Identification of a Large Pre-Lysosomal Compartment in the Pathogenic Protozoon Trypanosoma Cruzi

Journal of Cell Science 102, 157-167 (1992) 157 Printed in Great Britain © The Company of Biologists Limited 1992

Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi

MAURILIO J. SOARES1'2, THAIS SOUTO-PADR6N1 and WANDERLEY DE SOUZA1*

1 Departamento de Parasitologia e Bioflsica Celular, Institute/ de Biofisica Carlos Chagas Filho, UFRJ, 21949 Rio de Janeiro, RJ, Brazil 2Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, 20001 Rio de Janeiro, RJ, Brazil •Author for correspondence

Summary

Epimastigote forms of the pathogenic parasite Trypano- from Lowicryl-embedded cells demonstrated that the soma cruzi were used to study the endocytic process in a reservosomes are acidic compartments (pH 6.0, as protozoon. These elongated unicellular organisms are shown using DAMP as a pH probe) with no acid highly polarized cells: endocytosis occurs only at the phosphatase or typical lysosome-associated membrane anterior region through the cytostome and the flagellar proteins (LAMP 1, LAMP 2 and lgp 120), but rich in pocket membrane, areas of the plasma membrane where cysteine proteinase. These data suggest that the reservo- the cell cytoskeleton, formed by sub-peUicular micro- some is a pre-lysosomal compartment. Since cysteine tubules, is absent. When the cells were incubated at 4°C proteinase of T. cruzi contains no phosphorylated or 28°C with gold-labeled transferrin, fixed and pro- mannose residues and the cation-independent mannose cessed for routine transmission electron microscopy our 6-phosphate receptor could not be immunocytochemi- observations show that this ligand initially binds to the cally detected hi the reservosomes, it is possible that cytostome and the membrane lining the flagellar pocket lysosomal enzymes hi the epimastigote forms of T. cruzi and is subsequently ingested through a clathrtn-indepen- are targeted to compartments related to the endocytic dent receptor-mediated endocytotic process, with for- pathway through a mechanism different from that which mation of uncoated pits and vesicles. Ingested complexes occurs hi other eukaryotic cells. are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the Key words: endocytosis, Trypanosoma cruzi, posterior end of the cell. Immunocytochemical data immunocytochemistry, pre-lysosomal compartment.

Introduction movement, the ingested material to an inner late endosomal compartment (Matteoni and Kreis, 1987; Eukaryotic cells continuously ingest macromolecular Griffiths et al. 1989; Gruenberg et al. 1989). It has been complexes from the extracellular environment through recently suggested that late endosomes correspond to invaginations of the plasma membrane, either by non- pre-lysosomal compartments (PLCs) adjacent to, but selective fluid-phase pinocytosis or by concentrative functionally distinct from, the compartments of the receptor-mediated endocytosis, where the efficiency of frans-Golgi network (Griffiths et al. 1988). Although the process is greatly enhanced due to receptors present lysosomal enzymes could be detected in the PLCs, they on the cell surface that bind to specific ligand molecules, are both structurally and functionally distinct from the leading to their selective uptake. dense lysosomes, which, it has been suggested, either Recent studies on the endocytic pathway of eukary- bud off or mature from the late endosomes (Geuze et otic cells led to the proposition that distinct, pre- al. 1988; Griffiths et al. 1988; Griffiths et al. 1990). Such existing, cytoplasmic compartments are involved in the a vesicle shuttle model differs from the earlier matu- endocytic process and in the recycling of the plasma ration model, which assumes that no permanent membrane (Schmid et al. 1988; Gruenberg and Howel, endosomal compartments exist (reviewed by Hubbard, 1989; Gruenberg et al. 1989; Kornfeld and Mellman, 1989). 1989): ingested particles are first delivered to a The main goal of our work was to analyze the peripheral, tubulo-vesicular, early endosomal compart- structures involved in the endocytic process of epimasti- ment (Geuze et al. 1983; Mueller and Hubbard, 1986; gote forms of the protozoon Trypanosoma cruzi, a Schmid et al. 1988). Carrier vesicles bud off from this pathogenic uniflagellate organism that causes Chagas' structure and transport, by microtubule-dependent disease, which affects about 8 million people in South 158 M. J. Soares and others

America. This parasite presents three morphologically antibodies, were obtained through Dr. Gareth Griffiths distinct forms: in the final vertebrate host, the protozoa (European Molecular Biology Laboratories, Heidelberg, can be found either intracellularly (dividing amasti- Federal Republic of Germany). gotes) or free-swimming in the blood (non-dividing Bovine transferrin was purchased from Sigma Chemical Co (Saint Louis, MO, USA). Gold particles 15 nm in diameter trypomastigotes); in the digestive tract of the invert- were prepared according to Frens (1973) and conjugated to ebrate hosts (triatomid blood-sucking bugs), the para- transferrin as described by Bendayan et al. (1987). sites appear mostly as the multiplying epimastigote form. Epimastigotes can be easily maintained in vitro in Parasites axenic culture media, and therefore have been largely Five-day-old culture forms (epimastigotes) of the T. cruzi Y used to study the cell biology of this parasite (reviewed strain were used. Parasites were in the mid log phase of by De Souza, 1984). growth, when the number of reservosomes was maximal (Paulin et al. 1983; Soares, unpublished observations). The Data on fluid-phase pinocytosis of peroxidase (De C Souza et al. 1978) and on receptor-mediated endo- cells were kept at 28 C in Warren's liquid medium (Warren, cytosis of gold-labeled albumin, peroxidase, transferrin 1960) supplemented with 10% fetal calf serum. and LDL (Soares and De Souza, 1991) by T. cruzi Ingestion of transferrin-gold showed that the ingested material entered the cells Parasites were collected by centrifugation at 1,500 g for 5 through the cytostome and/or the flagellar pocket minutes, washed in phosphate buffered saline (PBS), pH 7.2, region. In the epimastigote forms, the proteins accumu- and then incubated for 30 minutes at 4 or 28CC in the presence lated in a cytoplasmic organelle, previously designated of a gold-labeled transferrin solution diluted 1:5 in PBS as the reservosome (Soares and De Souza, 1988). (absorbance at 520 nm=0.5). The cells were then washed in Stereological measurements suggested that these nutri- PBS, fixed for 2 hours in a solution containing 1.5% ents are degraded during metacyclogenesis, a process glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate by which non-infective epimastigotes are transformed buffer, pH 7.2, washed in this same buffer and then post-fixed into infective trypomastigotes (Soares et al. 1989). In for 1 hour with 1% osmium tetroxide/0.8% potassium addition, recent studies have shown that the most ferricyanide in cacodylate buffer, pH 7.2, containing 5 mM important T. cruzi antigens, which are being used for calcium chloride. Thereafter, the cells were washed in cacodylate buffer, dehydrated in a graded ethanol series and diagnosis of Chagas' disease, are located in the embedded in Epon. Ultra-thin sections were stained with reservosomes (Krieger et al. 1990; Goldenberg et al. uranyl acetate and lead citrate, and observed by transmission unpublished data). electron microscopy. In this work we used gold-labeled transferrin to define more clearly the compartments involved in the Cytochemical detection of acid phosphatase activity endocytic pathway of a pathogenic protozoon. Our Parasites were collected by centrifugation, washed in PBS, C observations confirm previous studies showing that briefly fixed for 20 minutes at 4 C with 1% glutaraldehyde in trypanosomatids are highly polarized in terms of 0.1 M cacodylate buffer, pH 7.2, washed in 0.1 M cacodylate endocytic activity, and that transferrin concentrates in buffer, pH 7.2, and in 0.1 M Tris-maleate buffer, pH 5.0, and then incubated for 1 hour at 28°C in a medium containing 2 the reservosome, an acidic compartment rich in cys- mM cerium chloride, 5% sucrose, 0.1 M Tris-acetate buffer, teine proteinase, but which does not contain acid pH 5.0, and 2 mM cytidine 5'-monophosphate or 2 mM phosphatase or other lysosomal membrane proteins. sodium /S-glycerophosphate as substrate (Robinson and Karnovsky, 1983; Robinson, 1985). The cells were then washed in Tris-maleate and cacodylate buffers, refixed with Materials and methods 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer, post- fixed in 1% osmium tetroxide, dehydrated in a graded acetone Materials series and embedded in Epon. As a control, some cells were DAMP [3-(2,4-dinitroanihno)-3'-amino-7V-methyldipropyl- incubated in medium without substrate. Ultra-thin sections amine] and a monoclonal mouse antibody directed against were observed unstained or briefly stained with lead citrate by DNP (dinitrophenol) were obtained from Dr. R. G. W. transmission electron microscopy. Anderson (University of Texas, Dallas, TE, USA). Poly- clonal rabbit antibody against T. cruzi cysteine proteinase Lowicryl embedding for immunocytochemistry (Campetella et al. 1990) was obtained from Dr. J. J. Cazzulo For the localization of antigens, cells were collected by (Instituto de Investigations Bioqufmicas Fundati6n Campo- centrifugation at 1,500 g, washed in PBS and then fixed for 2 mar, Buenos Aires, Argentina). Polyclonal goat anti-bovine hours with 0.1% glutaraldehyde and 4% paraformaldehyde clathrin antibody was purchased from Sigma Chemical Co diluted in 0.1 M phosphate buffer, pH 7.2. The cells were then (Saint Louis, MO, USA). Polyclonal rabbit anti-human washed in buffer, dehydrated in a graded methanol series cation-independent mannose 6-phosphate receptor (CI- (without post-fixation in osmium tetroxide) and embedded at M6PR) was obtained from Dr. S. Kornfeld (St. Louis -20°C in Lowicryl K4M (Bendayan et al. 1987). University Medical Center, St. Louis, MO, USA). Rabbit For the colocalization of transferrin and cysteine protein- anti-mouse and goat anti-rabbit IgGs labeled with 5 nm gold ase, or transferrin and DAMP, the cells were collected by particles and rabbit anti-goat IgG labeled with 10 nm gold centrifugation, washed in PBS and then incubated for 60 particles were purchased from E-Y Laboratories (San Mateo, minutes at 28°C in PBS containing either (a) 15 nm gold- CA, USA). labeled transferrin (1:5 dilution, absorbance at 520 nm=0.5) Monoclonal rat antibodies against the lysosomal membrane or (b) gold-labeled transferrin (1:5 dilution) and 50 ,uM of proteins lgp 120 (Lewis et al. 1985), LAMP 1 and LAMP 2 DAMP. Thereafter, the cells were washed in PBS and fixed (Mane et al. 1989), as well as polyclonal rabbit anti-rat and embedded as described above. Pre-lysosomal compartments in T. cruzi 159

Immunocytochemical localization of antigens incubated for 60 minutes with 5 nm gold-labeled goat anti- Ultra-thin sections of Lowicryl-embedded cells were collected rabbit or rabbit anti-mouse IgGs diluted 1:20 in PMT. The on nickel grids, incubated in phosphate buffered saline (PBS), grids were then washed in PBS, pH 7.2, rinsed in distilled pH 8.0, containing 5% non-fatty milk and 0.01% Tween 20 water, stained with uranyl acetate and lead citrate, and (PMT) for 30 minutes at room temperature and then for 1 observed by transmission electron microscopy. hour in the same medium containing a 1:20 dilution of monoclonal rat anti-LAMP 1, LAMP 2 or lgp 120 antibodies. Quantification of DAMP labeling After washing in PBS/l% bovine serum albumin/0.01% The internal pH of the reservosomes was estimated according Tween 20 (PAT), the grids were incubated firstly with a 1:75 to Anderson and Orci (1988): pH 7.0 - log (density of gold dilution of rabbit anti-rat IgGs and then with 5 nm gold particles in organelle/density of gold particles in cytoplasm). labeled-goat anti-rabbit IgGs diluted 1:20 in PMT. After The cytoplasm was assumed to be a pH 7.0 compartment. The washing in PBS, pH 7.2, and distilled water, the sections were number of 5 nm gold particles in each compartment was stained for transmission electron microscopy with uranyl determined in cells photographed and enlarged to diameters acetate and lead citrate. of 65,000 or 39,000. The surface area of the cytoplasm and For the detection of clathrin and the CI-M6PR, the ultra- reservosomes was recorded with an electronic pen on a graphic tablet coupled to a microcomputer programmed to thin sections were incubated with polyclonal goat anti-clathrin 2 or rabbit anti-CI-M6PR antibodies diluted 1:20 in PMT, calculate and express areas in ^m . The density of the gold washed in PAT and then incubated with 10 nm gold-labeled particles was determined by dividing the number of gold rabbit anti-goat or 5 nm gold-labeled goat anti-rabbit IgGs particles by the measured areas of the cytoplasm or of the diluted 1:20 in PMT. The grids were washed in PBS and in reservosomes. distilled water and then stained for transmission electron microscopy. As a positive control, mouse peritoneal macrophages were fixed and embedded in Lowicryl and the ultra- Results thin sections obtained were incubated as described above. General morphology of Epon-embedded cells Immunocytochemical colocalization of Epimastigotes of T. cruzi are elongated, polarized cells transferrin/cysteine proteinase or transferrin/DAMP presenting one flagellum at the anterior extremity, a Ultra-thin sections of Lowicryl-embedded cells were collected on nickel grids, incubated in PMT for 30 minutes at room centrally located nucleus, and an accumulation of large temperature and then for 1 hour in the same medium rounded membrane-bound bodies, the reservosomes, containing a 1:20 dilution of rabbit anti-7". cruzi cysteine at the posterior end of the cell. Lipid inclusions and proteinase (cells pre-incubated only with transferrin-gold) or glycosomes (peroxisome-like organelles) are other mouse anti-DNP (cells pre-incubated with transferrin-gold typical cytoplasmic structures. The cell body is encased and DAMP) antibodies. After washing in PAT the grids were in a cytoskeleton of subpellicular microtubules, which •hi

L_ 1

Figs 1 and 2. General morphology of Epon-embedded cells. Fig. 1. Longitudinal section through the anterior end of an epimastigote form of T. cruzi. The flagellum (F) arises from the basal body located close to the kinetoplast (K) and runs inside a flagellar pocket. A Golgi complex (G) and a network of vesicles and tubules (*) are found associated to the flagellar pocket. Fig. 2. Transverse section through the anterior end of the parasite. The tubular network (*) is clearly seen near the flagellar pocket (FP). Bar, 0.25 fxm. 160 M. J. Soares and others

* R • . "« * FP 8 9 Pre-lysosomal compartments in T. cruzi 161

Figs 3 to 9. Parasites were incubated for 30 minutes in a resulted in some unspecific labeling on the cell profiles, gold-labeled transferrin solution diluted 1:5 in PBS, fixed but no specific labeling on the plasma membrane or on and embedded in Epon. Figs 3 and 4. In epimastigotes the vesicle membranes (data not shown). No transfer- incubated at 4°C, transferrin-gold complexes are found only rin-gold and no positive reaction for clathrin could be in the cytostome (C; Fig. 3) or in the flagellarpocke t (arrow, Fig. 4). K, kinetoplast; N, nucleus; R, observed in the tubular network found near the reservosome. Figs 5 to 9. Cells incubated at 28°C. Fig. 5. flagellar pocket region. Detail of the cytostome (C). Gold particles (arrows) can be Reaction product indicative of acid phosphatase seen at the bottom of the structure. F, flagellum; FP, activity could be observed on the cell surface, in the flagellar pocket. Fig. 6. Note the absence of a coat in a cytostome and in the tubular network near the flagellar vesicle (arrow) budding off from the bottom of the pocket region, but not in the reservosomes (Fig. 13). In cytostome. G, Golgi complex. Fig. 7. Ingestion of gold- these experiments, either /J-glycerophosphate or cyti- labeled transferrin occurs through uncoated pits formed at dine monophosphate were used as substrate. In ad- the bottom of the flagellar pocket (arrow). K, kinetoplast. dition, no specific labeling of the reservosomes was Fig. 8. Ingested gold particles are seen inside an uncoated observed when ultra-thin sections of Lowicryl-embed- vesicle near the tubular network (*) found close to the flagellar pocket (FP). F, flagellum.Fig . 9. Ingested ded cells were incubated in the presence of antibodies transferrin-gold is transported to and accumulated into the that recognize the cation-independent mannose 6- reservosomes (R). Bar, 0.25 /jm. phosphate receptor (a marker for pre-lysosomal compartments) and lysosomal membrane proteins (LAMP 1, LAMP 2 and lgp 120), and subsequently in the run in a parallel array along the major axis of the presence of a secondary gold-labeled antibody (data not parasite. The flagellum arises near a specialized region shown). of the tubular mitochondrion, the kinetoplast, where a large number of DNA molecules is concentrated. Colocalization of transferrin-DAMP or transferrin- Before emerging at the anterior portion of the parasite, cysteine proteinase the flagellum runs inside an invagination of the plasma Incubation of living parasites in the presence of 15 nm membrane, the flagellar pocket, where the subpellicular gold-labeled transferrin allowed the colocalization of microtubules are missing (Fig. 1). The cytostome and some antigens in the reservosome, in Lowicryl ultra- the Golgi complex are always found near this region. A thin sections incubated with anti-cysteine proteinase or tubular network is also found associated with the anti-DNP IgGs. Sites of accumulation of DAMP in the flagellar pocket region, frequently very close to the epimastigote forms of T. cruzi were identified by Golgi complex (Fig. 2). indirect immunocytochemistry, using a first anti-DNP antibody, followed by a second antibody coupled to 5 Ingestion of transferrin-gold complexes nm gold particles. After incubation of the parasites with Gold-labeled transferrin can be used as a tracer for the DAMP for 60 minutes at 28°C, labeling of the endocytic pathway in epimastigotes of T. cruzi, as reservosomes with the 5 nm gold particles could be previously demonstrated (Soares and De Souza, 1991). observed (Fig. 14). With the simultaneous incubation of Incubation of the parasites at 4°C resulted in labeling of the parasites in the presence of transferrin/15 nm gold only the cytostome (Fig. 3) and the membrane lining and DAMP (detected with 5 nm gold particles), the flagellar pocket (Fig. 4), with no labeling either on colocalization of the markers could be observed in the other regions of the cell surface or inside the cell. When reservosomes (Fig. 15). No labeling against DAMP was the incubation temperature was raised to 28°C, gold- found in the Golgi complex, in vesicles budding from labeled proteins were internalized through uncoated the cytostome and the flagellar pocket, in the tubular vesicles arising from the cytostome (Figs 5 and 6) and network or inside the flagellar pocket (Fig. 16). DAMP the flagellar pocket membrane (Fig. 7). Ingested gold could rarely be detected in transferrin-containing particles were observed either entrapped in uncoated vesicles near the flagellar pocket (Fig. 16) and in the transport vesicles dispersed from the flagellar pocket small transport vesicles that fused with the reservo- region (Fig. 8) to the posterior part of the cell or somes (Fig. 17). Few 5 nm gold particles were accumulated in the reservosomes. The gold particles unspecifically located in the cytoplasm and nucleus. were usually dispersed in the dense matrix of the The estimation of the pH of the reservosomes was reservosomes (Fig. 9). accomplished by a quantification of the distribution of the 5 nm gold particles in the cells. The density of gold Cytochemical characterization of the compartments particles in the reservosomes was about 8-fold higher Transmission electron microscopy of parasites routinely than in the cytoplasm. (Table 1). By applying the fixed and embedded in Epon snowed that vesicles bud formula shown in Materials and methods, a pH of 6.0 off from the bottom of the cytostome (Fig. 6) and of the was found for the reservosomes. The number of gold flagellar pocket (Figs 7 and 10). Morphological obser- particles/jan2 varied in a wide range from one reservo- vations of such vesicles (Fig. 11), and of the transport some to another, independent of the size of the vesicles found near the flagellar pocket region (Fig. 12), organelle (Table 1). suggested the absence of a clathrin coat on these The enzyme cysteine proteinase could also be structures. Incubation of Lowicryl ultra-thin sections colocalized in sections of parasites previously incubated with a polyclonal antibody that recognizes clathrin with transferrin/15 nm gold compjexes. No labeling was 162 M. J. Soares and others

Figs 10 to 13. Epon-embedded epimastigotes. Figs 10 to 12. Parasites were incubated for 30 minutes in a gold-labeled transferrin solution diluted 1:5 in PBS, fixed and embedded in Epon; K, kinetoplast; N, nucleus. Fig. 10. Detail of the anterior end of a parasite showing the formation of an uncoated pit (arrow) at the bottom of the flagellar pocket. Several uncoated vesicles are also found near this region. Fig. 11. An ingested gold particle can be seen inside a small uncoated vesicle (arrow) close to the bottom of the flagellar pocket. Fig. 12. An uncoated vesicle containing a gold particle is observed near the basis of the flagellum (F). Fig. 13. Cytochemical detection of acid phosphatase activity. Positive reaction is found at the tubular network near the flagellar pocket (arrowheads), but not in the reservosomes (R). Bar, 0.25 jxm. found in the tubular network located near the flagellar Some 5 nm gold particles could also be detected on the pocket. Vesicles near the flagellar pocket region and parasite plasma membrane. transport vesicles (Fig. 18) were occasionally labeled. Intense labeling with the 5 run gold particles was Discussion detected inside the reservosomes, together with the transferrin/15 nm gold particles complexes (Fig. 19). The results presented in this paper show that epimasti- Pre-lysosomal compartments in T. cruzi 163 gote forms of the protozoon T. cruzi can be used as an Souza, 1988), suggests that the tubular network of interesting model for studies on endocytosis. These epimastigotes of T. cruzi, which is frequently observed elongated unicellular organisms are highly polarized near the Golgi complex, may represent a secretory cells: uptake of macromolecules takes place only at the compartment. Further experiments are necessary to anterior region, through the cytostome and the flagellar clarify the nature of this network. pocket membrane, and the ingested material is accumu- The uncoated endocytic vesicles coming from the lated into a specific organelle, the reservosome, found cytostome and the flagellarpocke t are carrier structures mostly at the posterior end of the cell. Endocytosis shuttled directly to the reservosomes, where they fuse occurs at the flagellar pocket region because it is the and discharge their contents. The absence of a coat in only area of the plasma membrane where the cytoskel- these vesicles suggests that little or no recycling of eton, formed mainly by subpellicular microtubules, is membranes occurs. Indeed, this helps to explain the absent. increase in number and size of the reservosomes during In previous work we demonstrated that some pro- the early and mid log phases of growth of the parasites teins may be ingested by epimastigotes of T. cruzi (Paulin et al. 1983; Soares, unpublished observations). through receptor-mediated endocytosis (Soares and De If reservosomes represent late endosomes (pre- Souza, 1991). However, no coated pits or coated lysosomal compartments), accumulating nutrients vesicles can be morphologically or immunocytochemi- ingested by the parasites, they should present at least cally (using goat anti-chlathrin antibodies) detected in two characteristics: (a) they should possess an internal the cytoplasm of the parasites. Our data suggest that acidic pH, close to 5.5-6.0, to allow the dissociation of receptor-mediated endocytosis in the epimastigotes the ligand-receptor complexes and of the lysosomal occurs through specialized, but non-clathrin-coated, enzymes from their receptors (Tycko et al. 1983; Fuchs portions of the plasma membrane, a phenomenon that et al. 1989); and (b) they should contain some lysosomal has also been observed in procyclic (invertebrate stage) enzymes, as it has been shown that some lysosomal trypomastigotes of Trypanosoma brucei (Langreth and proteinases can be also detected in endosomal struc- Balber, 1975), as well as in other eukaryotic cell types tures (Diment and Stahl, 1985; Roederer et al. 1987; (reviewed by Sandvig and Van Deurs, 1991). Coated Rodman et al. 1990). vesicles have been observed only in bloodstream In order to determine the pH of the reservosomes, we trypomastigote forms of the African trypanosome T. incubated the epimastigotes of T. cruzi in the presence brucei (Langreth and Balber, 1975; Webster and Grab, of DAMP, a weak base that accumulates in acidic 1988; Webster, 1989; Seyfang et al. 1990). A 77 kDa compartments and can be immunocytochemically protein has been isolated from such vesicles, but it did detected by antibodies against dinitrophenol (Ander- not cross-react with mammalian clathrin (Shapiro and son et al. 1984; Anderson and Pathak, 1985; Orci et al. Webster, 1989; Webster and Shapiro, 1990). Clathrin 1986; Anderson and Orci, 1988; Antoine et al. 1988; could also not be detected in trypomastigotes of T. Griffiths et al. 1988). Our observations showed a wide brucei using a chicken anti-clathrin antibody (unpub- range of variation in the concentration of DAMP in the lished results, quoted by Frevert and Reinwald, 1988). reservosomes, as evaluated by immunocytochemistry, It has been suggested that in the bloodstream trypomas- suggesting a heterogeneity of pH among different tigote forms of T. brucei a protein evolutionarily organelles. It has been shown that DAMP accumu- different from clathrin is involved in receptor-mediated lation is directly proportional to the H+ concentration endocytosis (Shapiro and Webster, 1989). and therefore the density of gold particles due to anti- In this work we used gold-labeled transferrin as a DNP IgG binding can be used to estimate the pH marker of the endocytic pathway in epimastigotes of T. (Anderson and Orci, 1988). Quantification of the cruzi- Transferrin was observed only in two cytoplasmic DAMP-associated gold particles showed that the compartments: in small uncoated transport vesicles and reservosomes have an internal pH of 6.0, which is in in the large reservosomes, found mostly at the posterior accordance with the values expected for a pre- end of the cells. Vesicles containing gold-labeled lysosomal compartment. These data suggest that the transferrin were occasionally observed near a tubular reservosomes contain a membrane-associated proton network found at the flagellar pocket region. It was not pump to maintain a low internal pH, thus allowing clear whether the vesicles were part of the network, ligand-receptor uncouphng after fusion of the vesicles which could then represent an early endosomal com- resulting from receptor-mediated endocytosis, as oc- partment, as was suggested for T. brucei (Webster, curs in endosomal compartments of a variety of cells 1989). These tubules are similar in appearance to the (Galloway et al. 1983; Mellman et al. 1986; Yamashiro collecting canals of the contractile vacuole already and Maxfield, 1987). described in other trypanosomatids (Brooker, 1971; In order to determine the presence of lysosomal Linder and Staehelin, 1979; Attias and De Souza, proteins, ultra-thin sections of Lowicryl-embedded 1985), and also to the tubular network involved in epimastigotes of T. cruzi were incubated with an anti- the exocytosis of the variable surface glycoproteins cysteine proteinase antibody. It has been shown that all of bloodstream trypomastigote forms of T. brucei stages of T. cruzi contain a major cysteine proteinase, (Duszenko et al. 1988; Frevert and Reinwald, 1988). whose activity is developmentally regulated, being 10- Detection of acid phosphatase activity in these tubules, fold higher in epimastigotes than in other stages as well as on the surface of the cells (Soares and De (Campettela et al. 1990; North et al. 1990). It is a high- 164 M. J. Soares and others

•r- *r»i.,

N v

I '• • •' '

FP 16 17

19 Pre-lysosomal compartments in T. cruzi 165

Figs 14 to 19. Lowicryl-embedded cells. cysteine proteinase antibody resulted in intense labeling Immunolocalization of antigens. Figs 14 to 17. Cells were of the reservosomes, suggesting that the enzyme is incubated for 60 minutes at 28°C with transferrin/15 nm transported to this compartment. This result, associated gold complexes and with 50 /an DAMP, fixed and with the low pH found inside this organelle, and the fact embedded in Lowicryl. Ultra-thin sections were incubated with mouse anti-DNP antibodies and then with rabbit anti- that reservosomes are accumulated in the cell cyto- mouse antibodies coupled to 5 nm gold particles. plasm during the early and mid log phase of growth in Fig. 14. DAMP accumulation occurred in the reservosomes the culture medium (Bretana and O'Daly, 1976; Paulin (R), located at the posterior end of the cell. Rare gold et al. 1983; Soares, unpublished observations), suggest particles could be found in the nucleus (N) or the that this organelle is a pre-lysosomal compartment, as kinetoplast (K). Fig. 15. DAMP (small gold particles) and denned for other eukaryotic cells (Griffiths et al. 1988; transferrin (large gold particles) could be colocalized in the Griffiths et al. 1990). Unlike what has been observed in reservosome. Fig. 16. No DAMP labeling could be found lysosomal enzymes of other eukaryotic cells, the in the flagellar pocket (FP), in the Golgi complex (G), or cysteine proteinase of T. cruzi presents no phosphoryl- in the tubular network (*) near the flagellar pocket. ated mannose residues (Cazzulo et al. 1990b), sugges- Fig. 17. DAMP could be immunolocalized in the ting that in this protozoon lysosomal enzymes are not reservosomes (R), but not in the transferrin-containing transport vesicles (arrows), which fuse with the targeted to the pre-lysosomal compartment (the reser- reservosomes. Figs 18 and 19. Cells were incubated for 60 vosome) through the mannose 6-phosphate receptor minutes at 28°C with transferrin-15 nm gold complexes, (M6PR) pathway. In addition, antibodies that recog- fixed and embedded in Lowicryl. Ultra-thin sections were nize the cation-independent M6PR, a marker of pre- incubated with rabbit anti-7! cruzi cysteine proteinase and lysosomal compartments (Griffiths et al. 1988; Griffiths then with goat anti-rabbit antibodies coupled to 5 nm gold et al. 1990), did not label the reservosomes. A mannose particles. Fig. 18. Small transport vesicles (large arrows) 6-phosphate-independent targeting of the proteinase containing transferrin (large gold particles) are seen passing cathepsin D to the lysosomes has also recently been along the nucleus (N), in the central region of the cell. reported (Rijboutt et al. 1991). These vesicles were poorly labeled with the anti-cysteine proteinase antibody (small arrow). Fig. 19. Posterior end Two additional observations support the view that of an epimastigote cell showing the colocalization of the reservosome is a pre-lysosomal, rather than a transferrin (large gold particles) and cysteine proteinase lysosomal, compartment: (a) no reaction product (small gold particles) in the reservosomes (R). The indicative of acid phosphatase activity could be local- reservosome in the lower left part of the micrograph shows ized in the reservosomes; and (b) no labeling of the high-density labeling for cysteine proteinase, but appears reservosome was observed when ultra-thin sections of not to contain transferrin. Bar, 0.25 /an. Lowicryl-embedded epimastigotes were incubated in the presence of antibodies that recognize lysosomal membrane proteins such as LAMP 1, LAMP 2 and lgp 120. However, we cannot exclude the possibility that Table 1. Quantification of gold particles/'\an2 after these antibodies, generated against mammalian lyso- incubation of the parasites in the presence of 50 piM somal proteins (Lewis et al. 1985; Mane et al. 1989), do DAMP not cross-react with equivalent proteins of protozoa. Number of In summary, our data suggest that in epimastigote Total ton2 gold particles forms of T. cruzi receptor-mediated endocytosis occurs measured counted Gold//m2 in specialized, but non-clathrin-coated, regions of the Cytoplasm 6.811 63 9.25 (4-20)* plasma membrane. Ingested proteins are accumulated Reservosomes 3.368 260 77.20 (37-224) in the reservosomes, which are acidic organelles containing cysteine proteinase. The cation-independent Cells were incubated for 60 minutes at 28°C with 50 /iM DAMP, mannose 6-phosphate receptor could not be immuno- fixed and embedded in Lowicryl. Ultra-thin sections were incubated with mouse anti-DNP antibodies and then with rabbit cytochemically detected in the membrane of the anti-mouse antibodies labeled with 5 nm gold particles. reservosomes, suggesting that lysosomal enzymes are *Numbers in parenthesis indicate the range of variation in the not targeted to these compartments via mannose 6- number of gold particles//zm2 in each of the measured cells. phosphate receptors. The lysosomal membrane proteins LAMP 1, LAMP 2 and lgp 120, as well as acid phosphatase, could not be detected in the reservo- mannose type of glycoprotein with a molecular mass of somes, thus suggesting that these organelles are pre- 60 kDa and a 65% homology with papain and cathepsin lysosomal compartments. Our data suggest some L (Cazzulo et al. 1989; Cazzulo et al. 1990b; Meirelles et similarities between the endocytic process in epimasti- al. 1990; Murta et al. 1990), being designated as gote forms of T. cruzi and the vesicle shuttle model cruzipain (Cazzulo et al. 1990a). Biochemical evidence proposed by Griffiths et al. (1988, 1990). However, first suggested that the cysteine proteinase of T. cruzi although a typical late endosomal, pre-lysosomal, was a lysosomal enzyme (Bontempi et al. 1989), but it compartment (the reservosome) could be well denned, was recently immunolocalized in elements of the no early endosomal structure could be clearly ident- endosomal-lysosomal system of epimastigotes (Murta ified. Our data suggest that uncoated transport vesicles et al. 1990; Souto-Padr6n et al. 1990). Our observations are shuttled directly from the plasma membrane to the show that incubation of the epimastigotes with the anti- reservosomes. 166 M. J. Soares and others

The authors thank Dr. R. G. W. Anderson, Dr. J. J. Geuze, H. J., Slot, J. W., Strous, G. J., Lodish, H. F. and Schwartz, Cazzulo, Dr. Gareth Griffiths and Dr. S. Kornfeld for A. L. (1983). Intracellular site of asialoglycoprotein receptor-ligand generously providing the antibodies. This has been supported uncoupling: double-label immunoelectron microscopy during by UNDP/World Bank/WHO Special Programme for Re- receptor-mediated endocytosis. Cell 32, 277-287. search and Training in Tropical Diseases, Conselho Nacional Geuze, H. J., Stoorvogel, W., Strous, G. J., Slot, J. W., Bleekemolen, J. and Mellman, I. (1988). Sorting of mannose 6-phosphate de Desenvolvimento Cientffico e Tecnol6gico (CNPq) e receptors and lysosomal membrane proteins in endocytic vesicles. Financiadora de Estudos e Projetos (FINEP). /. Cell Biol. 107, 2491-2501. Griffiths, G., Back, R. and Marsh, M. (1989). A quantitative analysis of the endocytic pathway in baby hamster kidney cells. J. Cell Biol. References 109, 2703-2720. Griffiths, G., Hoflack, B., Simons, K., Mellman, I. and Kornfeld, S. Anderson, R. G. W., Falck, J. R., Goldstein, J. L. and Brown, M. S. (1988). The mannose 6-phosphate receptor and the biogenesis of (1984). Visualization of acidic organelles in intact cells by electron lysosomes. Cell 52, 329-341. microscopy. Proc. Nat. Acad. Sci. U.S.A. 81, 4838-4842. Griffiths, G., Matteoni, R., Back, R. and Hoflack, B. (1990). Anderson, R. G. W. and Orel, L. (1988). A view of acidic intracellular Characterization of the cation-independent mannose 6-phosphate compartments. J. Cell Biol. 106, 539-543. receptor-enriched prelysosomal compartment in NRK cells. J. Cell Anderson, R. G. W. and Pathuk, R. K. (1985). Vesicles and dstemae Sci. 95, 441^61. in the trans Golgi apparatus of human flbroblasts are acidic Gruenberg, J., Griffiths, G. and Howel, K. E. (1989). compartments. Cell 40, 635-643. Characterization of the early endosome and putative endocytic Antolne, J. C, Jouanne, C, Ryter, A. and Benlchon, J. C. (1988). carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J. Leishmania amazonensis: acidic organelles in amastigotes. Exp. Cell Biol. 108, 1301-1316. ParasUol. 67, 287-300. Gruenberg, J. and Howel, K. E. (1989). Membrane traffic in Attias, M. and De Souza, W. (1985). Fine structure and cytochemistry endocytosis: insights from cell-free assays. Annu. Rev. Cell Biol. 5, of Phytomonas davidii. J. Submicrosc. Cytol. 17, 205-212. 453^81. Bendayan, M., Nanci, A. and Kan, F. W. K. (1987). Effect of tissue Hubbard, A. L. (1989). Endocytosis. Curr. Opin. Cell Biol. 1, 675- processing on colloidal gold cytochemistry. J. Histochem. 683. Cytochem. 35, 983-996. Kornfeld, S. and Mellman, I. (1989). The biogenesis of lysosomes. Bontempl, E., Martinez, J. and Cazzulo, J. J. (1989). Subcellular Annu. Rev. Cell Biol. 5, 482-525. localization of a cysteine proteinase from Trypanosoma cruzi. Mol. Krieger, M. A., Salles, J. M., Almeida, E., Linss, J., Bonaldo, M. C. Biochem. ParasUol. 33, 43-48. and Goldenberg, S. (1990). Expression and polymorphism of a Bretafia, A. and O'Daly, J. A. (1976). Uptake of fetal proteins by Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen. Trypanosoma cruzi. Immunofluorescence and ultrastructural Exp. Parasitol. 70, 247-254. studies. Int. J. Parasitol. 6, 379-386. Langreth, S. G. and Balber, A. E. (1975). Protein uptake and Brooker, B. E. (1971). The fine structure of Crithidia fasciculate with digestion in bloodstream and culture forms of Trypanosoma brucei. special reference to the organelles involved in the ingestion and J. Protozool. 22, 40-53. digestion of protein. Z. Zellforsch. mikrosk. Anat. 116, 532-563. Lewis, V., Green, S. A., Marsh, M., Vihko, P., Helenius, A. and Campetella, O., Martinez, J. and Cazzulo, J. J. (1990). A major Mellman, I. (1985). Glycoproteins of the lysosomal membrane. /. cysteine proteinase is developmentally regulated in Trypanosoma Cell Biol. 100, 1839-1847. cruzi. FEMS Microbiol. Lett. 67, 145-150. Under, J. C. and Staehelin, L. A. (1979). A novel model for fluid Cazzulo, J. J., Couso, R., Raimondi, A., Wemstedt, C. and Hellman. secretion by the trypanosomatid contractile vacuole apparatus. /. U. (1989). Further characterization and partial amino acid Cell Biol. 83, 371-382. sequence of a cysteine proteinase from Trypanosoma cruzi. Mol. Mane, S. M., MarzeUa, L., Bain ton, D. F., Holt, V. K., Cha, Y., Biochem. Parasitol. 33, 33^2. Hildreth, J. E. K. and August, J. T. (1989). Purification and Cazzulo, J. J., Franke, M. C. C, Martinez, J. and Franke de Cazullo, characterization of human lysosomal membrane glycoproteins. B. (1990a). Some kinetic properties of a cysteine proteinase Arch. Biochem. Biophys. 268, 360-378. (cruzipain) from Trypanosoma cruzi. Biochim. Biophys. Acta 1037, Matteoni, R. and Kreis, T. E. (1987). Translocation and clustering of 186-191. endosomes and lysosomes depends on microtubules. J. Cell Biol. Cazzulo, J. J., Hellman, U., Couso, R. and Parodi, A. J. A. (1990b). 105, 1253-1265. Amino acid and carbohydrate composition of a lysosomal cysteine Meirelles, M. N. L., Jullano, L., Carmona, E., Costa, E. M., SUva, S. proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues. Mol. Biochem. Parasitol. 38, 41-48. G., Lima, A. T. V. C, Amholdt, A. V., Leme, V. M. C, De Souza, W. (1984). Cell biology of Trypanosoma cruzi- Int. Rev. GuimarSes, E. S. P., Berro, O. J. and Scharfstein, J. (1990). Cytol. 86, 197-285. Functional and antigenic properties of the major cysteine De Souza, W., Carvalho, T. U. and Benchlmol, M. (1978). proteinase (GP57/51) of Trypanosoma cruzi. Mem. Inst. Oswaldo Trypanosoma cruzi: ultrastructural, cytochemical and freeze- Cruz 85, 533-538. fracture studies of protein uptake. Exp. Parasitol. 45, 101-115. Mellman, I., Fuchs, R. and Helenins, A. (1986). Acidification of the Diment, S. and Stahl, P. D. (1985). Macrophage endosomes contain endocytic and exocytic pathways. Annu. Rev. Biochem. 55, 663- proteases which degrade endocytosed protein ligands. /. Biol. 700. Chem. 260, 15311-15317. Mueller, S. C. and Hubbard, A. (1986). Receptor-mediated Duszenko, M., Ivanov, I. E., Ferguson, M. A. J., Plesken, H. and endocytosis of asialoglycoproteins by rat hepatocytes: receptor- Cross, G. A. M. (1988). Intracellular transport of a variant surface positive and receptor-negative endosomes. /. Cell Biol. 102, 932- glycoprotein in Trypanosoma brucei. J. Cell. Biol. 106, 77-86. 942. Frens, G. (1973). Controlled nucleation for the regulation of the Murta, A. C. M., Persechini, P. M., Souto-Padron, T., De Souza, W., particle size in monodisperse gold suspensions. Nature 241, 20-22. Guimaraes, J. A. and Scharfstein, J. (1990). Structural and Frevert, U. and Reinwald, E. (1988). Endocytosis and intracellular functional identification of GP57/51 antigen of Trypanosoma cruzi occurrence of the variant surface glycoprotein in Trypanosoma as a cysteine proteinase. Mol. Biochem. Parasitol. 43, 27-38. congolense. J. Ultrastruct. Mol. Struct. Res. 99, 137-149. North, M. J., Mottram, J. C. and Coombs, G. H. (1990). Cysteine Fuchs, R., Schmid, S. and Mellman, I. (1989). A possible role for proteinases of parasitic protozoa. Parasitol. Today 6, 270-275. sodium potassium-ATPase in regulating ATP-dependent endosome Orel, L., Ravazzola, M., Amherdt, M., Madsen, O., Perrelet, A., acidification. Proc. Nat. Acad. Sci. U.S.A. 86, 539-543. Vassal!, J. D. and Anderson, R. G. W. (1986). Conversion of Galloway, C. J., Dean, G. E., Marsh, M., Rudnick, G. and Mellman, proinsuhn to insulin occurs coordinately with acidification of I. (1983). Acidification of macrophage and fibroblast endocytic maturing secretory vesicles. J. Cell Biol. 103, 2273-2281. vesicles in vitro. Proc. Nat. Acad. Sci. U.S.A. 80, 3334-3338. Paulin, J. J. Jr, White, R. and Agosin, M. (1983). Ultrastructural Pre-lysosomal compartments in T. cruzi 167

modifications during the metabolism of metronidazole by trypanosomatids. A cytochemical and stereological study. J. Trypanosoma cruzi. J. Submicrosc. Cytol. 15, 951-964. Submicrosc. Cytol. Pathol. 20, 349-363. Rijnboutt, S., Aerts, H. M. F. G., Geuze, H. J., Tager, J. M. and Soares, M. J. and De Souza, W. (1991). Endocytosis of gold-labeled Strous, G. J. (1991). Mannose 6-phosphate-independent proteins and LDL by Trypanosoma cruzi. Parasitol. Res. 77, 461- membrane association of cathepsin D, glucocerebrosidase, and 469. sphingolipid-activating protein in HepG2 cells. /. Bio/. Chem. 266, Soares, M. J., Souto-Padr6n, T., Bonaldo, M., Goldenberg, S. and De 4862-4868. Souza, W. (1989). A stereological study of the differentiation Robinson, J. M. (1985). Improved localization of intracellular sites of process in Trypanosoma cruzi. Parasitol. Res. 75, 522-527. phosphatases using cerium and cell permeabilization. J. Histochem. Souto-Padron, T., Campctella, O. E., Cazzulo, J. J. and De Souza, W. Cytochem. 33, 749-754. (1990). Cysteine proteinase in Trypanosoma cruzi: Robinson, J. M. and Karnovsky, M. J. (1983). Ultrastructural immunocytochemical localization and involvement in parasite-host localization of several phosphatases with cerium. /. Histochem. cell interaction. J. Cell Sci. 96, 485-490. Cytochem. 31, 1197-1208. Tycko, B., Keith, C. H. and Maxfleld, F. R. (1983). Rapid Rodman, J. S., Levy, M. A., Dlment, S. and Stahl, P. D. (1990). acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line. J. Cell Biol. 97, 1762-1776. Immunolocalization of endosomal cathepsin D in rabbit alveolar Warren, L. (1960). Metabolism of Schyzotrypanum cruzi Chagas. I. macrophages. J. Leuk. Biol. 48, 116-122. Effect of culture age and substrate concentration on respiratory Roederer, M., Bowser, R. and Murphy, R. F. (1987). Kinetics and rate. J. Parasitol. 46, 529-539. temperature-dependence of exposure of endocytosed material to Webster, P. (1989). Endocytosis by African trypanosomes: I. Three- proteolytic enzyme and low pH: evidence for a maturation model dimensional structure of the endocytic organelles in Trypanosoma for the formation of lysosomes. J. Cell. Physiol. 131, 200-209. brucei and Trypanosoma congolense. Eur. J. Cell Biol. 49, 295-302. Sandvig, K. and Van Deurs, B. (1991). Endocytosis without clathrin Webster, P. and Grab, D. J. (1988). Intracellular colocalization of (a minireview). Cell Biol. Int. Rep. 15, 3-6. variant surface glycoprotein and transferrin-gold in Trypanosoma Schmld, S. L., Fuchs, R., Male, P. and Mellraan, I. (1988). Two brucei. J. Cell Biol. 106, 279-288. distinct sub-populations of endosomes involved in membrane Webster, P. and Shapiro, S. Z. (1990). Trypanosoma brucei: a recycling and transport to lysosomes. Cell 52, 73-83. membrane-associated protein in coated endocytotic vesicles. Exp. Seyfang, A., Mecke, D. and Duszenko, M. (1990). Degradation, Parasitol. 70, 154-163. recycling, and shedding of Trypanosoma brucei variant surface Yamashlro, D. J. and Maxfield, F. R. (1987). Acidification of glycoprotein. J. Protozool. 37, 546-552. morphologically distinct endosomes in mutant and wild-type Shapiro, S. and Webster, P. (1989). Coated vesicles from the Chinese hamster ovary cells. J. Cell Biol. 105, 2723-2733. protozoan parasite Trypanosoma brucei: purification and characterization. /. Protozool. 36, 344-349. (Received 14 October 1991 - Accepted, in revised form, Soares, M. J. and De Souza, W. (1988). Cytoplasmic organelles of 29 January 1992)