) of T. foetus; (c) scanning electron microscopy of cationized ferritin-coated 500 nm microspheres bound to the surface (asterisk) of T. vaginalis. Areas of bead internalization leave a depression at parasite surface (arrow); (d) laminin-coated 500 nm microspheres were internalized in vacuoles dispersed in the cytoplasm of T. vaginalis; (e) the presence of active acid phosphatase in the vacuoles (stars) of T. foetus was demonstrated by cytochemistry. Bars: 500 nm (a, c); 150 nm (b); 300 nm (d); 600 nm (e). After Affonso et al. (1997) (b), Benchimol et al. (1986) (a, e) and Benchimol et al. (1990) (c, d).

ARTICLE IN PRESS 74

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

important to point out that endocytic activity may take place throughout the protozoan surface. Some of these events are receptor-mediated endocytic processes (Affonso et al., 1994; Peterson and Alderete, 1984a, b). Trichomonads are able to ingest large particles. In the case of T. foetus, latex particles as large as 4.4 mm in diameter can be ingested (Benchimol et al., 1990). The extent of ingestion is higher when the particles are previously coated with fibronectin and laminin (Benchimol et al., 1990).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

75

All available data indicate that some strains of T. vaginalis, especially when recently isolated from patients, have an intense endocytic activity. Studies carried out with trichomonads are good examples of how different microscopy techniques can be used to investigate the endocytic pathway in protozoa. These include the use of (a) fluorescent labelled molecules (Fig. 2b); (b) small latex particles, which are ingested through typical pinocytic processes (Fig. 2a); (c) large latex particles, which can be seen both by scanning (Fig. 2c) and transmission (Fig. 2d) electron microscopy; (d) gold-labelled macromolecules, which can be seen by transmission electron microscopy of thin sections (Fig. 3c–f) as well as in freeze-fracture replicas (Fig. 3a, b); and (e) the use of the DAMP technique (Anderson et al., 1984) to estimate the pH of intracellular compartments (Fig. 3g, h). Indeed, genomic analysis has revealed a dramatic expansion of the genes coding for proteins involved in the membrane trafficking machinery (Carlton et al., 2007). These parasites are even able to ingest bacteria, yeast cells, erythrocytes, leukocytes and remnants of their host cells through a typical phagocytosis process (Benchimol et al., 1990; Francioli et al., 1983; Street et al., 1984; Juliano et al., 1991; GonzalezRobles et al., 1995; Rendon-Maldonado et al., 1998; Pereira-Neves and Benchimol, 2007). As with typical endocytic processes that occur in the well-characterized mammalian phagocytic cells, the phagocytic process that takes place in trichomonads involves the assembly of surface projections that envelop the particles (Fig. 4a, c, d), their ingestion and the formation of a typical phagocytic vacuole (Fig. 4d). Particles can also be ingested by a sinking process (Fig. 4b), however, which does not involve any apparent participation from plasma membrane extensions, as recently shown by scanning electron microscopy for the ingestion of yeast cells (PereiraNeves and Benchimol, 2007). It has also been shown that previous treatment of the parasites with cytochalasin D, a drug that interferes with actin polymerization, inhibits the endocytosis of yeast cells in trichomonads (Pereira-Neves and Benchimol, 2007). In addition, the assembly of actin filaments takes place in the protozoan at an area that is involved in the endocytic activity (Pereira-Neves and Benchimol, 2007). Proteins such as actin and alpha-actinin have been characterized in trichomonads (Brugerolle et al., 1996; Bricheux and Brugerolle, 1997; Addis et al., Fig. 3. T. foetus endocytic compartments – (a, b) the label fracture technique was very suitable to show binding (a) and the beginning of internalization (b) of gold-labelled lactoferrin forming clusters (arrows) at the surface of T. foetus; (c, d) after 5 min of incubation, goldlabelled transferrin was found in budding vesicles (arrowhead) and tubule vesicular compartments (arrows); (e) cationized ferritin could also be found in clusters attached to parasite surface (arrow) or inside peripheral tubules (arrowhead); (f) after 30 min at 37 1C, several compartments are full with the tracers (arrows) or present them concentrated near the periphery (arrowhead); (g–h) immunocytochemical detection of DAMP, a weak base, with 5 nm gold labelled anti-DNP antibodies (arrowheads), demonstrated that the endocytic compartments occupied by 15 nm gold-labelled lactoferrin (arrows) are acid. Bars: 120 nm (a, b); 80 nm (c, e, f, h); 150 nm (d, g). After Affonso et al. (1994) (a–d) and Affonso et al. 1997 (e–h).

ARTICLE IN PRESS 76

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Fig. 4. Phagocytosis by T. vaginalis – (a–b) Scanning electron microscopy is suitable to show that T. vaginalis is able to project pseudopods (asterisks) to ingest a yeast cell (a) or, alternatively, ingest it by simply soaking the yeast cell in the parasite membrane (b); (c, d) transmission electron microscopy further demonstrated pseudopod (arrows) emission by T. vaginalis engulfing an yeast (Y). AF, anterior flagella; Ax, axostyle; N, nucleus; H, hydrogenosome; RF, recurrent flagellum. Bars: 4 mm (a, b); 500 nm (c, d). After PereiraNeves and Benchimol (2007).

1998; Bricheux et al., 1998). Coronin, a protein known to interact with F-actin, has also been identified and localized in T. vaginalis (Bricheux et al., 2000). In addition, the genome of T. vaginalis predicts the presence of many genes that encode cytoskeletal-associated proteins, such as kinesin and dynein. No genes that encode myosin have been found, however (Carlton et al., 2007). It has also been shown that all materials ingested through an endocytic process are delivered to compartments that resemble the early and the late endosome, as well as typical lysosomal structures. These have been identified based on their characteristic morphology, the accumulation of gold particles and the cytochemical localization of acid phosphatase (Fig. 2e) (Benchimol et al., 1986; Affonso et al., 1994, 1997). Analysis of the T. vaginalis genome indicates that this organism has one of the most complex degradomes described, with more than 400 peptidases (Carlton et al., 2007).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

77

3.2. Endocytosis in Giardia lamblia The first analysis of the fine structure of trophozoites of G. lamblia revealed the presence of a large number of vesicles at the periphery; these were designated peripheral vesicles (Fig. 5a–c) (Bockman and Winborn, 1968, Tai et al., 1993; Lanfredi-Rangel et al., 1998). These vesicles are not distributed throughout the cell surface. Indeed, they are not seen in the ventral region, where the protozoan attaches to the substrate (either epithelial cells or the glass/plastic substrate where they grow in vitro), and are mainly concentrating in the dorsal region. The incubation of trophozoites in the presence of native ferritin shows that all molecules that are

Fig. 5. Giardia lamblia endocytic compartments – (a) Transversal thin section showing G. lamblia peripheral vesicles (arrowheads) just beneath the plasma membrane, the two nuclei (n), the adhesive disk (ad) and endoplasmic reticulum cisternae (arrows); (b) peripheral tubule (arrows) with juxtaposed vesicles (arrowheads); (c) peripheral vesicle budding from or fusing with plasma membrane (arrow); (d–e) Nomarski (d) and confocal fluorescence (e) images of a G. lamblia trophozoite previously incubated with acridine orange, a weak base that diffuses through membranes and accumulates inside proton-rich compartments, showing that peripheral vesicles are acidic. Bars: 500 nm (a–c); 5 mm (d, e). After Lanfredi-Rangel et al. (1998).

ARTICLE IN PRESS 78

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Fig. 6. Giardia lamblia peripheral vesicles – Trophozoites that have ingested horseradish peroxidase presented the tracer inside peripheral vesicles (arrows) as soon as after only 5 min of incubation (inset in a) or as long as after 2 h (a), as revealed by diaminobenzidine cytochemistry in transmission electron microscopy; (b) acid phosphatase cytochemistry demonstrated the acidic character of G. lamblia peripheral vesicles (arrows); (c–d) 3D-reconstruction from serial ultrathin sections of acid phosphatase (c) and glucose-6phosphatase (d) labelled trophozoites, evidenced continuity among peripheral vesicles (magenta in c) and endoplasmic reticulum (magenta in d). Bars: 500 nm. After LanfrediRangel et al. (1998).

ingested can be observed within the peripheral vesicles (Fig. 6a); this result confirms the endocytic nature of these vesicles (Bockman and Winborn, 1968). Acid phosphatase activity has been detected in the vesicles using a cytochemical approach

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

79

(Fig. 6b) (Feely and Dyer, 1987; Lindmark, 1988; Kattenbach et al., 1991; LanfrediRangel et al., 1998). Subsequent studies have shown that (a) all macromolecules ingested by the protozoan (ferritin particles and horseradish peroxidase (Fig. 6a), which were localized using the diaminobenzidine technique; proteins such as albumin and transferrin coated on colloidal gold particles; LDL; fluorescent lipid analogues; virus particles, etc.) concentrate in the peripheral vesicle; (b) the vesicles are acidic, displaying an orange to red staining when incubated in the presence of acridine orange and observed by fluorescence microscopy (Fig. 5d, e); (c) some of the vesicles are labelled when trophozoites are incubated in a cytochemical medium designed for the localization of acid phosphatase, a classical marker of lysosomes (Fig. 6b); (d) some vesicles are labelled when incubated in the presence of a cytochemical medium designed for the localization of glucose-6-phosphatase, an enzyme marker of the endoplasmic reticulum cisternae; (e) some vesicles, as well as the ER cisternae, are labelled when the cells are submitted to the zinc iodide-osmium tetroxide technique, which reveals the presence of proteins; (f) the three-dimensional (3D) reconstruction of serial thin sections (Fig. 6c, d) revealed the existence of continuity between some profiles of the endoplasmic reticulum (identified based on their morphology and staining for glucose-6-phosphatase) and some of the peripheral vesicles, explaining the labelling of some vesicles with ER markers; (g) some vesicles seem to fuse to each other to form tubules (Fig. 5b). Taken together, these observations have led to the suggestion that the peripheral vesicles of G. lamblia constitute a primitive endocytic system, in which small endocytic vesicles, early and late endosomes and lysosomes co-exist in the same organelle, the peripheral vesicle system (Lanfredi-Rangel et al., 1998). In addition, observations have indicated that not all portions of the protozoan cell surface are able to support endocytosis. Additional studies have shown that the formation of at least some of the vesicles requires the participation of clathrin (Hernandez et al., 2007) and a dynamin-related protein (Gaechter et al., 2008). Dynamin co-localizes with clathrin in the peripheral vesicles and plays some role in the assembly of encystation vesicles that are required for the encystation process (Gaechter et al., 2008). An adaptor protein was found to mediate the transport of an encystation specific cysteine proteinase to the peripheral vesicles using a tyrosine-based sorting system (Touz et al., 2003) as well as acid phosphatase (Touz et al., 2004). 3.3. Endocytosis in Entamoeba histolytica Even by light microscopy it is possible to note the presence of a large number of vesicles located in the cytoplasm of trophozoites of E. histolytica. They have acidic character (Fig. 7a, b) and accumulate external molecules uptaken by endocytic process (Fig. 8a, b). It is also possible to notice, especially in samples isolated directly from patients infected with pathogenic strains, that remnants of epithelial cells and erythrocytes can be seen within the cytoplasmic vacuoles, indicating their phagocytic nature. Transmission electron microscopy has been performed on thin sections of E. histolytica incubated in the presence of native ferritin, cationized ferritin, colloidal gold particles coated with albumin, transferrin, lactoferrin (Fig. 7d–f) and LDL,

ARTICLE IN PRESS 80

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Fig. 7. Endocytosis in Entamoeba histolytica – Phase contrast (a) and acridine orange fluorescence (b) images showed many acidic vesicles and vacuoles in the cytoplasm of Entamoeba. Ultrathin section (c) of a trophozoite confirmed that the cytoplasm is filled with vacuoles (v); gold-labelled lactoferrin could be found bound to parasite surface (arrowheads in d), inside peripheral tubules (arrowheads in e) and vesicles (arrowheads in e). Bars: 17 mm (a, b); 1.6 mm (c); 400 nm (d); 500 nm (e); 170 nm (f). After Batista et al. (2000).

fluid-phase tracers (Fig. 8a–c), latex particles of variable diameters, bacteria (Bracha et al., 1982; Francioli et al., 1983; Batista et al., 2000), erythrocytes (Tsutsumi et al., 1992) and even epithelial cells (Huston et al., 2003) and cells from the immune system (Ravdin, 1989). These work show clearly the intense endocytic activity that takes place in this protozoan. Transferrin-binding proteins have been identified (Welter et al., 2006). Cytochemical analysis has shown that endocytic vesicles can be formed anywhere along protozoan surface and that many of them are coated with clathrin (Hernandez et al., 2007; Gaechter et al., 2008). The vesicles fuse with each to form structures that can be recognized as early and late endosomes; later, their cargo is delivered to typical lysosomes. Small GTPases that are involved in the control of vesicle trafficking along the endocytic pathway have been identified. In contrast to

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

81

Fig. 8. Endocytosis in Entamoeba histolytica – Trophozoites uptake fluid-phase tracers like Lucifer yellow (a, phase contrast, b, fluorescence), or peroxidase (c). Inset: acid phosphatase cytochemistry (white arrows) demonstrated that lactoferrin gold (black arrows) containing compartments are acid. A nearby vacuole may be unlabelled (asterisk). Bars: 17 mm (a, b); 1.7 mm (c); 300 nm (inset). After Batista et al. (2000).

mammalian cells, in which there is only one single gene for Rab7, E. histolytica has nine Rab7 genes (reviews in Temesvari et al., 1999; Saito-Nakano et al., 2005; Okada and Nozaki, 2006). EnRab7A is localized to a non-acidic compartment that fuses with lysosomes (Nakada-Tsukui et al., 2005), while EhRab7B is exclusively localized in acidic vacuoles containing lysosomal proteins such as amoebapore-A and cysteine proteinase (Saito-Nakano et al., 2007). Rab11B, which is involved in secretion of cysteine proteinase, has been located to non-acidic vesicles, which may correspond to recycling endosomes (Mitra et al., 2006, 2007). Rab5A, Rab7 and Rab11B were identified in isolated E. histolytica phagosomes (Rodriguez and Orozco, 2000; SaitoNakano et al., 2004; Okada et al., 2005). The lysosomes have been identified based

ARTICLE IN PRESS 82

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

on localization of acid phosphatase activity (Fig. 8c inset) (Batista et al., 2000). The kinetics of acidification of the lysosomes varies according to the pathogenicity of the protozoan strain. Phagosomes from attenuated strains acidify rapidly, within 2 min of formation (Mitra et al., 2006). It has been shown that the endocytic activity in E. histolytica is a process that involves cell-signalling events and the participation of protein kinases and phosphatases (Boettner et al., 2008). Indeed, treatment of the trophozoites with genistein, staurosporin and Wortmannin, well-known inhibitors of protein kinases and PI-3 kinase, significantly inhibits the ingestion of erythrocytes (Batista and De Souza, 2004). Cytoskeletal components of the E. histolytica trophozoite are also involved in this endocytic activity. A prior treatment of the parasite with cytochalasins inhibits endocytic activity (Bailey et al., 1987; Marion et al., 2004). Rapid actin polymerization is observed upon contact of E. histolytica with target cells (Bailey et al., 1987). Rho proteins are also involved in actin rearrangement; it was shown that antibodies recognizing EhRhoA1 translocate from cytoplasmic vesicles to the protozoan plasma membrane (Franco-Barraza et al., 2006). A gene coding for an unconventional myosin in E. histolytica has been described and characterized (Vargas et al., 1997; Voigt et al., 1999). The involvement of cell surface signalling and cytoskeleton involvement in the early steps of the phagocytic process have been well established using several approaches (Marion et al., 2005). A calcium binding protein (EhCaBP1) has been shown to participate in cellular processes involving actin filaments. The overexpression of this protein inhibits phagocytic activity but not of fluid-phase pinocytosis (Orozco et al., 1983; Sahoo et al., 2004; Jain et al., 2008).

4. Endocytosis in Apicomplexa The Apicomplexa Phylum includes a large number of species. Some of these organisms are agents of important human diseases, such as malaria (Plasmodium genus) and toxoplasmosis (Toxoplasma gondii). There are also species that cause disease in animals (Eimeria in chickens and in cattle, Theileria in cattle). Also included in this phylum is Cryptosporidium, a parasite that infects animals and is also an opportunistic human parasite. Very few studies have been conducted on the endocytic activity of this group of organisms. The data that exist are mainly on Plasmodium. This parasite lives within a special vacuole, known as the parasitophorous vacuole, which is found in the cytoplasm of the host cells. In order to survive and multiply, molecules from the host cell cytoplasm must first cross the parasitophorous vacuole membrane and then trigger a process of ingestion by the protozoa. Since the first studies on the fine structure of the Plasmodium species, it has been shown that while living within the erythrocytes of their hosts, these organisms are able to ingest portions of the cytoplasm through a process called intracellular

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

83

phagotrophy. The presence of structures with the same electron density as the erythrocyte cytoplasm, usually very dense due to the presence of haemoglobin, was seen within the intracellular protozoan (Rudzinska et al., 1965; Aikawa et al., 1966; Aikawa, 1971). Subsequent studies showed clearly that as the parasites progresses within the erythrocyte, massive amounts of haemoglobin is internalized and degraded (Slomianny, 1990; Goodyer et al., 1997; Saliba and Kirk, 2001). It has also been shown that internalization of at least part of the haemoglobin takes place through the cytostome (Fig. 9a, b), a structure found on the protozoan surface corresponding to an invagination of the plasma membrane with an interruption of

Fig. 9. Endocytosis in Plasmodium falciparum – (a) Intraerythrocytic trophozoite presenting three cytostomes (CYT) in the same thin section up taking haemoglobin from red blood cell (RBC) cytosol; (b) a cytostome (CYT) penetrates into parasite and interacts with its food vacuole (FV). In both sections (a, b), parasite plasma membrane (PPM) is clearly detached from parasitophorous vacuolar membrane (PVM). (c–h) A serial thin section sequence demonstrating that cytostome and haemoglobin containing compartments are continuous. Bars: 100 nm. After Lazarus et al. (2008).

ARTICLE IN PRESS 84

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

the inner membrane complex (Rudzinska et al., 1965; Aikawa et al., 1966; Langreth et al., 1978; Olliaro et al., 1989). It has a circular shape of approximately 150 nm in diameter and penetrates a few micrometers into the cytoplasm (Langreth et al., 1978; Trager, 1986). The mechanism of ingestion of haemoglobin has been further analysed more recently using three-dimensional reconstruction of serial thin sections by transmission electron microscopy. The data suggest the existence of four distinct processes (Elliott et al., 2008). One process uses the cytostome in its classical description, leading to the formation of small endocytic vesicles with localized Rab5a. A second process also involves the participation of the cytostome but with the formation of long thin tubes; this process depends on the participation of actin filaments, since it can be inhibited by drugs like cytochalasin D. The third process resembles phagocytosis but does not require the participation of actin. It resembles the intracellular phagotrophy process that was previously discussed by Trager (1986). The fourth process, designated as the ‘‘Big Gulp’’, involves the folding of the protozoan onto itself to cause the internalization of a large portion of the erythrocyte cytoplasm. Independent of the mechanism used to ingest haemoglobin, this large portion of the cytoplasm is delivered to a vacuole known as the food vacuole. There, it is digested and the toxic heme moiety is released and detoxified by its polymerization and sequestration as an inert crystalline deposit known as hemozoin (Egan et al., 2002). As previously discussed, cytoskeletal structures are involved in the trafficking of the endocytic vesicles in the cytoplasm. In the case of Plasmodium, microtubules do not appear until the schizont stage (Schuler et al., 2005; Taraschi et al., 1998). Recently, it has been shown that the treatment of Plasmodium falciparum with jasplakinolide inhibits endocytosis and leads to the accumulation of vesicles close to the plasma membrane and a concentration of actin in the parasite cortex (Smythe et al., 2008). From serial sections of controls as well as cells treated with cytochalasin D or jasplakinolide, it was recently suggested that actin dynamics plays a role in cytostome organization and haemoglobin transport. In addition, the existence of a vesicle-independent model for haemoglobin ingestion and transport to the food vacuole has been proposed (Lazarus et al., 2008). According to this model, small cytostomes mature into larger structures that extend into the parasite cytosol, establish contact and subsequently fuse with the food vacuole (Fig. 9c–h). The process of fusion between the cytostome and the food vacuole would require the nucleation of actin filaments.

5. Endocytosis in Trypanosomatids Protozoa of the Trypanosomatidae family are agents of parasitic diseases that have a high incidence of occurrence and a negative economic impact on developing countries. In the case of leishmaniasis, caused by several species of Leishmania, about 16 million people are infected in Africa, Asia, parts of Europe and Latin

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

85

America. Sleeping sickness, caused by the Trypanosoma brucei group, affects about 3 million people in Africa. Chagas’ disease, caused by T. cruzi, infects about 16–18 million individuals, and more than 80 million are at risk of infection (Schmunis and Cruz, 2005; WHO, 1991). Some trypanosome species are also important in veterinary medicine, since they seriously affect animals of economic interest, such as horses and cattle. Diseases caused by plant trypanosomatids are increasing in importance, due to the serious problems they have caused in coconut and oil palm plantations in South America.

5.1. Structural organization Several structures and organelles of trypanosomatids are unique and interesting. Here, we will emphasize only those structures that are involved in the endocytic pathway. The cell surface of trypanosomatids is a functional assembly of two components: the plasma membrane and a stable corset of subpellicular microtubules. The association of the cytoskeleton with the plasma membrane is now well established in most eukaryotic cells, but in trypanosomatids it is particularly strong; membrane and subpellicular microtubules remain associated even after lysis of the protozoan. Short filaments (6 nm thick) connect the regularly spaced subpellicular microtubules with each other and with the plasma membrane of all trypanosomatids (De Souza, 2002). Microfilaments were never observed in the cytoplasm of T. cruzi. Cytochalasin, however, a drug that interferes with actin microfilaments among other things, induces changes in the morphology of bloodstream trypomastigotes and inhibits movement. In epimastigotes, cytochalasin causes a 48% decrease in peroxidase uptake (Bogitsh et al., 1995). Correˆa et al. (2008) demonstrated that cytochalasin B treatment leads to morphological alterations in the cytoskeletal elements associated with the cytostome–cytopharynx complex, which is responsible for transferrin uptake. Comparative genomic analysis identified a potential role for an actin–myosin system in T. cruzi, as this protozoa presents, in addition to an actin gene, an expanded myosin family and a CapZ F-actin capping complex; these are not found in T. brucei or Leishmania (El-Sayed et al., 2005). These authors suggested that an actin–myosin system might function at the cytostome, the main cytoskeleton element that distinguishes parasites from the Schyzotrypanum sub-genus, including T. cruzi from the other trypanosomes. Actin and actin-binding proteins have been characterized recently in T. cruzi (De Melo et al., 2008). TcActin was observed to exist in several patch-like cytoplasmic structures, spread along the T. cruzi stages, similar to the actin distribution in Leishmania (Sahasrabuddhe et al., 2004). In contrast to actin in Leishmania, TcActin is not associated with subpellicular microtubules. Although, actin of T. cruzi has a similar structure as higher eukaryote actin, homology modelling has revealed fundamental differences, predominantly in the loops responsible for the oligomerization and interactions with actin-binding proteins. As a consequence, actin filaments have never been detected in T. cruzi.

ARTICLE IN PRESS 86

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

In several cell types, events like phagocytosis, intracellular traffic and cell adhesion are controlled by Rho (Welsh and Assoian, 2000). TcRho1 is the only member of the Rho family that has been identified in T. cruzi (De Melo et al., 2004). It is expressed in all developmental forms and localized to the Golgi complex in epimastigotes. TcRho1 has a role in metacyclogenesis, as epimastigotes transfected with a gene encoding a modified inactive protein die synchronously at the metacyclogenesis process. It is still difficult, however, to correlate TcRho1 function with the cytoskeleton, as canonical actin filaments have never been visualized in trypanosomatids. Recent reports in Leishmania have described the existence of uncommon oligomeric forms of actin that are neither stained by phalloidin nor affected by cytochalasin (Sahasrabuddhe et al., 2004). Those actin forms are distributed in patches all over the cell body and flagella, co-localizing partially with subpellicular microtubules. In a subsequent study, the same group identified an actin-binding protein similar to coronin that presents the same subcellular distribution pattern (Nayak et al., 2005). When Leishmania actin and coronin are co-expressed in mammalian cells they do form filaments. In bloodstream forms of T. brucei, RNAi demonstrated that actin is essential for parasite survival. Although filaments have not been observed, it co-localizes with the endocytic pathway (Garcia-Salcedo et al., 2004); additionally, flagellar pocket enlargement, which occurs as a result of actin depletion, strongly indicates that actin may play a role in endocytosis. Recently, it was demonstrated that the ablation of actin does not affect the transport of secretory proteins to plasma membrane, suggesting that, at least in T. brucei, the secretory and the endocytic pathways are independent (Nolan and Garcia-Salcedo, 2008). The plasma membrane of T. cruzi epimastigotes consists of four domains, each of them with particular exposed components and functional properties: the membrane of the main cell body; the flagellar membrane; the membrane that lines the flagellar pocket and the membrane around the cytostome entry. The latter two domains are relevant for endocytosis and will be discussed below. 5.2. Endocytic pathway in trypanosomatids Endocytosis has been extensively described in mammalian cells and yeast. It involves nutrient uptake from the plasma membrane and an orderly flow of endocytic vesicles that traffic en route to digestive or recycling pathways (Mukherjee et al., 1997). In trypanosomatids, endocytosis has been reported to exist exclusively in T. cruzi, Leishmania and, mainly, in T. brucei. No endocytic activity has been found in monoxenic trypanosomatids. Conserved pathways, organelles, molecular markers and the molecular machinery that governs fusion and fission processes have been found (McConville et al., 2002). Nonetheless, there are particularities that set trypanosomatids apart from higher eukaryotes, and as such, they are becoming an attractive model for understanding the endocytic mechanisms. In contrast to most eukaryotes, trypanosomatids have high polarized endocytic and exocytic systems that place organelles in conserved positions of the cytoplasm. The difference is striking when considering the topographic distribution of the endocytic compartments in the developmental forms. Here, we will review the

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

87

ultrastructural organization of the endocytic compartments in trypanosomes, focusing on the T. cruzi endocytic pathway. Although trypanosomatids are similar in some aspects of endocytosis, a divergence in the structural organization between them has been reported (Fig. 10) and will be detailed later. In T. brucei, the flagellar pocket and the whole endocytic and exocytic structures, including lysosomes and the Golgi complex, are all localized in the posterior region of the parasite body. Leishmania has a long endosomal/lysosomal multivesicular tubule spanning from the anterior to the posterior region. In T. cruzi epimastigotes, however, the cytostome

Fig. 10. Comparing trypanosomes endocytic pathway – (a) Trypanosoma brucei bloodstream trypomastigotes endocytic pathway is restricted to the parasite body posterior region, where early endosome, multivesicular body, lysosomes and transport vesicles coming from Golgi complex are packed (Field and Carrington, 2004); (b) in Leishmania the early endosome and Golgi complex are close to flagellar pocket at promastigote anterior region, both contributing to the formation of a long multivesicular tubule that spans longitudinally sustained by a cytoplasmic microtubule (Waller and McConville, 2002); (c) in the endocytic pathway of T. cruzi epimastigotes cargo enters the cytostome and flagellar pocket at the anterior region, continues through a branched network of vesicles and tubules that spreads until the posterior extremity before travelling in the opposite direction and finally fuse with reservosomes, positioned at the nuclear vicinity. Golgi-derived enzymes also comes from the anterior region and are added somewhere along the pathway (Porto-Carreiro et al., 2000). Adapted from Field and Carrington (2004) (a), Waller and McConville (2002) (b) and Porto-Carreiro et al. (2000) (c).

ARTICLE IN PRESS 88

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

and flagellar pocket entry sites are situated in the anterior region, whereas the accumulation/digestion sites are at the posterior region. Somewhere along this pathway, hydrolytic enzymes supplied by the Golgi complex, which are seen at the epimastigote anterior region, are added. Leishmania, T. brucei and T. cruzi have at least one developmental form with high endocytic activity. In T. brucei, the endocytic process is well established and similarities to that of high eukaryotes have been found, indicating the conserved function of the endocytic molecular machinery. This subject has been reviewed (Morgan et al., 2002a, b; Field and Carrington, 2004; Overath and Engstler, 2004). In T. brucei and Leishmania, endocytosis is related to nutrition and host immune response evasion and is a mechanism for parasite survival during infection. In T. brucei, endocytosis and the expression of certain proteins that are involved in the endocytic pathway is developmentally regulated. This regulation is thought to be established because the host stage bloodstream forms have a higher nutritional requirement than the insect vector procyclic forms (Overath and Engstler, 2004). Little information has been acquired on Leishmania and T. cruzi; their processes remain controversial. Nevertheless, the data on the T. cruzi epimastigote endocytic pathway suggest a very interesting model that differs from those of both the mammalian and T. brucei endocytic pathways. It is well established that trypanosomatids are able to ingest macromolecules from the external environment via a typical endocytic process; this process occurs in a well-defined region of the cell, known as the flagellar pocket. Studies in T. brucei have shown that this process follows the same basic mechanisms that have been well characterized in mammalian cells; it involves the presence of surface receptors, an assembly of clathrin-coated vesicles, the fusion of endocytic vesicles, early and late endosomes and lysosomes. Particularities have been found in other members, however. In Leishmania promastigotes, there is a remarkable, peculiar endocytic multivesicular tubule that spans the parasite body (Weise et al., 2000). In T. cruzi, an organelle specialized in nutrient storage, which concentrates lysosomal hydrolases as well, has been the subject of several studies that will be reviewed later. Fig. 11. Leishmania promastigote endocytic compartments – (a) transmission electron microscopy of the anterior region of a L. mexicana promastigote preserved by high pressure freezing and processed by freeze substitution showed electron-lucent vesicles (v) between Golgi complex (g) and the flagellar pocket (fp), and electron dense vesicles forming a budding zone (bz) between Golgi and endoplasmic reticulum (er); the inset showed coated pit-like invaginations of the flagellar pocket membrane; (b) the trans side of Golgi complex exhibited special structures, as multivesicular bodies (asterisk) and coated vesicles (arrowheads); (c) longitudinal section of a tubule (t) communicating with vesicles (v); the inset shows a transverse section of such a tubule; (d, e) L. major promastigotes expressing a GFP-chimera; (d) overlay of fluorescence and DIC; (e) fluorescence; (f–g) L. mexicana with FM4-64 red staining of early endosome (f) and multivesicular tubule (g), plasma membrane was stained green with concanavalin-A – FITC; (h) the tubular portion of the multivesicular tubule (m) accompanied by a rarely observed cytoplasmic microtubule (arrowheads). Bars: 500 nm (a–c); 100 nm (insets); 2 mm (d, e); 5 mm (f, g); 500 nm (h). After Weise et al. (2000) (a–c, h), Ghedin et al. (2001) (d–e) and Mullin et al. (2001) (f–g).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

89

ARTICLE IN PRESS 90

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

5.3. Ultrastructural organization of endocytic pathway in Leishmania Endocytosis in Leishmania has been demonstrated in promastigote and amastigote forms. In this parasite, endocytosis is related to nutrition and host immune system evasion. Promastigote forms uptake external macromolecules through the flagellar pocket (Fig. 11a), which is a plasma membrane invagination that is devoid of microtubules and comprises 3% of the total cell surface; it is a unique site of endocytic and exocytic activity. GPI-anchored surface proteins (ex. GP63), as well as transmembrane proteins (ex. acid phosphate), first accumulate inside the flagellar pocket before being ingested, destined for the endo-lysosomal system (Weise et al., 2000; Ghedin et al., 2001). Weise et al. (2000) demonstrated for the first time the presence of coated vesicles budding off from flagellar pocket and (Fig. 11a) the Golgi complex (Fig. 11b) and in promastigotes, suggesting the existence of a clathrindependent endocytic process. In silico analysis confirmed the presence of the clathrin heavy chain (LmCHC) in the Leishmania genome. LmCHC has been immunolocalized to vesicular structures spread along the parasite body but concentrated at the anterior half of promastigotes and amastigotes (Denny et al., 2005). The presence of clathrin molecules indicates receptor-mediated endocytosis from the flagellar pocket. Transferrin receptor has also been characterized in promastigotes of Leishmania as a 70 kD glycoprotein that is distinct from the mammalian cell transferrin receptor (Voyiatzaki and Soteriadou, 1992). A 46 kDa transmembrane haemoglobin receptor was localized to the flagellar pocket (Singh et al., 2003). Interestingly, Singh et al. (2003) studying Rab5-dependent homotypic fusion between early endosomes performed for the first time a cell-free endosome–endosome fusion assay for a member of the trypanosomatid family. In promastigotes distinct structures that belong to the endocytic pathway have been identified: (i) multivesicular bodies, (ii) tubular structures and (iii) multivesicular tubules. Multivesicular bodies are electron lucent, membrane-bounded and rounded structures with diameter 200 300 nm (Fig. 11b). They are filled with several vesicles (45 nm) and are adjacent to the Golgi complex (Weise et al., 2000). The function of these structures remains unknown. Tubular structures (TS) are clusters of regular tubules of 60 nm in diameter localized next to the Golgi complex and flagellar pocket (Fig. 11c). It is believed that they are connected to each other and may correspond to the early endosomes of promastigotes. Transmission electron micrographs show that the tubular structures are constantly bound to the electron-lucent vesicles. Fluid-phase endocytic tracer accumulation indicates that they are related to the promastigote endocytic pathway. In addition, TSs concentrate GPI-anchored and transmembrane proteins (Weise et al., 2000). A homologue of mammalian Rab5 has been identified in Leishmania (LmRab5). LmRab5 is localized to early endosomes (EE) at the anterior region of the parasite; these endosomes are also positive for the haemoglobin that is taken up by the parasite (Marotta et al., 2006). Leishmania early endosomes are dynamic organelles that are capable of performing LmRab5-dependent homotypic fusion (Singh et al., 2003).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

91

In Leishmania, there is a remarkable long endosomal/lysosomal multivesicular tubule (MVT) that spans from the anterior to the posterior region (Fig. 11d–h). Multivesicular tubules were first described in Leishmania mexicana overexpressing dolichol phosphate mannose synthesis (DPMS) (Ilgoutz et al., 1999) (Fig. 11d, e), an ER resident protein involved in GPI biosynthesis. For this reason, MVTs were incorrectly classified as a sub-compartment of the ER. Later, Weise et al. (2000) performed a fine morphological characterization of promastigotes by transmission electron microscopy using high-pressure frozen and freeze-substituted promastigotes. The tubular structures were characterized as large membrane-bounded tubules containing several small inner vesicles of heterogeneous size and morphology, with a median diameter of 100–200 nm (Fig. 11h). They participate in the endocytic pathway of promastigotes, as shown by fluid-phase endocytic tracer accumulation (Fig. 11f, g) (Weise et al., 2000; Ghedin et al., 2001); it is the final organelle in this pathway. Their acid character can be detected by Lysotracker accumulation, a weak base that accumulates in acidic organelles (Ghedin et al., 2001). MVT also accumulates overexpressed transmembrane cell surface and GPI-anchored proteins; the former are more accessible to MVT than GPI-anchored proteins. A concentration of ceramide was also observed, suggesting the incorporation of sphingolipid. Interestingly, Weise et al. (2000) demonstrated the presence of two new microtubules that are distinct from subpellicular microtubules (SPMTs) and the SPMT-associated quadruplet found close to the flagellar pocket. It has been inferred that these microtubules are required for the structural organization of the MVT, since promastigotes treated with a microtubule-depolymerization agent lose their MVT organization (Ghedin et al., 2001). Together, it has been proposed that MVTs are the site of accumulation of the protein and lipid that is ingested by the parasite and the final compartment of the endocytic pathway of Leishmania promastigotes. Multivesicular tubules have also been observed in Leishmania chagasi (Alberio et al., 2004). Amastigote forms, which use the intracellular environment to escape from the host cell immune response, ingest and degrade MHC class II and co-factors in infected macrophages, avoiding the antigen presentation and thus their elimination (De Souza Leao et al., 1995; Antoine et al., 1999). Collectively, intracellular amastigotes are capable of ingesting macromolecules through the flagellar pocket, which has access to the parasitophorous vacuole (De Souza Leao et al., 1995: Borges et al., 1998; Antoine et al., 1999). This process seems to be mediated by clathrin molecules (Denny et al., 2005). Later, the ingested materials are degraded in the megasomes, the final organelles in the amastigote endocytic route. Megasomes were first described in the complex Leishmania mexicana (Alexander and Vickerman, 1975). More recently, megasomes have also been described in L. chagasi (Alberio et al., 2004). Megasomes are large membrane-bound electron-dense organelles that vary in shape and size. In some cases, they can have a diameter that is larger than the nucleus (Fig. 12a–b). Because of their large size, they were named megasomes. The volume, size and number of megasomes can vary depending on their origin, as shown by 3D reconstruction (Fig. 12e, f) and morphometric analysis. While in axenically cultivated Leishmania amazonensis they occupy 5% of the parasite’s total volume,

ARTICLE IN PRESS 92

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

lesion-derived L. mexicana megasomes occupy 15% of the volume (Coombs et al., 1986). Due to their acidic character (Antoine et al., 1988) and the accumulation of lysosomotropic agents (Antoine et al., 1989), megasomes are thought to have lysosomal characteristics. Additionally, they are rich in lysosomal hydrolases, such as arylsulphatase, beta-glycosidase and cysteine protease; these enzymes are regulated according to developmental stage, being more active in amastigotes (Pupkis et al., 1986). Cysteine proteases, which are linked to parasite infectivity and survival, have been considered a molecular marker for the megasome (Fig. 12c, d) (Traub-Cseko et al., 1993). The biogenesis of megasomes has been described in the in vitro differentiation of promastigotes to amastigotes (Ueda-Nakamura et al., 2001, 2002). Promastigotes have several small electron-lucent vesicles that accumulate cysteine protease and are therefore considered megasome precursors. During the differentiation process these precursors gradually become amastigote megasomes (Ueda-Nakamura et al., 2001). 5.4. Ultrastructural organization of the endocytic pathway in T. brucei The largest dataset available on endocytosis in trypanosomes is in T. brucei. The work in this field has been largely facilitated by the completed genome sequence and the ability to introduce interference RNA (RNAi), which has revolutionized protein characterization and has been extensively used to study the role of macromolecules in the parasite life cycle. Due to life in the extracellular environment, parasite survival is associated with the uptake of extracellular nutrients and host cell immune system evasion. In T. brucei, endocytosis is regulated according to developmental stage. In bloodstream forms (BSF), the endocytic activity is 10-fold higher than in procyclic forms (PCF), the insect vector stage (Morgan et al., 2001). During differentiation it has been observed that the expression of some proteins involved in endocytosis is altered, such as clathrin (Morgan et al., 2001) and the small GTPase TbRab 11 (Jeffries et al., 2001). It has been postulated that this difference in expression correlates with the increased nutritional requirements of BSF compared to PCF. The endocytic pathway in T. brucei involves a complex system of highly polarized organelles. In T. brucei, all endocytic and secretory machinery is confined to the posterior half of the cell, perhaps because rapid recycling is required for parasite survival in the host cell bloodstream. External macromolecule uptake is Fig. 12. Leishmania megasomes – L. amazonensis amastigotes (a) from axenic culture showing a big megasome (arrow), (b) lesion-derived showing several megasomes (arrows); (c–d) megasomes concentrate cysteine protease, as shown by immunocytochemistry in an axenic amastigote (c) or extracted from lesion (d); N, nucleus, M, mitochondrion, K, kinetoplast, FP, flagellar pocket, L, lipid inclusion; (e–f) megasome distribution was demonstrated by 3D reconstruction of serial sections of axenic (e) or lesion-derived (f) L. amazonensis amastigotes. White, plasma membrane; yellow, nucleus; green, mitochondrion; magenta, megasomes. Bars: 500 nm. After Ueda-Nakamura et al. (2001) (a–d) and Ueda-Nakamura et al. (2007) (e–f). For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

93

restricted to the flagellar pocket (FP), a flask-shaped membrane invagination from which the flagellum emerges (Fig. 13a–c) (Webster, 1989; Landfear and Ignatushchenko, 2001). The total area of the flagellar pocket is completely internalized in about 2 min (Coppens et al., 1987). From the FP, clathrin-coated vesicles bud off

ARTICLE IN PRESS 94

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

(Fig. 13d, e), carrying ingested materials that are delivered to the early endosomes, a system of cisternae and tubules. In this parasite, the clathrin heavy chain (TbCLH), as well as AP-2 beta-adaptin subunit has been characterized. The expression of TbCLH in the bloodstream is about 10-fold higher than in the insect stage; this difference reflects the degree of nutritional requirements. TbCLH is localized to the tubular structures at the anterior region of the parasite body, as well as to the TGN (Morgan et al., 2001). Ultrastructural analysis has been used to classify clathrincoated vesicles into two classes: (i) CCV class I and (ii) CCV class II (Grunfelder et al., 2003). CCV class I are large vesicles with a diameter of 135 nm; they are rich in macromolecules ingested from cell surface (ex. VSG) and originate from the coated pits in the flagellar pocket (Fig. 13e). It is estimated that 6–7 class I vesicles bud from the flagellar pocket per second (Engstler et al., 2004). CCV class I vesicles then fuse with endosomal cisternae. CCV class I has not been found in procyclic forms (Hung et al., 2004). CCV class II are small vesicles with diameters ranging from 50 to 60 nm; they concentrate fluid-phase tracers but exclude material that is destined for recycling, such as VSG (Engstler et al., 2004). They bud off from the endosomal cisternae rims (Grunfelder et al., 2003) and from recycling endosomes and finally fuse with late endosomes (Fig. 14c–e). Clathrin-coated vesicles have also been observed to bud off from the trans-Golgi network. The ablation of clathrin is lethal, indicating that this molecule is essential for parasite survival (Allen et al., 2003). Furthermore, TbCLH RNAi leads to a severe swelling of the flagellar pocket, generating a phenotype called ‘‘BigEye’’. While the impairment of TbCLH expression results in the inability to uptake nutrients from the external medium, which remains concentrated at the FP, the exocytic process remains unaltered. Therefore, the ‘‘BigEye’’ phenotype may be a consequence of the loss in membrane homeostasis that is maintained by endocytosis and exocytosis. In PCF, TbCLH RNAi leads to a drastic decrease in macromolecule uptake, abolishing their degradation and inhibiting the transport of molecules from the ER to the flagellar pocket (Hung et al., 2004). These observations, together with the absence of the caveolin gene in the T. brucei genome, strongly suggest that clathrin-mediated

Fig. 13. T. brucei flagellar pocket – (a) T. brucei bloodstream forms incubated for 5 min at 37 1C with transferrin-gold conjugates previously bound to the parasite surface at 0 1C for 30 min presented the tracer inside the flagellar pocket (fp) and bound to flagellar membrane (F); (b) using diaminobenzidine to cytochemically detect peroxidase, the tracer was found bound to the surface (S) of bloodstream forms at 0 1C, including flagellar membrane; (c) 5 min after warming to 37 1C, peroxidase filled flagellar pocket and nearby vesicles (arrows); (d) the excellent preservation of parasites that were high-pressure frozen and embedded in Epon after freeze substitution, evidenced the VSG surface coat (SC) lining flagellar pocket membrane. The coat was internalized in clathrin-coated pits (arrowhead) that formed typical endocytic vesicles (arrows), with VSG coating the vesicle membrane at the luminal side; (e) anti-clathrin antibodies recognized the coat (CCP) of the vesicle budding at the flagellar pocket membrane of a T. brucei cryosection. The inset shows a typical coated vesicle in Epon ultrathin section. Bars: 200 nm (a); 500 nm (b, c); 250 nm (d, e). After Webster (1989) (a–c and inset) and Grunfelder et al. (2003) (d–e).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

95

endocytosis is the primary method of nutrient ingestion. In T. brucei, the uptake of transferrin through an unconventional transferrin receptor has been reported (Grab et al., 1993). The T. brucei Tf receptor is a complex formed by the products of two expression site-associated genes (ESAGs), ESGA 6 and ESGA 7, which are binding

ARTICLE IN PRESS 96

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Tf with high affinity. ESGA 6 is a GPI-anchored protein of 50–60 kDa that is heterogeneously glycosylated. ESGA 7 is a 42 kDa glycoprotein with an unmodified C-terminus (Steverding et al., 1994). It has been demonstrated that the expression of the Tf receptor varies according to the concentration of Tf that is available in the host blood (Mussmann et al., 2004). A dynamin-like protein (TbDLP) has also been characterized in T. brucei (Morgan et al., 2004; Chanez et al., 2006). RNAi has been used to assess the function of TbDLP in procyclic and bloodstream forms. In bloodstream forms, TbDLP is active in mitochondrial fission and not endocytosis (Morgan et al., 2004). In procyclic stages, however, it is reported to have a role in both mitochondrial fission and endocytosis (Chanez et al., 2006). Early studies on the T. brucei endocytic pathway have used Tf and BSA coupled to gold particles or horseradish peroxidase (HRP) (Webster, 1989; Webster and Fish, 1989). When these tracers were incubated with parasites at 0 1C, they were found to concentrate at the cell surface and flagellar membrane (Fig. 13a). Parasites incubated in the presence of HRP at 0 1C and the endocytic tracer was mainly concentrated in the cell surface (Fig. 13b). After warming the parasites to 37 1C for 5 min, the endocytic tracers entered through the flagellar pocket and were observed in vesicles, cisternal structures and lysosomes (Fig. 13 a, b) (Webster, 1989). Parasites incubated simultaneously with Tf and BSA coupled to gold particles of different sizes accumulate both tracers together inside the same endocytic organelles (Fig. 14a). In a similar experimental design, however, gold-labelled tracers were not able to access a large part of the HRP-containing structures (Webster and Fish, 1989). It was estimated that HRP filled organelles comprised 5% of the total cell volume (Fig. 14b), while gold-labelled tracers comprised 3%. In addition, gold-labelled endocytic tracers were excluded from VSG-containing organelles. These results are consistent with the existence of distinct pathways for different molecules. While this Fig. 14. T. brucei and T. congolense endosomes – (a) after 5 min of incubation at 37 1C, peroxidase previously bound to cell surface was found inside the endosomal network of T. congolense; (b) although T. brucei bloodstream forms have been simultaneously incubated with all tracers, gold-labelled Tf (large particles) and BSA (small particles) did not occupy all endosomal cisterna filled with peroxidase after 30 min at 37 1C; (c) immunocytochemistry on cryosections of T. brucei showed that clathrin-coated vesicles budding from endosomal cisterna did not contain VSG (immunogold); (d) double labelling of a long endosomal cisternae is positive for VSG (larger gold) and negative for clathrin (smaller gold) in its core, whereas the reverse occurs in budding vesicles (arrows); (e) T. brucei that had been incubated with BSA-gold (small) for one hour before fixation were cryosectioned and labelled with antiVSG (larger gold). Anti-VSG (arrows) bound to parasite surface (s), flagellar pocket (fp) membrane and vesicles (v), filled or not with BSA-gold. (f–g) BSA-gold containing vesicles communicated directly with thin tubules positive for VSG but that did not contain BSA both in T. brucei (f) and in T. congolense (g); (h) after one hour at 37 1C, Tf-gold was found inside T. brucei lysosomes; (i) together with endosomal compartments, exosomal carriers (EXC), positive for TbRab11 (immunogold) close to the flagellar pocket (FP) had a higher VSG coat density. Bars: 500 nm (a–h); 250 nm (i); 125 nm (inset). After Webster and Fish (1989) (a, e–g), Webster (1989) (b, h) and Grunfelder et al. (2003) (c–d, i).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

97

work highlights the morphologic similarities between the endocytic organelles of higher eukaryotes and T. brucei, the shapes of the organelles are different between the two classes, with the majority of the organelles in the trypanosome being tubules and cisternae (Fig. 14a–d). The GPI-anchored variant surface glycoprotein (VSG) is the major surface protein of the bloodstream stage. VSG has been extensively used to investigate endocytic and recycling pathways (Overath and Engstler, 2004). VSG is endocytosed by the flagellar pocket and accumulates to high levels in the CCV class I, the class of clathrin-coated vesicles that are involved in the ingestion of nutrients from the FP. CCVs class I bud off from recycling endosomes and fuse with late endosomes. After,

ARTICLE IN PRESS 98

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

these vesicles fuse with circular cisternae, which are the early endosomes (Fig. 14c). T. brucei EEs are located in a perinuclear region juxtaposed to late endosomes. According to Engstler et al. (2007) an individual parasite does not possess more than four EE. Like in mammalian cells, in T. brucei, EEs are defined by the localization of the small GTPase Rab5. Two homologues of Rab5 have been characterized in T. brucei (Pal et al., 2002), TbRab5A and TbRab5B. In PCF, Rab5 homologues colocalize in early endosomes. In contrast, TbRab5A and TbRab5B have distinct localizations in BSF, suggesting that there is a division in the pathway and that the two have distinct functions. TbRAb5A-positive endosomes concentrate GPIanchored proteins, such as VSG and Tf receptor, indicating that they may have a role in the recycling process. It is thought that proteins that are destined for recycling (ex. Tf receptor and VSG) are able to access TbRAb5A-positive endosomes and are positively sorted for recycling to the flagellar pocket via a TcRab11-exocytic carrier (Fig. 14i) (Grunfelder et al., 2003) that will be described later; this pathway is a rapid recycling route, demonstrating that there is a specialized transport of GPI-anchored proteins in bloodstream stages that is required for survival in the mammalian host. On the other hand, TbRab5B compartments have transmembrane proteins (ex. ISG100) and exclude VSG and transferrin receptor. While TbRab5A and -5B have distinct sets of cargo, both function in fluid-phase endocytosis. The ablation of these proteins causes an impairment in endocytosis and parasite death, demonstrating that they are vital to parasite. BSF that are incubated at 12 1C retain endocytic tracers in the early endosomes (Brickman et al., 1995). The core of EEs is rich in VSG, but their rims are without such molecules (Fig. 14d). CCV class II buds off from the EE filled with fluid-phase tracers. VSGs are not incorporated in such vesicles, however. From the EE, the endocytosed cargo is exclusively delivered to TbRab7-positive late endosomes (LE) by CCV class II. Pleomorphic LEs are localized close to lysosomes and early endosomes but remain distinct from these vesicles. It is believed that such vesicles are responsible for the unusual transport to LE and lysosomes in T. brucei. From late endosomes, there is a distinct and slower route toward recycling endosomes. According to Engstler et al. (2007), a 3D model of recycling endosomes has arisen from analysis of transmission electron micrographs that show these organelles as unique large, flat and fenestrate compartments that are confined to the parasite’s posterior end. Recycling endosomes seem to give rise to exocytic carry vesicles (EXC), which are small cisternae spread around the posterior region of the parasite and rich in TbRab 11 and VSG destined for recycling (Fig. 14i). Finally, EXCs fuse with the flagellar pocket, and VSG returns to the parasite’s surface. T. brucei lysosomes are mainly observed as unique round organelles located at the posterior region of the parasite. They can also be found as a cluster of organelles (Fig. 14h) (Webster, 1989; Alexander et al., 2002). The transport of endocytosed cargo to lysosomes remains unclear. Lysosomes in T. brucei are often determined by the presence of p67, a type I glycoprotein that is structurally similar to mammalian LAMPs (lysosomal markers). While p67 RNAi impairs the normal growth of BSF, it seems to have no effect on PCF. In addition, p67 ablation leads to abnormal lysosome morphology (Peck et al., 2008).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

99

6. Endocytic pathway in Trypanosoma cruzi Studies of T. cruzi have shown that this protozoan exhibits certain peculiarities in its endocytic pathway that distinguish it from other cells. First, endocytosis only occurs in the epimastigote form, which is found in the digestive tract of the invertebrate host and in axenic cultures maintained in vitro (Fig. 15); endocytosis is low or absent in metacyclic or bloodstream trypomastigotes and in intracellular amastigotes forms. Second, epimastigotes have two sites of macromolecule ingestion: the flagellar pocket, as described for T. brucei and Leishmania, and a highly specialized structure known as the cytostome. Third, the cargo of the endocytic vesicles is delivered to unusual structures, designated the reservosomes, which are localized at the posterior region of the protozoan. These remarkable differences

Fig. 15. Schematic representation of T. cruzi life cycle – The biological cycle may start when the invertebrate host (Hemiptera, Reduvidae) feeds on the infected vertebrate host by sucking blood. During feeding, the trypomastigote forms in the blood of the infected vertebrate host are ingested by the insect. It is assumed that in the stomach of the insect most of the bloodstream trypomastigotes differentiate into epimastigotes and some rounded forms. In the intestine the epimastigotes divide repeatedly by a process of binary fission and can attach to the intestinal cells by hemidesmosomes. In the rectum a certain proportion of the epimastigotes transform into metacyclic trypomastigotes, which are eliminated with the feces and are able to infect the vertebrate host (Garcia and Azambuja, 1991; Kollien and Schaub, 2000). The trypomastigotes are able to penetrate into vertebrate cells where they differentiate into amastigote forms. Amastigotes proliferate and give rise to trypomastigotes, which are liberated to the intercellular space, may reach the bloodstream, and can penetrate into other cells initiating a new cycle. Black arrows represent infection events, curved arrows represent proliferation within one host, and white arrows represent differentiation.

ARTICLE IN PRESS 100

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

make T. cruzi an interesting model for the study of the evolution of the endocytic pathway.

6.1. Endocytic activity in T. cruzi: site of entry As mentioned above, T. cruzi epimastigotes use two specialized regions of its cell surface to ingest macromolecules from the environment, the flagellar pocket and the cytostome. The flagellar pocket is formed by a lateral depression at the anterior region of the cell from where the flagellum emerges. The flagellar pocket membrane is a large invagination of the plasma membrane that directly contacts the membrane of the flagellum. The physical contact between the cell body membrane and the membrane of the flagellum around its emergence point makes the flagellar pocket a special extracellular compartment. The flagellar pocket is a highly specialized region of the surface of trypanosomatids; its membrane differs both from the membrane that lines the cell body and the flagellar membrane in terms of the distribution of intramembranous particles and localization of proteins and enzymes. Furthermore, it is the only area that lacks subpellicular microtubules, allowing for intense endocytic and exocytic activity (De Souza, 2002). Epimastigote and amastigote forms the Schizotrypanum sub-genus, such as T. cruzi, Trypanosoma vespertilionis and Trypanosoma dionisii, have an additional surface specialization, the cytostome complex. This structure is an invagination of the plasma membrane coupled to a few special microtubules that penetrate so deep into the cell that they can reach the nuclear region. The opening of this complex, which is known as the cytostome, has a diameter up to 0.3 mm; this diameter is significantly smaller in the deeper portion, the cytopharynx, so that the structure resembles a funnel. There is a specialized region of the membrane lining the parasite that starts in the opening of the cytostome and projects towards the flagellar pocket (Fig. 16a–b). Fundamental freeze-fracture studies showed that this area is delimited Fig. 16. T. cruzi endocytosis: sites of entry – The membrane between the cytostome and the flagellar pocket is a special membrane domain. In freeze-fracture replicas (a), the membrane domain that projects from the cytostome opening to the flagellar pocket is almost devoid of intramembranous particles and limited by a linear array of particles on both sides (arrowheads). If the replica is flipped (b) to expose the real cell surface, the same domain appears rugous. The same rugous aspect can be observed by high-resolution field emission scanning electron microscopy (c). This peculiar aspect correlates with a great concentration of surface glyconjugates, as have already been demonstrated by ruthenium red staining (d), that may have a receptor function, as indicated by the binding of gold-labelled transferrin (e) observed by transmission electron microscopy. The cytostome opening is indicated by an arrow and the special membrane domain by an asterisk in all micrographs. F, flagella; FP, flagellar pocket; K, kinetoplast. Bars: 200 nm. After Martinez-Palomo et al. (1976) (a), Pimenta et al. (1989) (b), Vatarunakamura et al. (2005) (c), De Souza et al. (1978) (d) and Porto-Carreiro et al. (2000) (e).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

101

by a palisade-like array of closely associated particles that correspond to transmembrane proteins; these proteins remain unidentified, even 30 years after the first study (Martinez-Palomo et al., 1976). The delimited area is almost devoid of transmembrane proteins, appearing smooth in freeze-fracture replicas (Fig. 16a). Upon flipping the replicas, however (Fig. 16b) (Pimenta et al., 1989), the actual surface was exposed, and the membrane lining the cytostome appeared very rugous. Cytochemical studies using ruthenium red (Fig. 16d) or concanavalin A – peroxidase in transmission electron microscopy or gold-labelled concanavalin A in field emission scanning electron microscopy (Fig. 16c) showed a great concentration of

ARTICLE IN PRESS 102

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

surface carbohydrates in this area. Together, the freeze-fracture and cytochemical data indicate that the membrane lining the cytostome is rich in glycoconjugates that are not inserted in the membrane. Indeed, cytochemical techniques have shown that the surface coat of T. cruzi is thicker in the region lining the cytostome (Fig. 16d). The morphological and functional characteristics of the membrane lining the cytostome entry therefore suggest the existence of a specialized membrane domain (Vatarunakamura et al., 2005). Quantitative analysis of ingestion of gold-labelled macromolecules has shown that in epimastigote forms of T. cruzi about 85% of gold particles associate with the cytostome (Fig. 16e) (Porto-Carreiro et al., 2000); this result is in contrast to that obtained with T. brucei, where the particles are seen only within the flagellar pocket. Epimastigotes that are incubated with acridine orange, a weak base that concentrates in acidic compartments, accumulate the dye in the cytopharynx (Fig. 17a), suggesting that this compartment has an acidic character (Porto-Carreiro et al., 2000). Additionally, the presence of a P-type H+-ATPase has been demonstrated, further supporting the acidic nature of the cytostome (Vieira et al., 2005).

6.2. Initial steps of internalization Following binding to the cytostome and flagellar pocket, macromolecules are rapidly internalized and appear in small endocytic vesicles, which bud from the deeper regions of this structure. Early transmission electron microscopy work on ultrathin sections showed that the small vesicles do not have a cytoplasmic coat (Soares and De Souza, 1991), which would be indicative of the presence of clathrin. In T. brucei (Morgan et al., 2001; Allen et al., 2003) and Leishmania major (Denny et al., 2005), clathrin and adaptin complexes have been well characterized. In T. cruzi, evidence of receptor-mediated endocytosis has been previously reported in epimastigote forms (Soares and De Souza, 1991; Porto-Carreiro et al., 2000). The molecular machinery governing this process needs to be better defined, however. Fig. 17. T. cruzi endosomal pH – The entire endocytic pathway is an acidic network. Acridine orange accumulates inside reservosomes as result of low pH of these organelles, as shown by overlay images (a, b) of acridine orange fluorescence (red channel) and phase contrast (green channel). If the contrast of fluorescence images is enhanced, a tiny line at the cytostome position (arrow in a) and a tubule at the cell posterior extremity (arrow in b) can be observed. Fluorescein-labelled transferrin is found inside tubules and vesicles spanning along parasite body after 5–10 min of incubation (d–e, in different focal planes) and concentrates in reservosomes after 20 min (f). Reservosomes were compared to late endosomes because of the pH and the kinetics of protein uptake, but TcRab7 (c), similar to mammalian late endosome marker, was immunolocalized at Golgi complex (green channel), next to kinetoplast (propidium iodide at red channel). Phase contrast images were captured in the blue channel in (c–f). Bars: 5 mm. After Porto-Carreiro et al. (2000) (a, b) and Araripe et al. (2004) (c). For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

103

Coated vesicles, suggestive of a clathrin coat, were first reported to be budding from Golgi complex (Sant’Anna et al., 2004). Correˆa and co-workers (2008) demonstrated that transferrin uptake is dependent on membrane cholesterol and on cytoskeletal elements that are associated with the cytostome (Correˆa et al., 2008). In silico analysis revealed the presence of clathrin, adaptin and clathrin self-assembly genes (Correˆa et al., 2007). Moreover, clathrin expression in T. cruzi was demonstrated with Western blots using polyclonal antibodies raised against bovine clathrin heavy chain. TcClathrin has been localized to the Golgi complex and flagellar pocket region. Curiously, agents that disturb receptor-mediated endocytosis do not impair transferrin uptake in epimastigotes (Correˆa et al., 2008). Unlike T. brucei (Morgan et al., 2002b), T. cruzi has the TcAP2 complex in its genome, indicating that there is

ARTICLE IN PRESS 104

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Fig. 18. T. cruzi endocytic pathway. Gold-labelled BSA is found inside a tubule (a) at the posterior extremity of an epimastigote (delineated area at the inset) after 5–10 min of uptake. It is possible to visualize the electron dense gold particles filling the tubule in whole unfixed cells using energy loss spectroscopy under the transmission electron microscope. After 20 min, cargo concentrates inside reservosomes at the posterior cell region as peroxidase (arrow) in (b). Reservosomes appear as multivesicular bodies (MV) in freeze-fracture replicas (c), but may present crystalloid electron-lucent inclusions (asterisks in d) in routine ultrathin sections. BSA coupled to 40 nm gold particles (arrow) are concentrated in the same reservosomes that were already full of BSA coupled to 5 nm gold particles (arrowhead) uptaken 4 h before (e). Bars7 0U2 mm. (a) After Porto-Carreiro et al. (2000); (c) after De Souza et al. (1978).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

105

likely to be endocytic activity. Moreover, the presence of a dynamin gene may help unravel the mechanism of endocytic vesicle fission (Correˆa et al., 2007). In the African trypanosome, dynamin has been implicated in mitochondrial fission, endocytosis and cytokinesis (Chanez et al., 2006). The fusion of endocytic vesicles with the tubule-vesicular network can be observed from the perinuclear region to posterior tip of the protozoan. Using acridine orange to probe the pH of intracellular compartments, this tubular structure has been shown to be acidic (Fig. 17b). The cargo pathway kinetics and pH suggest that this compartment may correspond to the epimastigote early endosome (Porto-Carreiro et al., 2000). The spatial distribution and morphology of the EE have been detailed with a 3D reconstruction of a sequence of ultrathin sections (Porto-Carreiro et al., 2000). Weak bases are known to block vesicle fusion in mammalian cells. The incubation of parasites with ammonium chloride or chloroquine slows endocytosis and facilitates the observation of an intricate and branched network of tubular structures that contain gold-labelled tracers and span from the bottom of the cytostome to the parasite posterior extremity (Figs. 17d–f and 18a) (Porto-Carreiro et al., 2000). In mammalian cells, it was demonstrated that incubation at 12 1C retains cargo inside the early endosome and arrests the progression of the endocytic pathway. Epimastigotes that are incubated at 12 1C retain gold-labelled transferrin at the cytostome (De Figueiredo and Soares, 2000), suggesting that epimastigotes might not have a corresponding early endosome compartment. Since cargo containing vesicles that bud from the bottom of the cytostome do not fuse directly with reservosomes, however, the temperature blockage of vesicle traffic may not be the same for parasites that live at temperatures that are much lower than the physiological temperature of mammals. A gene homologous to mammalian Rab5, an early endosome molecular marker, has already been identified in T. brucei (Field et al., 1998) and Leishmania (Singh et al., 2003). The TcRab5 gene was cloned sequenced, and its expression has been measured in all T. cruzi developmental stages (Araripe et al., 2005). Its localization, however, has not been examined. 6.3. Intersection with the ER-Golgi The major protease of T. cruzi, cruzipain, belongs to the cysteine protease family; it is very active in epimastigote forms and concentrates in reservosomes. The enzyme is a glycoprotein synthesized in the ER-Golgi system in a proenzyme form and addressed to the endocytic pathway. The pro-peptide sequence is necessary and sufficient to drive cruzipain to reservosomes (Souto-Padron et al., 1990). The epimastigote Golgi complex is localized close to the flagellar pocket, on the opposite side of the T. cruzi final endocytic organelle, called the reservosome. It is not clear in which compartment cruzipain joins the endocytic pathway. The enzyme may reach reservosomes either aboard special vesicles that travel along the parasite body in an independent way or together with the cargo. In the latter case, cruzipain transport vesicles would either fuse with the flagellar pocket membrane, being subsequently

ARTICLE IN PRESS 106

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

internalized together with the cargo, or is incorporated into the endocytic pathway by fusion with the tubular early endosomal network.

6.4. Reservosomes Macromolecules from the extracellular medium or from the ER-Golgi system are concentrated in structures known as reservosomes. These organelles are particularly interesting because they are found exclusively in the Schizotrypanum sub-genus, such as T. vespertilionis, T. dionisii and T. cruzi. Reservosomes are unique organelles that have a pivotal role in the life cycle of T. cruzi. They were named for their unusual capacity to accumulate all of the macromolecules that are ingested by the parasite through an endocytic process. In particular, T. cruzi epimastigotes specify a class of endocytic organelles, the reservosomes, whose main function is to store macromolecules, although they also concentrate lysosomal hydrolases. Thus, reservosomes are also considered the main site of protein degradation and regulation. Two lysosomal hydrolases have been well characterized in T. cruzi: cruzipain and serine carboxypeptidase. The former is synthesized as a zymogen (Eakin et al., 1992), which matures in the Golgi complex (Engel et al., 1998, 2000), accumulates to high levels (Souto-Padron et al., 1990; Soares et al., 1992) and becomes active in reservosomes (Cunha-e-Silva et al., 2002). Cruzipain is considered a fundamental virulence factor for T. cruzi during parasite host cell invasion and for intracellular survival. Additionally, reservosomes concentrate the cruzipain natural inhibitor chagasin, suggesting that there is an endogenous modulation of cruzipain activity (Santos et al, 2005). Serine carboxypeptidase catalyses the hydrolysis of the carboxyterminal bond in peptides and proteins; biochemically, it appears to localize to reservosomes (Parussini et al., 2003). We further demonstrated the reservosomal localization of serine carboxypeptidase by immunofluorescence and immunogold labelling (Fig. 22) (Sant’Anna et al., 2008b). Recently, T. cruzi autophagic processes were investigated in parasites under nutritional stress (Alvarez et al., 2008a, b). Autophagy seems to be essential for parasite differentiation and survival. It has been proposed that the reservosome functions as an important regulator of protein concentrations and Fig. 19. T. cruzi reservosomes ultrastructure. Ultrathin section of the posterior half of an epimastigote where it is possible to observe reservosome distribution and morphology (a). Cytochemical detection of basic proteins in a parasite treated with ethanolic phosphotungstic acid (E-PTA); positive reaction is observed in reservosome matrix (b). Reservosome lipid inclusions were identified by using Os-imidazole buffer incubation (b, inset). Random distribution of intramembrane particles was evidenced by freeze-fracture at the E-face of reservosomes (c); particles with distinct diameter were observed in a high magnification of a reservosome (d, inset). The purity of reservosome (e) and total reservosome membrane (f) fractions from Dm28c epimastigotes was assessed by ultrathin section. K, kinetoplast; N, nucleus; R, reservosome. Bars: 500 nm (a, inset in b); 600 nm (b, d); 90 nm (e); 45 nm (inset in e); 1 mm (e, f). After Sant’Anna et al. (2008a) (a, d, e), Soares et al. (1989) (b, c) and Sant’Anna et al. (2009) (f, g).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

107

ARTICLE IN PRESS 108

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

organelles during the metacyclogenesis process. Also, reservosome morphology is abnormal in epimastigotes after a long period of starvation. Each epimastigote form has several reservosomes, mainly in the posterior region of the cell (Figs. 18b and 19a). These organelles are surrounded by a unit membrane, with a mean diameter of 0.6 mm; it is usually globular but may appear asymmetrical. Ultrastructural cytochemical studies have shown that the electron-dense portion of the reservosome matrix is mainly composed of proteins while the electron-lucent inclusions are likely to be lipid (Fig. 19b inset). Proteins, especially basic proteins, can be localized using the ethanolic phosphotungstic acid technique in cells (Fig. 19b) that have been previously fixed in glutaraldehyde but not post-fixed in osmium tetroxide (Bloom and Aghajanian, 1968; Soares and De Souza, 1988). Lipids were initially localized using the imidazole-buffered osmium tetroxide technique (Angermu¨ller and Fahimi, 1982; Soares and De Souza, 1988). Initially, the presence of inner membranes was controversial. The existence of inner vesicles by ultrastructural cytochemistry in HRP-loaded reservosomes (Fig. 18b) was reported (Boiso et al., 1977; De Souza et al., 1978) and also by freeze-fracture (Fig. 18c) (De Souza et al., 1978). Guided by the morphology of late endosomes in mammalian cells, the T. cruzi storage organelles were first designated as multivesicular bodies (MVBs). Later, Soares and De Souza (1988) failed to find a reservosome inner membrane using transmission electron microscopy on ultrathin sections. It was then recognized that the term ‘‘multivesicular bodies’’ was incorrect. In 2005, Vieira et al. showed that inner membranes existed in isolated reservosomes. Recently, using different TEM approaches, our group performed a detailed description of reservosome morphology (Fig. 20) (Sant’Anna et al., 2008a). We demonstrated the presence of internal vesicles both in isolated (Fig. 20a) reservosomes and in situ; we also observed long membrane profiles transversing the reservosome lumen (Fig. 20b). The inner vesicles (Fig. 20e–f), always present in low levels, do not have access to endocytosed macromolecules. Their nature remains to be determined. Another noticeable structure that we observed was a rod-shaped electron-lucent structure bound by a membrane monolayer (Figs. 18d and 20c–d). The large amount of lipids in the organelle may cause heterogeneous arrangements of the membrane and lead to differentiated lipid regions, which could be functional. In addition, we have determined the distribution of the reservosome transmembrane proteins by freeze fracture (Fig. 19c, d). Using the DAMP technique to evaluate pH at the level of electron microscopy, reservosomes were found to be comparable to mammalian late endosomes (prelysosomes), with a pH of 6.0, the presence of acid hydrolases and the lack of a lysosomal molecular marker (Soares et al., 1992). Typically, the pH of endocytic compartments is kept low by the action of V-type (vacuolar) proton ATPases. Surprisingly, the acidification of reservosomes, as well as of the entire epimastigote endocytic pathway, is a result of the activity of P-type proton ATPases (Vieira et al., 2005); this phenomenon is unique among all eukaryotic cells. A P-type proton pump has been characterized in plants, yeast and, recently, in trypanosomatids (Vieira et al., 2005). In the case of T. cruzi, two tandemly arranged genes, TcHA1 and TcHA2, encode two pumps: TcHA1 is present in the plasma membrane and

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

109

Fig. 20. T. cruzi reservosomes ultrastructure. Ultrathin section of isolated reservosome showing small inner membrane profiles in organelle core (a). Isolated reservosome uranyl acetate-stained where is possible to observe large internal membranes transversing the reservosome lumen (b). Internal electron-lucent rod-shaped inclusions (asterisks) surrounded by a membrane monolayer (arrowheads) are remarkable structures in the reservosome (c). After cytochemistry using Os-imidazole buffer, electron-lucent rods showed no reaction (d, arrows). However, positive reaction was evidenced in a lipid inclusion (asterisk). Reservosomal inner vesicles were shown by ultrathin section (e, arrows) and transmembrane proteins distribution was determined by freeze fracture (f, arrows). Bars: 150 nm (a); 90 nm (b); 200 nm (c); 100 nm (inset in c); 300 nm (d); 20 nm (e, f). After Sant’Anna et al. (2008a).

ARTICLE IN PRESS 110

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

endocytic pathway and TcHA2 acidifies reservosomes exclusively. Due to their absence in mammals and presence in trypanosomatids, the P-type H+-ATPases may be a potential chemotherapy target against trypanosomiasis. Reservosomes can be isolated in a purified subcellular fraction (Fig. 19e) (Cunhae-Silva et al., 2002). The first biochemical analysis showed that reservosomes accumulate lipids; the protein/lipid ratio of the purified fraction is 1:1, while the ratio in whole parasite extracts is 2:1. Cholesteryl ester and ergosterol are massively concentrated in this organelle. A transporter of the ABC family, ABCA1, has been characterized and is predicted to localize to reservosomes (Torres et al., 2004). This transporter belongs to ABC transporter family, which is involved in the ATPdependent transport of substrates through the plasma membrane. They are also involved in cholesterol and phospholipid flux in the endocytic and secretory pathways. Importantly, it was not possible to identify lysobisphosphatidic acid (LBPA), a mammalian inner membrane late endosome molecular marker, by TLC. Attempts have been made to identify a protein profile for reservosomes by SDSPAGE; however, these were not successful (Cunha-e-Silva et al., 2002). Few reservosomal resident proteins have been described so far. To obtain a protein profile, understand its function and identify molecular marker candidates, we have recently performed a subcellular proteomic analysis of a purified reservosome fraction (Fig. 19e) and total reservosome membrane (Fig. 19f) using liquid chromatography coupled to mass spectrometry (LC–MS/MS) (Sant’Anna et al., 2009). Using this approach, we identified around 700 proteins with predicted or unknown functions. The presence of previously characterized proteins was confirmed, such as of cruzipain, serine carboxypeptidase, P-type H+-ATPase isoforms and ABC transporter. A P-glycoprotein was also found. A similar transporter was recently suggested to function in heme uptake through the epimastigote plasma membrane (Lara et al., 2007), as its fast internalization is impaired by the typical inhibitors. In untreated control parasites, heme concentrates in reservosomes a few minutes after entry. A second and still undetermined transporter would be responsible for heme storage in reservosomes. We could also identify PRL-1, a member of the tyrosine phosphatase family; PRL-1 was localized Fig. 21. Ultrastructure of lysosome related (TcLROs) organelles in T. cruzi trypomastigotes and amastigotes. Round organelles presenting epimastigote reservosomes typical morphology, electron dense core and electron lucent inclusions, are evidenced by ultrathin sections in trypomastigotes (a, d). TcLROs placed at the trypomastigote posterior region containing inner vesicles could be visualized (b). Figure (c) is a higher magnification of organelles in (b). Polymorphic organelles presenting slightly electron dense material (asterisk) surrounded by a membrane monolayer (arrowhead) are mainly observed concentrated at the amastigote posterior region (e). Figure (f) is a higher magnification of amastigote organelles shown in (e). Tridimensional reconstruction from serial ultrathin sections showed the special distribution of LROs in trypomastigotes, which are rounded structures confined between the nucleus and kinetoplast (g, h). In the case of amastigotes, TcLROs are found as polymorphic organelles with different shape and size that span from perinuclear region to posterior tip of the parasite. Bars: 500 nm (a, b); 230 nm (c); 400 nm (d); 600 nm (e); 200 nm (f). After Sant’Anna et al. (2008b).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

111

in intracellular compartments in trypomastigotes and amastigotes, as well as in the epimastigote endocytic pathway, including reservosomes (Cuevas et al., 2005). Additionally, new lysosomal hydrolases were described, demonstrating that a variety of substrates can be digested in reservosomes. Also, proteins were involved in signal transduction and lipid metabolism were found in the reservosome proteome. Endosomal integral membrane proteins and proteins involved in membrane trafficking, especially small GTPases from the Rab family (Rab 1, Rab2b, Rab 7 and Rab18), were also detected.

ARTICLE IN PRESS 112

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Molecular markers have been extensively used to identify endocytic compartments and understand trafficking systems in eukaryotic cells, including trypanosomes. Rab proteins, which have a central role in endocytic vesicle fusion (Jordens et al., 2005), are the best studied endomembrane molecular markers in trypanosomes. While there are about 60 Rab subfamily members that have been characterized in mammalian cells, the T. cruzi genome has only six Rab homologues (TcRabs) that can be related to endocytosis; only three of them had been studied experimentally prior to our proteomic study (Mauricio de Mendonca et al., 2000; Araripe et al., 2004, 2005). TcRab7, a small GTPase homologue of the mammalian late endosome marker Rab 7, has been found in T. cruzi; this protein, however, was found to be localized by immunocytochemistry to the Golgi complex, rather than reservosomes (Fig. 17c) (Araripe et al., 2004). The discovery of TcRAb7 in the reservosome proteome may indicate that there is trafficking between these two organelles. Assuming that there is a direct correlation between sequence similarity and function, the localization of TcRab11 (Mauricio de Mendonca et al., 2000), which is similar to the mammalian recycling endosome marker Rab11, to reservosomes may indicate that this organelle has recycling ability (Cunha-e-Silva et al., 2006). By searching the T. cruzi genome, it is possible to identify other proteins and protein complexes that participate in membrane fusion in eukaryotic cells: N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAP) and SNAP receptors (SNAREs), molecular components involved in the regulation of vesicle budding from donor compartments and specific fusion with acceptor organelles (Leabu, 2006). We did not identify these proteins in the reservosome proteome, however. Two promising candidates for a reservosome molecular marker were identified: p67 and TcHA3. The former is a transmembrane lysosomal glycoprotein with a structure similar to that of lysosome associated membrane proteins (LAMPs) in mammalian cells, although they differ in amino acid sequence. p67 has been well characterized in T. brucei and is considered a lysosome molecular marker (Alexander et al., 2002; Peck et al. 2008). Among the P-type H+-ATPase isotypes that have been identified in trypanosomatids (HA1, HA2 and HA3), only HA1 and HA2 were found and characterized in T. cruzi strain Y (Luo et al., 2002; Vieira et al., 2005). While tagged TcHA1 has been detected in the epimastigote plasma membrane and endocytic compartments, TcHA2 seems to be restricted to reservosomes (Vieira et al., 2005). The presence of HA3, an isoform very similar to HA2, was demonstrated in our proteomic analysis of isolated reservosomes from the Dm28c clone; due to its high coverage, TcHA3 might be a suitable reservosomal molecular marker. The molecular and cellular characterization of p67 and TcHA3 are under investigation. Reservosomes have been described as an exclusive structure of epimastigote forms. While lipid and protein uptake have never been demonstrated in either trypomastigotes or amastigotes, intracellular organelles that share many reservosomal features were recently described in the T. cruzi mammalian stages (Fig. 21a–f) (Sant’Anna et al., 2008b). Like reservosomes, they are concentrated in the parasite’s posterior region; they accumulate cruzipain, its natural inhibitor chagasin and serine carboxypetidase (Fig. 21a–e). They are acidic and have the P-type H+-ATPase.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

113

Interestingly, rod-shaped electron-lucent lipid bodies, similar to those that were recently characterized in the reservosome lumen (Sant’Anna et al., 2008a), were also found in trypomastigote and amastigote hydrolase-rich compartments (Fig. 21e–f) (Sant’Anna et al., 2008b). Collectively, these results indicate that these compartments are closely related. Nonetheless, they differ from reservosomes in the ability to store external macromolecules. Because of the low internal pH and accumulation of lysosomal hydrolases, we have proposed that epimastigote reservosomes and trypomastigotes and amastigotes organelles be considered lysosomal-related organelles (LROs), a group of organelles that share fundamental properties with mammalian lysosomes (Sant’Anna et al., 2008b). Further experiments are underway to investigate whether trypomastigote compartments are derived from epimastigote reservosomes in the metacyclogenesis process. Reservosomes have been considered the ultimate fate of macromolecules captured from the extracellular medium and also the site of the accumulation of parasite major proteases; this organelle probably has lysosomal functions, as classical lysosomes have never been identified in T. cruzi (reviewed in Cunha-e-Silva et al., 2006). Nevertheless, arylsulphatase activity, which is characteristic of lysosomes, has been detected inside small vesicles distributed all over the cell body of epimastigotes and trypomastigotes (Adade et al., 2007). The digestive function of these compartments has not been addressed. In conclusion, reservosomes have been implicated mainly in macromolecule storage; however, a complex role for these organelles in the T. cruzi life cycle has been demonstrated by its pivotal function in digestive, autophagic and recycling processes. Furthermore, an improved understanding of the key genes and proteins in the T. cruzi genome and reservosome proteome, respectively, as well as additional high-resolution electron microscopy techniques, may allow us to gain insight into the endocytic pathway that exists in this peculiar cell biology model. The recent identification in the T. cruzi infective stages of organelles that share characteristics with reservosomes, organelles that are so dissimilar from mammalian cell compartments, may provide new potential chemotherapy targets for the treatment of Chagas’s disease.

7. Concluding remarks Parasitic protozoa represent a small eukaryotic group. Nevertheless, the diversity of endocytic characteristics is very high. Anaerobic parasitic protozoa, as Trichomonas, Entamoeba and Giardia, considered to be early divergent eukaryotes, are very efficient phagocytes. Among this restricted group, endocytic process evolved towards polarization: while trichomonads and amoeba are able to form endocytic vesicles at any place of their cell surface, in Giardia vesicle budding is restricted to dorsal surface. In Apicomplexa, ultrastructural data concerning endocytosis does not support any conclusion yet. In trypanosomatids, the endocytic process is extremely polarized, with cargo uptake confined to the flagellar pocket in Leishmania

ARTICLE IN PRESS 114

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Fig. 22. Immunolocalization of cruzipain and serine carboxypeptidase in T. cruzi trypomastigotes and amastigotes. Serine carboxypeptidase was observed in round and elongated TcLROs confined between nucleus and kinetoplast in trypomastigotes (a). Colocalization between cruzipain (5 nm-gold particle) and carboxypeptidase (15 nm-gold particle) was found in trypomastigotes LROs (b, c). In amastigotes, carboxypeptidase is concentrated in polymorphic TcLROs (d), where it (15 nm-gold particle) co-localizes with cruzipain (5 nmgold particle). Bars: 500 nm (a); 300 nm (b); 100 nm (c); 400 nm (d); 200 nm (e). After Sant’Anna et al. (2008b).

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

115

Fig. 23. Schematic representation of T. cruzi epimastigotes endocytic pathway. Endocytic compartments present a very polarized distribution, flagellar pocket (FP) and cytostome (C) invaginates from anterior region plasma membrane (in grey), close to Golgi complex (GC) and kinetoplast (K). Cytopharynx (Cx) spans from cytostome opening until nucleus (N) vicinity. Early endosome corresponds to a collection of long tubules and vesicles (purple). Reservosomes (R) are shown as round organelles with inclusions. This scheme is the first frame of an animated endocytic model (Supplementary video).

ARTICLE IN PRESS 116

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

and T. brucei or distributed between the pocket and the cytostome in T. cruzi epimastigotes. The knowledge about T. cruzi endocytic pathway is still poor when compared with T. brucei and Leishmania, mainly due to the absence of molecular markers. Nevertheless, morphological data already allow a proposition on how the epimastigote endocytic compartments are distributed (Fig. 22) and on the first dynamic model of cargo route from the sites of entry to storage and digestion (Fig. 23 and Supplementary video). As an essential process for parasite survival, differences between molecules and organelles involved in the endocytic pathway of parasite and host cells surely promise to be a good source of chemotherapy targets. Acknowledgements The authors are grateful to Drs. Julia R. Araripe, Evander J.O. Batista, Marlene Benchimol, Adriana Lanfredi-Rangel, Kildare Miranda, Paulo F.P. Pimenta, Isabel Porto-Carreiro, Maurilio J. Soares, Tania Ueda-Nakamura and Celso Vatarunakamura for kindly providing photographs. They also thank Dirceu Esdras, Luiz Leonardo and Breno Alcaˆntara for beautiful drawing and animation. The authors are deeply indebted to Miria G. Pereira, Daniela Lourenc¸o and Drs. Ana Paula C.A. Lima, Tecia Ulisses de Carvalho and Thaı¨s Souto-Padro´n for helpful discussion. The authors receive financial support from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o Carlos Chagas Filho de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nivel Superior (CAPES).

Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.proghi.2009.01.001

References Adade CM, de Castro SL, Soares MJ. Ultrastructural localization of Trypanosoma cruzi lysosomes by aryl sulphatase cytochemistry. Micron 2007;38:252–6. Addis MF, Rappelli P, Delogu G, Carta F, Cappuccinelli P, Fiori PL. Cloning and molecular characterization of a cDNA clone coding for Trichomonas vaginalis alpha-actinin and intracellular localization of the protein. Infect Immun 1998;66:4924–31. Affonso AL, Benchimol M, Ribeiro KC, Lins U, De Souza W. Further studies on the endocytic activity of Tritrichomonas foetus. Parasitol Res 1994;80:403–13. Affonso AL, de Almeida JC, Benchimol M. Partial characterization of cytoplasmic compartments involved in the endocytic process of Tritrichomonas foetus. Eur J Cell Biol 1997;72:247–56. Aikawa M. Parasitological review. Plasmodium: the fine structure of malarial parasites. Exp Parasitol 1971;30:284–320.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

117

Aikawa M, Hepler PK, Huff CG, Sprinz H. The feeding mechanism of avian malarial parasites. J Cell Biol 1966;28:355–73. Alberio SO, Dias SS, Faria FP, Mortara RA, Barbieri CL, Freymuller Haapalainen E. Ultrastructural and cytochemical identification of megasome in Leishmania (Leishmania) chagasi. Parasitol Res 2004;92:246–54. Alexander DL, Schwartz KJ, Balber AE, Bangs JD. Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei. J Cell Sci 2002;115:3253–63. Alexander J, Vickerman K. Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages. J Protozool 1975;22:502–8. Allen CL, Goulding D, Field MC. Clathrin-mediated endocytosis is essential in Trypanosoma brucei. EMBO J 2003;22:4991–5002. Alvarez VE, Kosec G, Sant’Anna C, Turk V, Cazzulo JJ, Turk B. Autophagy is involved in nutritional stress response and differentiation in Trypanosoma cruzi. J Biol Chem 2008a;283:3454–64. Alvarez VE, Kosec G, Sant’Anna C, Turk V, Cazzulo JJ, Turk B. Blocking autophagy to prevent parasite differentiation: a possible new strategy for fighting parasitic infections? Autophagy 2008b;4:361–3. Anderson RG, Falck JR, Goldstein JL, Brown MS. Visualization of acidic organelles in intact cells by electron microscopy. Proc Natl Acad Sci USA 1984;81:4838–42. Andrews NW. Lysosomes and the plasma membrane: trypanosomes reveal a secret relationship. J Cell Biol 2002;158:389–94. Angermu¨ller S, Fahimi HD. Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy. Histochem J 1982;14:823–35. Antoine JC, Jouanne C, Ryter A, Benichou JC. Leishmania amazonensis: acidic organelles in amastigotes. Exp Parasitol 1988;67:287–300. Antoine JC, Jouanne C, Ryter A. Megasomes as the targets of leucine methyl ester in Leishmania amazonensis amastigotes. Parasitology 1989;1:1–9. Antoine JC, Lang T, Prina E, Courret N, Hellio R. H-2M molecules, like MHC class II molecules, are targeted to parasitophorous vacuoles of Leishmania-infected macrophages and internalized by amastigotes of L. amazonensis and L. mexicana. J Cell Sci 1999;112:2559–70. Araripe JR, Cunha e Silva NL, Leal ST, De Souza W, Rondinelli E. Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes. Biochem Biophys Res Commun 2004;321: 397–402. Araripe JR, Ramos FP, Cunha e Silva NL, Urmenyi TP, Silva R, Leite Fontes CF, et al. Characterization of a RAB5 homologue in Trypanosoma cruzi. Biochem Biophys Res Commun 2005;329:638–45. Bailey GB, Day DB, Nokkaew C, Harper CC. Stimulation by target cell membrane lipid of actin polymerization and phagocytosis by Entamoeba histolytica. Infect Immun 1987;55:1848–53. Balzac F, Avolio M, Degani S, Kaverina I, Torti M, Silengo L, et al. E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function. J Cell Sci 2005;118:4765–83. Batista EJ, De Souza W. Involvement of protein kinases on the process of erythrophagocytis by Entamoeba histolytica. Cell Biol Int 2004;28:243–8. Batista EJ, de Menezes Feitosa LF, de Souza W. The endocytic pathway in Entamoeba histolytica. Parasitol Res 2000;86:881–90. Benchimol M, Batista C, De Souza W. Fibronectin- and laminin-mediated endocytic activity in the parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus. J Submicrosc Cytol Pathol 1990;22:39–45. Benchimol M, da Cunha e Silva NL, Elias CA, de Souza W. Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis. Exp Parasitol 1986;62:405–15. Bloom FE, Aghajanian GK. Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid. J Ultrastruct Res 1968;22:361–75. Bockman A, Winborn WB. Electron microscopic localization of exogenous ferritin within vacuoles of Giardia muris (sp.). J. Protozool. 1968;15:26–30. Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, et al. Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family. PLoS Pathog 2008;4:8. Bogitsh BJ, Ribeiro-Rodrigues R, Carter CE. In vitro effects of mannan and cytochalasin B on the uptake of horseradish peroxidase and [14C]sucrose by Trypanosoma cruzi epimastigotes. J Parasitol 1995;81:144–8. Boiso JF, Docampo R, Stoppani AOM. Investigacion citoquimica por microscopia electronica de la presencia de microperoxisomas en Trypanosoma cruzi. Medicina (Buenos Aires) 1977;37:365–70.

ARTICLE IN PRESS 118

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Bonifacino JS, Traub LM. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 2003;72:395–447. Borges VM, Vannier-Santos MA, de Souza W. Subverted transferrin trafficking in Leishmania-infected macrophages. Parasitol Res 1998;84:811–22. Bracha R, Kobiler D, Mirelman D. Attachment and ingestion of bacteria by trophozoites of Entamoeba histolytica. Infect Immun 1982;36:396–406. Bricheux G, Brugerolle G. Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences. FEMS Microbiol Lett 1997;153:205–13. Bricheux G, Coffe G, Bayle D, Brugerolle G. Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis. Eur J Cell Biol 2000;79:413–22. Bricheux G, Coffe G, Pradel N, Brugerolle G. Evidence for an uncommon alpha-actinin protein in Trichomonas vaginalis. Mol Biochem Parasitol 1998;95:241–9. Brickman MJ, Cook JM, Balber AE. Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in African trypanosomes. J Cell Sci 1995;108:3611–21. Bright NA, Gratian MJ, Luzio JP. Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol 2005;15:360–5. Brugerolle G. Mise en evidence du processus d’endocytose et de structures lysosomiques chez Trichomonas vaginalis. C R Acad Sci (Paris) 1971;272:2558–60. Brugerolle G, Bricheux G, Coffe G. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads. Biol Cell 1996;88:29–36. Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, Zhao Q, et al. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 2007;315:207–12. Chadda R, Howes MT, Plowman SJ, Hancock JF, Parton RG, Mayor S. Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway. Traffic 2007;8:702–17. Chanez AL, Hehl AB, Engstler M, Schneider A. Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest. J Cell Sci 2006;119: 2968–74. Coombs GH, Tetley L, Moss VA, Vickerman K. Three dimensional structure of the Leishmania amastigote as revealed by computer-aided reconstruction from serial sections. Parasitology 1986;92:13–23. Coppens I, Opperdoes FR, Courtoy PJ, Baudhuin P. Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei. J Protozool 1987;34:465–73. Correˆa JR, Atella GC, Batista MM, Soares MJ. Transferrin uptake in Trypanosoma cruzi is impaired by interference on cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis. Exp Parasitol 2008;119:58–66. Correˆa JR, Atella GC, Menna-Barreto RS, Soares MJ. Clathrin in Trypanosoma cruzi: in silico gene identification, isolation, and localization of protein expression sites. J Eukaryot Microbiol 2007;54: 297–302. Cuevas IC, Rohloff P, Sanchez DO, Docampo R. Characterization of farnesylated protein tyrosine phosphatase TcPRL-1 from Trypanosoma cruzi. Eukaryot Cell 2005;4:1550–61. Cullen PJ. Endosomal sorting and signalling: an emerging role for sorting nexins. Nat Rev Mol Cell Biol 2008;9:574–82. Cunha-e-Silva N, Sant’Anna C, Pereira MG, Porto-Carreiro I, Jeovanio AL, de Souza W. Reservosomes: multipurpose organelles? Parasitol Res 2006;99:325–7. Cunha-e-Silva NL, Atella GC, Porto-Carreiro IA, Morgado-Diaz JA, Pereira MG, De Souza W. Isolation and characterization of a reservosome fraction from Trypanosoma cruzi. FEMS Microbiol Lett 2002;214:7–12. De Figueiredo RC, Soares MJ. Low temperature blocks fluid-phase pinocytosis and receptor-mediated endocytosis in Trypanosoma cruzi epimastigotes. Parasitol Res 2000;86:413–8. De Matteis MA, Luini A. Exiting the Golgi complex. Nat Rev Mol Cell Biol 2008;9:273–84. De Melo LD, Nepomuceno-Silva JL, Sant’Anna C, Eisele N, Ferraro RB, Meyer-Fernandes JR, et al. TcRho1 of Trypanosoma cruzi: role in metacyclogenesis and cellular localization. Biochem Biophys Res Commun 2004;323:1009–16. De Melo LD, Sant’Anna C, Reis SA, Lourenco D, De Souza W, Lopes UG, et al. Evolutionary conservation of actin-binding proteins in Trypanosoma cruzi and unusual subcellular localization of the actin homologue. Parasitology 2008;135:955–65. De Souza Leao S, Lang T, Prina E, Hellio R, Antoine JC. Intracellular Leishmania amazonensis amastigotes internalize and degrade MHC class II molecules of their host cells. J Cell Sci 1995;108: 3219–31.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

119

De Souza W. Basic cell biology of Trypanosoma cruzi. Curr Pharm Des 2002;8:269–85. de Souza W, de Carvalho TU, Benchimol M, Chiari E. Trypanosoma cruzi: ultrastructural, cytochemical and freeze-fracture studies of protein uptake. Exp Parasitol 1978;45:101–15. Denny PW, Morgan GW, Field MC, Smith DF. Leishmania major: clathrin and adaptin complexes of an intra-cellular parasite. Exp Parasitol 2005;109:33–7. Eakin AE, Mills AA, Harth G, McKerrow JH, Craik CS. The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. J Biol Chem 1992;267:7411–20. Egan TJ, Combrinck JM, Egan J, Hearne GR, Marques HM, Ntenteni S, et al. Fate of haem iron in the malaria parasite Plasmodium falciparum. Biochem J 2002;365:343–7. Elliott DA, McIntosh MT, Hosgood III HD, Chen S, Zhang G, Baevova P, et al. Four distinct pathways of hemoglobin uptake in the malaria parasite Plasmodium falciparum. Proc Natl Acad Sci USA 2008;105:2463–8. El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, et al. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 2005;309:409–15. Engel JC, Doyle PS, Palmer J, Hsieh I, Bainton DF, McKerrow JH. Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi. J Cell Sci 1998;111:597–606. Engel JC, Torres C, Hsieh I, Doyle PS, McKerrow JH. Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi. J Cell Sci 2000;113:1345–54. Engstler M, Bangs JD, Field MC. Intracellular transport systems in trypanosomes: function, evolution and virulence. In: Barry JD, Mottram J, McCulloch R, Acosta-Serrano A, editors. Trypanosomes – after the genome. Horizon Press; 2007. Engstler M, Thilo L, Weise F, Grunfelder CG, Schwarz H, Boshart M, et al. Kinetics of endocytosis and recycling of the GPI-anchored variant surface glycoprotein in Trypanosoma brucei. J Cell Sci 2004;117:1105–15. Feely DE, Dyer JK. Localization of acid phosphatase activity in Giardia lamblia and Giardia muris trophozoites. J Protozool 1987;34:80–3. Field H, Farjah M, Pal A, Gull K, Field MC. Complexity of trypanosomatid endocytosis pathways revealed by Rab4 and Rab5 isoforms in Trypanosoma brucei. J Biol Chem 1998;273:32102–10. Field MC, Carrington M. Intracellular membrane transport systems in Trypanosoma brucei. Traffic 2004;5:905–13. Francioli P, Shio H, Roberts RB, Muller M. Phagocytosis and killing of Neisseria gonorrhoeae by Trichomonas vaginalis. J Infect Dis 1983;147:87–94. Franco-Barraza J, Zamudio-Meza H, Franco E, del Carmen Dominguez-Robles M, Villegas-Sepulveda N, Meza I. Rho signaling in Entamoeba histolytica modulates actomyosin-dependent activities stimulated during invasive behavior. Cell Motil Cytoskeleton 2006;63:117–31. Frick M, Bright NA, Riento K, Bray A, Merrified C, Nichols BJ. Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding. Curr Biol 2007;17:1151–6. Gaechter V, Schraner E, Wild P, Hehl AB. The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport. Traffic 2008;9:57–71. Garcia ES, Azambuja P. Development and interactions of Trypanosoma cruzi within the insect vector. Parasitol Today 1991;7:240–4. Garcia-Salcedo JA, Perez-Morga D, Gijon P, Dilbeck V, Pays E, Nolan DP. A differential role for actin during the life cycle of Trypanosoma brucei. EMBO J 2004;23:780–9. Ghedin E, Debrabant A, Engel JC, Dwyer DM. Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan. Traffic 2001;2:175–88. Gonzalez-Robles A, Lazaro-Haller A, Espinosa-Cantellano M, Anaya-Velazquez F, Martinez-Palomo A. Trichomonas vaginalis: ultrastructural bases of the cytopathic effect. J Eukaryot Microbiol 1995;42: 641–51. Goodyer ID, Pouvelle B, Schneider TG, Trelka DP, Taraschi TF. Characterization of macromolecular transport pathways in malaria-infected erythrocytes. Mol Biochem Parasitol 1997;87:13–28. Grab DJ, Shaw MK, Wells CW, Verjee Y, Russo DC, Webster P, et al. The transferrin receptor in African trypanosomes: identification, partial characterization and subcellular localization. Eur J Cell Biol 1993;62:114–26. Grassart A, Dujeancourt A, Lazarow PB, Dautry-Varsat A, Sauvonnet N. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2. EMBO Rep 2008;9:356–62. Grunfelder CG, Engstler M, Weise F, Schwarz H, Stierhof YD, Morgan GW, et al. Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers. Mol Biol Cell 2003;14:2029–40.

ARTICLE IN PRESS 120

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Hernandez Y, Castillo C, Roychowdhury S, Hehl A, Aley SB, Das S. Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia. Int J Parasitol 2007;37:21–32. Hung CH, Qiao X, Lee PT, Lee MG. Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei. Eukaryot Cell 2004;3:1004–14. Huston CD, Boettner DR, Miller-Sims V, Petri Jr WA. Apoptotic killing and phagocytosis of host cells by the parasite Entamoeba histolytica. Infect Immun 2003;71:964–72. Idrissi FZ, Grotsch H, Fernandez-Golbano IM, Presciatto-Baschong C, Riezman H, Geli MI. Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane. J Cell Biol 2008;180:1219–32. Ilgoutz SC, Mullin KA, Southwell BR, McConville MJ. Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana. EMBO J 1999;18:3643–54. Jain R, Santi-Rocca J, Padhan N, Bhattacharya S, Guillen N, Bhattacharya A. Calcium-binding protein 1 of Entamoeba histolytica transiently associates with phagocytic cups in a calcium-independent manner. Cell Microbiol 2008;10:1373–89. Jeffries TR, Morgan GW, Field MC. A developmentally regulated rab11 homologue in Trypanosoma brucei is involved in recycling processes. J Cell Sci 2001;114:2617–26. Jordens I, Marsman M, Kuijl C, Neefjes J. Rab proteins, connecting transport and vesicle fusion. Traffic 2005;6:1070–7. Juliano C, Cappuccinelli P, Mattana A. In vitro phagocytic interaction between Trichomonas vaginalis isolates and bacteria. Eur J Clin Microbiol Infect Dis 1991;10:497–502. Kaksonen M, Toret CP, Drubin DG. A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 2005;123:305–20. Kattenbach WM, Pimenta PF, de Souza W, Pinto da Silva P. Giardia duodenalis: a freeze-fracture, fracture-flip and cytochemistry study. Parasitol Res 1991;77:651–8. Kollien AH, Schaub GA. The development of Trypanosoma cruzi in triatominae. Parasitol Today 2000;16:381–7. Lafourcade C, Sobo K, Kieffer-Jaquinod S, Garin J, van der Goot FG. Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization. PLoS ONE 2008;3:e2758. Landfear SM, Ignatushchenko M. The flagellum and flagellar pocket of trypanosomatids. Mol Biochem Parasitol 2001;115:1–17. Lanfredi-Rangel A, Attias M, de Carvalho TM, Kattenbach WM, De Souza W. The peripheral vesicles of trophozoites of the primitive protozoan Giardia lamblia may correspond to early and late endosomes and to lysosomes. J Struct Biol 1998;123:225–35. Langreth SG, Jensen JB, Reese RT, Trager W. Fine structure of human malaria in vitro. J Protozool 1978;25:443–52. Lara FA, Sant’Anna C, Lemos D, Laranja GA, Coelho MG, Reis Salles I, et al. Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes. Biochem Biophys Res Commun 2007;30; 355:16–22. Lazarus MD, Schneider TG, Taraschi TF. A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum. J Cell Sci 2008;121:1937–49. Leabu M. Membrane fusion in cells: molecular machinery and mechanisms. J Cell Mol Med 2006;10: 423–7. Lindmark DG. Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites. Exp Parasitol 1988;65:141–7. Luo S, Scott DA, Docampo R. Trypanosoma cruzi H+–ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+–ATPases with different enzymatic properties. J Biol Chem 2002;277:44497–506. Luzio JP, Pryor PR, Bright NA. Lysosomes: fusion and function. Nat Rev Mol Cell Biol 2007;8: 622–32. Marion S, Laurent C, Guillen N. Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach. Cell Microbiol 2005;7:1504–18. Marion S, Wilhelm C, Voigt H, Bacri JC, Guillen N. Overexpression of myosin IB in living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis. J Cell Sci 2004;117:3271–9. Marotta DE, Gerald N, Dwyer DM. Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani. Mol Cell Biochem 2006;292:107–17.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

121

Martinez-Palomo A, De Souza W, Gonzalez-Robles A. Topographical differences in the distribution of surface coat components and intramembrane particles. A cytochemical and freeze-fracture study in culture forms of Trypanosoma cruzi. J Cell Biol 1976;69:507–13. Mauricio de Mendonca SM, Nepomuceno da Silva JL, Cunha-e-Silva N, de Souza W, Gazos Lopes U. Characterization of a Rab11 homologue in Trypanosoma cruzi. Gene 2000;243:179–85. Mayor S, Pagano RE. Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 2007;8:603–12. McConville MJ, Mullin KA, Ilgoutz SC, Teasdale RD. Secretory pathway of trypanosomatid parasites. Microbiol Mol Biol Rev 2002;66:122–54 table of contents. McGrory T, Meysick K, Lemchuk-Favel LT, Garber GE. The interaction of Lactobacillus acidophilus and Trichomonas vaginalis in vitro. J Parasitol 1994;80:50–4. Mitra BN, Kobayashi S, Saito-Nakano Y, Nozaki T. Entamoeba histolytica: differences in phagosome acidification and degradation between attenuated and virulent strains. Exp Parasitol 2006;114:57–61. Mitra BN, Saito-Nakano Y, Nakada-Tsukui K, Sato D, Nozaki T. Rab11B small GTPase regulates secretion of cysteine proteases in the enteric protozoan parasite Entamoeba histolytica. Cell Microbiol 2007;9:2112–25. Morgan GW, Allen CL, Jeffries TR, Hollinshead M, Field MC. Developmental and morphological regulation of clathrin-mediated endocytosis in Trypanosoma brucei. J Cell Sci 2001;114:2605–15. Morgan GW, Goulding D, Field MC. The single dynamin-like protein of Trypanosoma brucei regulates mitochondrial division and is not required for endocytosis. J Biol Chem 2004;279:10692–701. Morgan GW, Hall BS, Denny PW, Carrington M, Field MC. The kinetoplastida endocytic apparatus. Part I: a dynamic system for nutrition and evasion of host defences. Trends Parasitol 2002a;18: 491–6. Morgan GW, Hall BS, Denny PW, Field MC, Carrington M. The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system. Trends Parasitol 2002b;18:540–6. Mullin KA, Foth BJ, Ilgoutz SC, Callaghan JM, Zawadzki JL, McFadden GI, et al. Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana. Mol Biol Cell 2001;12:2364–77. Mussmann R, Engstler M, Gerrits H, Kieft R, Toaldo CB, Onderwater J, et al. Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei. J Biol Chem 2004;279:40690–8. Nakada-Tsukui K, Saito-Nakano Y, Ali V, Nozaki T. A retromerlike complex is a novel Rab7 effector that is involved in the transport of the virulence factor cysteine protease in the enteric protozoan parasite Entamoeba histolytica. Mol Biol Cell 2005;16:5294–303. Nayak RC, Sahasrabuddhe AA, Bajpai VK, Gupta CM. A novel homologue of coronin colocalizes with actin in filament-like structures in Leishmania. Mol Biochem Parasitol 2005;143:152–64. Nolan DP, Garcia-Salcedo JA. Loss of actin does not affect export of newly synthesized proteins to the surface of Trypanosoma brucei. Mol Biochem Parasitol 2008;157:233–5. Okada M, Huston CD, Mann BJ, Petri Jr WA, Kita K, Nozaki T. Proteomic analysis of phagocytosis in the enteric protozoan parasite Entamoeba histolytica. Eukaryot Cell 2005;4:827–31. Okada M, Nozaki T. New insights into molecular mechanisms of phagocytosis in Entamoeba histolytica by proteomic analysis. Arch Med Res 2006;37(2):244–52. Olliaro P, Castelli F, Milano F, Filice G, Carosi G. Ultrastructure of Plasmodium falciparum ‘‘in vitro’’. I. Base-line for drug effects evaluation. Microbiologica 1989;12:7–14. Orozco E, Guarneros G, Martinez-Palomo A, Sanchez T. Entamoeba histolytica. Phagocytosis as a virulence factor. J Exp Med 1983;158:1511–21. Orth JD, Krueger EW, Weller SG, McNiven MA. A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization. Cancer Res 2006;66:3603–10. Overath P, Engstler M. Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system. Mol Microbiol 2004;53:735–44. Pal A, Hall BS, Nesbeth DN, Field HI, Field MC. Differential endocytic functions of Trypanosoma brucei Rab5 isoforms reveal a glycosylphosphatidylinositol-specific endosomal pathway. J Biol Chem 2002;277:9529–39. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol 2007;8:185–94. Parussini F, Garcia M, Mucci J, Aguero F, Sanchez D, Hellman U, et al. Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi. Mol Biochem Parasitol 2003;131:11–23. Peck RF, Shiflett AM, Schwartz KJ, McCann A, Hajduk SL, Bangs JD. The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes. Mol Microbiol 2008;68:933–46. Pereira-Neves A, Benchimol M. Phagocytosis by Trichomonas vaginalis: new insights. Biol Cell 2007;99:87–101.

ARTICLE IN PRESS 122

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Peterson KM, Alderete JF. Selective acquisition of plasma proteins by Trichomonas vaginalis and human lipoproteins as a growth requirement for this species. Mol Biochem Parasitol 1984a;12: 37–48. Peterson KM, Alderete JF. Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors. J Exp Med 1984b;160:398–410. Pimenta PF, de Souza W, Souto-Padron T, Pinto da Silva P. The cell surface of Trypanosoma cruzi: a fracture-flip, replica-staining label-fracture survey. Eur J Cell Biol 1989;50:263–71. Porto-Carreiro I, Attias M, Miranda K, De Souza W, Cunha-e-Silva N. Trypanosoma cruzi epimastigote endocytic pathway: cargo enters the cytostome and passes through an early endosomal network before storage in reservosomes. Eur J Cell Biol 2000;79:858–69. Pupkis MF, Tetley L, Coombs GH. Leishmania mexicana: amastigote hydrolases in unusual lysosomes. Exp Parasitol 1986;62:29–39. Ravdin JI. Immunobiology of human infection by Entamoeba histolytica. Pathol Immunopathol Res 1989;8:179–205. Rendon-Maldonado JG, Espinosa-Cantellano M, Gonzalez-Robles A, Martinez-Palomo A. Trichomonas vaginalis: in vitro phagocytosis of lactobacilli, vaginal epithelial cells, leukocytes, and erythrocytes. Exp Parasitol 1998;89:241–50. Rodriguez MA, Orozco E. Characterization of the EhRabB recombinant protein of Entamoeba histolytica. Arch Med Res 2000;31:S171–2. Rudzinska MA, Trager W, Bray RS. Pinocytotic uptake and the digestion of hemoglobin in malaria parasites. J Protozool 1965;12:563–76. Sahasrabuddhe AA, Bajpai VK, Gupta CM. A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules. Mol Biochem Parasitol 2004;134:105–14. Sahoo N, Labruyere E, Bhattacharya S, Sen P, Guillen N, Bhattacharya A. Calcium binding protein 1 of the protozoan parasite Entamoeba histolytica interacts with actin and is involved in cytoskeleton dynamics. J Cell Sci 2004;117:3625–34. Saito-Nakano Y, Loftus BJ, Hall N, Nozaki T. The diversity of Rab GTPases in Entamoeba histolytica. Exp Parasitol 2005;110:244–52. Saito-Nakano Y, Mitra BN, Nakada-Tsukui K, Sato D, Nozaki T. Two Rab7 isotypes, EhRab7A and EhRab7B, play distinct roles in biogenesis of lysosomes and phagosomes in the enteric protozoan parasite Entamoeba histolytica. Cell Microbiol 2007;9:1796–808. Saito-Nakano Y, Yasuda T, Nakada-Tsukui K, Leippe M, Nozaki T. Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica. J Biol Chem 2004;279:49497–507. Saksena S, Sun J, Chu T, Emr SD. ESCRTing proteins in the endocytic pathway. Trends Biochem Sci 2007;32:561–73. Saliba KJ, Kirk K. Nutrient acquisition by intracellular apicomplexan parasites: staying in for dinner. Int J Parasitol 2001;31:1321–30. Sandvig K, Torgersen ML, Raa HA, van Deurs B. Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity. Histochem Cell Biol 2008;129:267–76. Sant’Anna C, De Souza W, Cunha-e-Silva N. Biogenesis of the reservosomes of Trypanosoma cruzi. Microsc Microanal 2004;10:637–46. Sant’Anna C, Pereira MG, Lemgruber L, de Souza W, Cunha e Silva NL. New insights into the morphology of Trypanosoma cruzi reservosome. Microsc Res Tech 2008a;71:599–605. Sant’Anna C, Parussini F, Lourenc¸o D, de Souza W, Cazzulo JJ, Cunha-E-Silva NL. All Trypanosoma cruzi developmental forms present lysosome-related organelles. Histochem Cell Biol 2008b;130: 1187–98. Sant’Anna C, Nakayasu ES, Pereira MG, Lourenc¸o D, De Souza W, Almeida IC, et al. Subcellular proteomics of Trypanosoma cruzi reservosomes. Proteomics 2009; in press, doi:10.1002/pmic. 200800730. Santos CC, Sant’Anna C, Terres A, Cunha-e-Silva NL, Scharfstein J, de ALAP. Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells. J Cell Sci 2005;118:901–15. Schmunis GA, Cruz JR. Safety of the blood supply in Latin America. Clin Microbiol Rev 2005;18:12–29. Scholtyseck E, Teras J, Kasakova I, Sethi KK. Electron microscope observations on the interaction of Mycoplasma fermentans with Trichomonas vaginalis. Z Parasitenkd 1985;71:435–42. Schuler H, Mueller AK, Matuschewski K. Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites. FEBS Lett 2005;579:655–60.

ARTICLE IN PRESS W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

123

Singh SB, Tandon R, Krishnamurthy G, Vikram R, Sharma N, Basu SK, et al. Rab5-mediated endosome–endosome fusion regulates hemoglobin endocytosis in Leishmania donovani. EMBO J 2003;22:5712–22. Slomianny C. Three-dimensional reconstruction of the feeding process of the malaria parasite. Blood Cells 1990;16:369–78. Smythe WA, Joiner KA, Hoppe HC. Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum. Cell Microbiol 2008;10:452–64. Soares MJ, De Souza W. Cytoplasmic organelles of trypanosomatids: a cytochemical and stereological study. J Submicrosc Cytol Pathol 1988;20:349–61. Soares MJ, De Souza W. Endocytosis of gold-labeled proteins and LDL by Trypanosoma cruzi. Parasitol Res 1991;77:461–8. Soares MJ, Souto-Padron T, Bonaldo MC, Goldenberg S, de Souza W. A stereological study of the differentiation process in Trypanosoma cruzi. Parasitol Res 1989;75:522–7. Soares MJ, Souto-Padron T, De Souza W. Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi. J Cell Sci 1992;102:157–67. Souto-Padron T, Campetella OE, Cazzulo JJ, de Souza W. Cysteine proteinase in Trypanosoma cruzi: immunocytochemical localization and involvement in parasite–host cell interaction. J Cell Sci 1990;96:485–90. Steverding D, Stierhof YD, Chaudhri M, Ligtenberg M, Schell D, Beck-Sickinger AG, et al. ESAG 6 and 7 products of Trypanosoma brucei form a transferrin binding protein complex. Eur J Cell Biol 1994;64:78–87. Street DA, Wells C, Taylor-Robinson D, Ackers JP. Interaction between Trichomonas vaginalis and other pathogenic micro-organisms of the human genital tract. Br J Vener Dis 1984;60:31–8. Tai JH, Ong SJ, Chang SC, Su HM. Giardiavirus enters Giardia lamblia WB trophozoite via endocytosis. Exp Parasitol 1993;76:165–74. Taraschi TF, Trelka D, Schneider T, Matthews I. Plasmodium falciparum: characterization of organelle migration during merozoite morphogenesis in asexual malaria infections. Exp Parasitol 1998;88: 184–93. Temesvari LA, Harris EN, Stanley Jr SL, Cardelli JA. Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases. Mol Biochem Parasitol 1999;103:225–41. Torres C, Perez-Victoria FJ, Parodi-Talice A, Castanys S, Gamarro F. Characterization of an ABCA-like transporter involved in vesicular trafficking in the protozoan parasite Trypanosoma cruzi. Mol Microbiol 2004;54:632–46. Touz MC, Kulakova L, Nash TE. Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia. Mol Biol Cell 2004;15:3053–60. Touz MC, Lujan HD, Hayes SF, Nash TE. Sorting of encystation-specific cysteine protease to lysosomelike peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif. J Biol Chem 2003;278:6420–6. Trager W. Living together. The biology of animal parasitism. New York: Plenum Press; 1986. Traub-Cseko YM, Duboise M, Boukai LK, McMahon-Pratt D. Identification of two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes using the polymerase chain reaction. Mol Biochem Parasitol 1993;57:101–15. Tsutsumi V, Ramirez-Rosales A, Lanz-Mendoza H, Shibayama M, Chavez B, Rangel-Lopez E, et al. Entamoeba histolytica: erythrophagocytosis, collagenolysis, and liver abscess production as virulence markers. Trans R Soc Trop Med Hyg 1992;86:170–2. Ueda-Nakamura T, Attias M, de Souza W. Megasome biogenesis in Leishmania amazonensis: a morphometric and cytochemical study. Parasitol Res 2001;87:89–97. Ueda-Nakamura T, Attias M, de Souza W. Comparative analysis of megasomes in members of Leishmania mexicana complex. Res Microbiol 2007;158:456–62. Ueda-Nakamura T, da Conceicao Rocha Sampaio M, Cunha-e-Silva NL, Traub-Cseko YM, de Souza W. Expression and processing of megasome cysteine proteinases during Leishmania amazonensis differentiation. Parasitol Res 2002;88:332–7. Van Meel E, Klumperman J. Imaging and imagination: understanding the endo-lysosomal system. Histochem Cell Biol 2008;129:253–66. Vargas M, Voigt H, Sansonetti P, Guillen N. Molecular characterization of myosin IB from the lower eukaryote Entamoeba histolytica, a human parasite. Mol Biochem Parasitol 1997;86:61–73.

ARTICLE IN PRESS 124

W. de Souza et al. / Progress in Histochemistry and Cytochemistry 44 (2009) 67–124

Vatarunakamura C, Ueda-Nakamura T, de Souza W. Visualization of the cytostome in Trypanosoma cruzi by high resolution field emission scanning electron microscopy using secondary and backscattered electron imaging. FEMS Microbiol Lett 2005;242:227–30. Vieira M, Rohloff P, Luo S, Cunha-e-Silva NL, de Souza W, Docampo R. Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi. Biochem J 2005;392:467–74. Voigt H, Olivo JC, Sansonetti P, Guillen N. Myosin IB from Entamoeba histolytica is involved in phagocytosis of human erythrocytes. J Cell Sci 1999;112(Pt 8):1191–201. Voyiatzaki CS, Soteriadou KP. Identification and isolation of the Leishmania transferrin receptor. J Biol Chem 1992;267:9112–7. Waller RF, McConville MJ. Developmental changes in lysosome morphology and function Leishmania parasites. Int J Parasitol 2002;32:1435–45. Webster P. Endocytosis by African trypanosomes. I. Three-dimensional structure of the endocytic organelles in Trypanosoma brucei and T. congolense. Eur J Cell Biol 1989;49:295–302. Webster P, Fish WR. Endocytosis by African trypanosomes. II. Occurrence in different life-cycle stages and intracellular sorting. Eur J Cell Biol 1989;49:303–10. Weise F, Stierhof YD, Kuhn C, Wiese M, Overath P. Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells. J Cell Sci 2000;113:4587–603. Welsh CF, Assoian RK. A growing role for Rho family GTPases as intermediaries in growth factor- and adhesion-dependent cell cycle progression. Biochim Biophys Acta 2000;1471:M21–9. Welter BH, Powell RR, Laughlin RC, McGugan GC, Bonner M, King A, et al. Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes. Exp Parasitol 2006;113:91–9. WHO. Control of Chagas disease. WHO Tech Series vol. 811, Geneva, 1991. p. 1–95.