THE CELL SURFACE OF TRYPANOSOMA CRUZI: CYTOCHEMISTRY AND FREEZE-FRACTURE

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1 J. Cell Sci. 33, (1978) 285 Printed in Great Britain Company of Biologists Limited 197S THE CELL SURFACE OF TRYPANOSOMA CRUZI: CYTOCHEMISTRY AND FREEZE-FRACTURE W. DE SOUZA,* A. MARTfNEZ-PALOMOf AND A. GONZALEZ-ROBLESf *Instituto de Biofisica, Universidade Federal do Rio de Janeiro, , Rio de Janeiro, Brazil, and fdepartamento de Biologia Celular, Centra de Investigacidn del IPN, Apartado Postal , Mexico, D.F. SUMMARY The ultrastructure of epimastigotes of Trypanosoma cruzi, obtained from, accllular cultures, and bloodstream, trypomastigotes, isolated from infected mice, were studied by thin-sectioning and freeze-fracturing techniques. Epimastigotes showed a thin (5 nm) surface coat when stained with, ruthenium red, while the surface coat of trypomastigotes was more prominent (15 nm thick). Both P and E faces of the plasma membrane of T. cruzi had roughly the same number of intramembranous particles (IMP) as seen by freeze-fracture. The plasma membrane of bloodstream trypomastigotes had less IMP than epimastigotes. Several differentiations of the plasma membrane were observed. In epimastigotes a cytostome appears as a particle-poor region delimited by a pallisade-like row of adjacent IMP. Bloodstream trypomastigotes did not have a cytostome. Instead, abundant pinocytic vesicles were observed. At the base of the flagellum of epimastigotes a ciliary necklace was found. At this region, the surface coat was differentiated as long, hair-like projections after staining with ruthenium red. The flagellar membrane had less IMP than the body membrane. Clusters of IMP were present on both faces of the flagellar membrane at the flagellar-body adhesion zone of epimastigotes. Linear arrays of IMP were also seen. In bloodstream trypomastigotes clusters of particles were observed both on the flagellar and cell body membranes. Our observations demonstrate the presence of considerable structural variations of the T. cruzi plasma membrane at the two stages of the life cycle studied. INTRODUCTION One approach to an understanding of the host-parasite interactions present in Chagas' disease is the study of the outer membrane of the causative agent, the protozoon Trypanosoma cruzi. A comparison between the surface of the invasive trypomastigote form with that of the non-invasive stage, the epimastigote, could give some information on the factors that determine the invasive behaviour of the parasite towards host cells. Previous studies on the cell surface of T. cruzi carried out by us have centred on the detection of specific carbohydrate components by means of lectin-binding studies (Chiari, De Souza, Romanha, Chiari & Brener, 1978), the measurement of total surface charge estimated both as binding of cationized ferritin and electrophoretic mobility (De Souza et al. 1977) and the study of the plasma membrane of epimastigote forms by ultrastructural cytochemistry, and electron microscopy of freeze-fracture replicas (Martinez-Palomo, De Souza & Gonzalez-Robles, 1976). * For all correspondence. 19 C E I. 33

2 286 W. De Souza, A. Martinez-Palomo and A, Gonzdlez-Robles The introduction of the freeze-fracture technique has increased our understanding of the structure and organization of animal membranes in general, and of various human parasites, in particular, such as Trypanosoma brucei (Smith, Njogu, Cayer & Jarlfors, 1974; Hogan & Patton, 1976), Entamoeba histolytica (Pinto da Silva, Martinez- Palomo & Gonzalez-Robles, 1975; Martinez-Palomo, Pinto da Silva & Chavez, 1976) and Onchocerca volvulus (Martinez-Palomo, 1978). We have now applied the freeze-fracture technique to study the fine structure of the plasma membrane of both invasive blood forms and non-invasive culture forms of T. cruzi. The parasite plasma membrane was found to present considerable variations at these 2 stages of the life cycle. MATERIALS AND METHODS Microorganisms Trypanosoma cruzi, strain Y, was cultivated in. LIT (liver infusion-tryptosc) medium (Camargo, 1964) for 4 days at 28 C. Under these conditions only epimastigote forms were obtained. Bloodstream trypomastigote forms were obtained from mice infected with the Y strain. The blood was collected in the presence of sodium citrate and centrifuged at 50 g for 10 min at 4 C. The pellet was discarded. The supernatant fluid, which contained the trypanosomes, was collected and centrifuged at 800 g for 15 min at 4 C. The pellet obtained was washed twice in cold 001 M phosphate-buffered 0-15 M saline (PBS), ph 72. Electron microscopy Cells were fixed in glutaraldehyde 25 % ( v / v ) in o-i M cacodylate buffer, ph 72, for 1 h at room temperature. After a rinse in buffer the cells were postfixed in 1 % (v/v) OsO. t in. O'i M cacodylate buffer, ph 72, for 1 h at room temperature. Then they were dehydrated through a graded ethanol series and embedded in Epon. Ruthenium red After fixation in glutaraldehyde the cells were washed twice in cacodylate buffer and postfixed in 1 % OsOi in o-i M cacodylate buffer + o-s mg/ml ruthenium red for 1 h in the dark at room temperature (Luft, 1971). Freeze-fracture Cells fixed in glutaraldehyde were washed twice in o-i M cacodylate buffer, ph 72, and gradually impregnated during 30 min with glycerol in cacodylate buffer up to 20 % concentration, where they were left for about 60 min at room temperature. They were then mounted on Balzer's support disks and rapidly frozen in the liquid phase of partially solidified Freon 22, Fig. 1. Epimastigote form of T. cruzi treated with ruthenium red. The surface coat of the flagellum (/) shows a local modification in the form of a hairy coat (arrows), k, kinetoplast. x Fig. 2. Cell surface of T. cruzi showing the uniform disposition of subpellicular microtubules. x Fig. 3. The cytostome (c) found in epimastigotes appears as a funnel-shaped depression covered by a ruthenium red-positive coat, x Fig. 4. Region of adhesion between the cell body (cb) and the flagellum (/) of epimastigotes. At the cytoplasmic side of the body surface, macular densities are seen (arrowheads), x

3 Cell surface of T. cruzi 2S7 \ \ X «5 1 \ > > ', -»!.. t 19-2

4 288 W. De Souza, A. Martinez-Palomo and A. Gonzalez-Robles

5 Cell surface of T. cruzi 289 cooled by liquid nitrogen, and stored in liquid nitrogen until used. Freeze-fracture was carried out at 115 C in a Balzer's 300 apparatus equipped with a turbomolecular pump. Replicas were produced by evaporation from a platinum-carbon source. The specimens were shadowed at 267 x io~' N m~ 2 within 2 s of fracturing and the knife edge positioned under conditions that minimize contamination. Replicas were recovered in distilled water, cleaned with Clorox and distilled water and mounted on 200-mesh grids coated with Formvar. Thin sections and replicas were studied with an EM9S-2 or an EM 10 Zeiss electron microscope. Micrographs are mounted with the shadow direction from bottom to top except when indicated otherwise. Shadows are white. RESULTS Epimastigote forms As a reference for interpretation of the freeze-fracture replicas, the general aspect of the epimastigote form of Trypanosoma cruzi in ultrathin sections, is shown in Figs The periplast is composed of a plasma membrane 8-10 nm thick, covering the cell body, the flagellum, and lining the flagellar pocket region. About 8 nm below the plasma membrane there is a single layer of microtubules in parallel array, arranged along the major axis of the cell (Fig. 2). With standard techniques, no surface coat can be detected on the plasma membrane (Fig. 2). However, ruthenium red staining reveals a 5-nm-thick surface coat (Fig. 1). In the region of the cytostome, a local differentiation of the cell surface and the plasma membrane has been described by us previously (Martinez-Palomo et al. 1976). The cytostome appears as a funnel-shaped depression lined by the cell membrane, close to the region where the Golgi complex is located (Fig. 3). At the junctions between body and flagellum faint macular densities can be seen only on the cytoplasmic side of the body surface membrane (Fig. 4). Trypanosomes studied by freeze-fracture replication may cleave through the cytoplasm (Figs. 5, 6), or through the hydrophobic region of the plasma membrane, as in other cells (Pinto da Silva & Branton, 1970), exposing large surfaces of the inner or outer membranes halves (Fig. 7). An overall picture of the cytoplasm of T. cruzi as seen in replicas obtained by freeze-fracture is shown in Fig. 5. The nucleus, the kinetoplast, the Golgi complex and the emergence of the flagellum are evident. In favourable fractures, the nuclear membrane can be seen with its characteristic pores (Fig. 6). At low magnifications, the plasma membrane of epimastigotes shows a uniform distribution of intraniembranous particles (IMP), except at the cytostome and the flagellar regions, where IMP are either sparse or absent (Fig. 7). Contrary to what has been found in most cells studied with the freeze-fracture technique, the difference between the number of IMP on the P face (which represents Figs General aspect of the epimastigote form of T. cruzi as seen by freezefracture. In Figs. 5 and 6 the cells were cleaved through the cytoplasm showing the nucleus (n) with its pores, the kinetoplast (k), Golgi complex (g) and the emergence of the flagellum (/). In Fig. 7 a large area of the cell body and the flagellar membrane is shown. Intramembranous particles are sparse or absent in the flagellar membrane (/) and cytostome (c). Figs. 5 and 7, x ; Fig. 6, x

6 290 W. De Souza, A. Martmez-Palomo and A. Gonzdlez-Robles the outer aspect of the inner membrane half) and IMP on the E face (which corresponds to the inner aspect of the outer membrane half) is not large. Estimations of the number of IMP per square micrometre of membrane area reveal a particle density of i83o//tm 2 on the P face and i45o//im 2 on the E face. IMP on both faces of T. cruzi plasma membrane are very heterogeneous, both in size and shape, as shown in Fig. 8. The flagellar membrane differs in structure when compared with the membrane of the cell body. In general, IMP are absent except in a few regions of the flagellar membrane (Fig. 9) where IMP are arranged in clusters spaced at irregular intervals. Such clusters of IMP are observed both on P and E faces of the flagellar membrane (Figs. 9, 10), but are lacking on P or E faces of the cell body plasma membrane. A second specialization of the flagellar membrane, associated with the region of attachment to the cell body, appears as a linear array of closely adjacent IMP located in the portion of the flagellum where it emerges from the base (Fig. 11). The linear arrays are found both in E and P faces of the flagellum, but are absent from the plasma membrane of the cell body. A third specialization of the flagellum is formed by circular concentrations of IMP at the base of theflagellum,forming 5-6 circular rows (Fig. 12). At this site the surface coat of the flagellum shows in thin sections a local modification in the form of a fuzzy collar (Fig. 1). Bloodstream trypomastigote forms The ultrastructure of the bloodstream trypomastigote forms of T. cruzi as seen in sections has been described previously (Maria, Tafuri & Brener, 1972). The fine structure is basically similar to that found in epimastigotes. However, various differences can be demonstrated: (a) the nuclei are more elongated; and (b) the kinetoplast appears as a spherical body, localized at one tip (called the posterior tip), and showing a different pattern in the array of the DNA-containing fibrils. The cytostome found in epimastigotes (Martinez-Palomo et al. 1976) was not detected in bloodstream forms of T. cruzi. In sections of trypomastigotes stained with ruthenium red, a prominent ruthenium red-positive surface coat is found (Fig. 13). At the zone of adhesion between body and flagellum, adjacent membranes run parallel and the converging surface coats seem to overlap, giving rise to a median dense line (Fig. 13). Observations of freeze-fracture replicas of plasma membranes reveal the presence of intramembranous particles in both E and P faces (Fig. 14). As found in epimastigotes, there is little difference in the number of IMP on the P face and E face. However, both faces show many fewer particles than the cell membrane of epimastigote forms. The average number of IMP per/tm 2 of cell membrane is 122 on P faces and 126 on E faces. Therefore, the plasma membrane of bloodstream forms roughly contains 10 times fewer IMP than the surface membrane of culture forms. The absence of a cytostome in bloodstream forms of T. cruzi was confirmed in freeze-fracture replicas. Instead, abundant pinocytotic vesicles are found on the plasma membrane (Fig. 14). As in epimastigotes, the flagellar membrane of trypomastigotes is almost devoid of IMP both on P and E faces, except at the region of contact with the cell body, where

7 Cell surface of T. cnisi Fig. 8. P (P) and E (E) faces of the cell membrane of two epimastigotes. The number of intramembranous particles in the two faces is roughly similar, x Fig. 9. Particles arranged in clusters (arrowheads) are seen in the flagellar membrane of epimastigotes. x

8 2Q2 W. De Sousa, A. Martinez-Palomo and. A. Gonzdlez-Robles Fig. 10. Micrography showing the difference in the number of particles between the cell body (cb) and the flagellar (/) membrane, x Fig. 11. Specialization of the flagellar membrane of epimastigote in the region of attachment of the flagellum to the body. Particles in linear array are observed, x Fig, 12. Ciliary necklace localized at the base of the flagellum (arrows), x

9 - *, Cell surface of T. cruzi 293 rows of IMP clusters are found. The flagellum is located over a specialized region of the cell body membrane which appears as a regular pit (Fig. 15). Linear arrays of IMP clusters are not limited to E and P faces of the flagellar membrane; they are also present on both fracture faces of the cell body membrane at the region of attachment of the flagellum (Figs. 15, 16). Furthermore, in replicas where the fracture jumps from the P face of the flagellar membrane to the E face of the cell body membrane, and vice versa, the rows of IMP on adjacent membranes are found to be in register (Fig. 15).! > " > Fig. 13. Thin section of bloodstream trypomastigote stained with ruthenium red. A prominent surface coat is evident. The stain penetrates at the zone of adhesion (arrow) between the body (cb) and the flagellum. (/). x DISCUSSION The fine structure of trypanosomatids has been adequately studied by electron microscopy using ultrathin sections. Although this technique gives important information concerning the organization and structure of cellular components, it does not permit detailed examination of the structure and organization of cell membranes. In contrast, in replicas of freeze-fractured specimens, the inner components of the cell membrane are exposed permitting examination of the outer aspect of the inner

10 W. De Sonza, A. Martinez-Palomo and A. Gonzdlez-Robles 14 Fig. 14. E (E) and P (P) faces of the cell membrane of bloodstream trypomastigotes, showing pinocytotic vesicles (arrowheads), x

11 Cell surface of T. cruzi 295 Figs. 15, 16. Region of attachment of the flagellum to the body of a bloodstream trypomastigote. Linear clusters of particles (arrowheads) are seen both in the cell body (cb) and flagellar membrane. Fig. 15, x ; Fig. 16, x

12 296 W. De Souza, A. Martinez-Palomo and A. Gonzdlez-Rohles membrane half (P face) and the inner aspect of the outer membrane half (E face) (Pinto da Silva & Branton, 1970; Branton et al. 1975). In most cells studied, fracture face P exhibits distinctly larger density of IMP than that observed on fracture face E. Usually IMP are randomly distributed over the cell membrane. IMP are assumed to represent integral proteins located in the hydrophobic domain of the membrane lipid bilayer. A special pattern in the arrangement of the particles is usually interpreted as a functionally specialized region of the cell membrane. Our results show that the plasma membrane of T. cruziis structurally more complex when compared with metazoan cell membranes such as those of fibroblasts, erythrocytes, etc. In contrast to other membranes, the number of IMP in both inner faces of the T. cruzi plasma membrane is roughly similar. Furthermore, differentiations of IMP distribution are characteristic of the epimastigote and trypomastigote stages of T. cruzi cell cycle. One of the specialized membrane regions of T. cruzi is the cytostome, found only in epimastigote forms. This structure plays an important role in the nutrition of epimastigote and intracellular spheromastigote forms. In the latter stage it participates in a process of intracellular phagotrophy (Meyer & De Souza, 1973). In freezc-fracture, the cytostome appears as a particle-poor region delimited by a pallisade-like row of adjacent IMP localized close to the flagellar pocket (Martinez-Palomo et al. 1976). In bloodstream trypomastigote forms, the cytostome is not present. Instead, a large number of pinocytic vesicles were observed. This fact can be understood in terms of the life cycle of T. cruzi. Intracellular spheromastigote forms always possess a cytostome. Trypomastigote forms, however, live in the blood of the vertebrate host and take nutrients from the blood by diffusion or pinocytosis. The presence of the cytostome in the epimastigote forms can be explained by the fact that they represent an intermediate stage between sphero- and trypomastigotes. A cytostome has also been found in epimastigotes of T. mega, T. conorhini, and T. raiae (Steinert & Novikoff, i960; Brooker & Preston, 1967; Milder & Deane, 1969), in choanomomastigotes of Critliidia fasciculata (Brooker, 1971) and in promastigotes of Herpetomonas samuelpessoai (De Souza et al. 1976). In salivarian trypanosomatids, cytostomes were not found in bloodstream forms of T. vivax, T. equinum and congolense, or in any of the developmental stages of T. brucei (Brown, Armstrong & Valentine, 1963; Vickerman, 1969a; Langreth & Balber, 1975). Another specialized region of the T. cruzi plasma membrane is located on the flagellum. At the base of the flagellum of epimastigotes, we have found a collar-like array of IMP similar to the ciliary necklace found in other flagella and cilia (Gilula & Satir, 1972; Linder & Staehelin, 1977). It has been suggested that the necklace may be involved in the control of local membrane permeability and therefore this region could represent an energy-transducing zone of the flagellum. The specialized region of T. en/.s7 trypanosomes is remarkable because the membrane rows of IMP appear to be related to a localized differentiation of the surface coat at the base of the flagellum in the form of hair-like surface projections. The paucity of IMP on the flagellar membranes of both epi- and trypomastigote forms has also been observed in Critliidia fasciculata (Easterbrook, 1971) and T. brucei

13 Cell surface of T. cruzi 297 (Smith et al. 1974; Hogan & Patton, 1976). These results suggest that, although the flagellar membrane is in continuity with the membrane of the cell body, it may have a different composition, particularly in terms of protein components. It is well known that the flagellum of the epi- and trypomastigote forms runs attached to the cell body from its point of origin, ending in a free flagellum. There are contacts between the flagellum and the cell membrane forming areas of specialized junctions. This region has been extensively studied in ultrathin sections and by freeze-fracture in T. brucei (Vickerman, 19696; Smith et al. 1974; Hogan & Patton, 1976). In T. cruzi the small desmosome-like junctions as seen in sections are not as clear as those reported for T. brucei. In this last trypanosomatid the presence of clusters of membrane particles spaced at intervals of nm, formed by groups of up to 6 particles was described as representing 'the only obvious specialization of the fractured surfaces of the cell body or flagellum that can be related to the junctional complexes' (Smith et al. 1974). The junctions show specialized structures on the P face of the flagellar membrane and not on the general cell membrane at the junctional area. However, in bloodstream trypomastigote forms of T. cruzi we observed regions of the cell membrane which show a special pattern in the organization of membrane particles on both E and P faces, as well as on both faces of theflagellarmembrane. The presence of IMP on both faces of flagellar and body membrane tends to confirm the suggestion that the rows of particles represent fractured protein components involved in the adhesion of the flagellum to the cell body. This assertion is supported by the fact that the IMP rows are found in register where the fracture plane deviates from the flagellum to the body plasma membrane. The cell membrane of bloodstream trypomastigote forms of T. cruzi present fewer membrane particles than epimastigote forms. Studies of the chemical nature of membrane particles in other membrane systems has demonstrated that the particles represent, in general, membrane-intercalated proteins of the integral type. It has been suggested that the frequency of the particles may be related to the physiological activity of the cell membrane. Other studies have shown the existence of some differences in the cell surface of epi- and trypomastigote forms of T. cruzi. Examination of net surface charge of epimastigotes from acellular cultures and bloodstream trypomastigotcs show that trypomastigote forms exhibit a much higher electrophoretic mobility and a more intense binding of cationized ferritin than epimastigote forms. Studies of mixed populations in acellular cultures demonstrate that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi (De Souza et al. 1977). Another difference is related to the susceptibility of T. cruzi to lysis induced by mammalian serum (Nogueira, Bianco & Cohn, 1975): while epimastigote forms are lysed, the trypomastigote forms remain unaltered. An interesting feature of the bloodstream forms of T. cruzi is the presence of a surface coat covering the entire plasma membrane. In T. brucei (Vickerman, 19696) the surface coat contains the variant antigens demonstrated by agglutinating reaction. Another characteristic of the surface coat of salivarian trypanosomes is its mobility in the form of streamers that are shed into the blood, probably accounting for the exoantigen of immune serum. The surface coat may contain also absorbed host serum

14 298 W. De Sonza, A. Martinez-Palomo and A. Gonzdlez-Robles proteins. The surface coat of salivarian components is of importance for the understanding of host-parasite relationships, because antigenic variation of coat antigens, shedding of antigenic components, and adsorption of host serum proteins may all represent effective means by which the protozoon may avoid the immune response of the host. In turn, the nature of the coat found in T. cruzi bloodstream forms is not known, except for the fact that carbohydrates are present (De Souza & Meyer, 1975). The coat is barely visible in culture forms of T. cruzi, where only specific cytochemical techniques selective for carbohydrates reveal a thin (5 nm) surface coat. The surface coat of bloodstream forms of T. cruzi was found to be more prominent than in culture forms, although less so than in T. brucei (Vickerman, 19696). In view of the considerable significance of the surface coat of salivarian trypanosomes, the elucidation of the nature and function of the surface coat of T. cruzi might be highly relevant to an understanding of the complex host-parasite interaction present in Chagas' disease. This research was supported in part by grants from. Bayer Laboratories, Mexico City, given to W.S. and A.M.P. through the National Academy of Medicine, Mexico, from the National Research Council of Brazil, to W.S., and from CONACYT, Mexico to A.M.P. REFERENCES BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., MUHLETHALER, K., NORTHCOTE, D. H., PACKER, L., SATIR, P., SATIR, B., SPETH, V., STAEHELIN, L. A., STEERE, R. L. & WEINSTEIN, R. S. (1975). Freeze-etching nomenclature. Science, N.Y. 190, BROOKER, B. E. (1971). The fine structure of Crithidia fasciculata with special reference to the organelles involved in the ingestion and digestion of proteins. Z. Zellforsch. mikrosk. Anat. 116, BROOKER, B. E. & PRESTON, T. M. (1967). The cytostome in trypanosomes and allied flagellates. J. Protozool. 14, Suppl., 41. BROWN, K. N., ARMSTRONG, J. A. & VALENTINE, R. C. (1963). The ingestion of protein molecules by blood forms of Trypanosoma rhodesiense. Expl Cell Res. 39, CAMARGO, E. P. (1964). Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev. Inst. Med. Trop. Sao Paulo 6, CHIARI, E., DE SOUZA, W., ROMANHA, A, J. CHIARI, C. A. & BRENER, Z. (1978). Concanavalin A receptors on the cell membrane of Trypanosoma cruzi. Ada trop. (in Press). DE SOUZA, W., ARCUELLO, C, MARTINEZ-PALOMO, A., TRISSL, D., GONZALEZ-ROBLES, A. & CHIARI, E. (1977). Surface charge of Trypanosoma crust. Binding of cationized ferritin and measurement of cellular electrophoretic mobility. J. Protozool. 24, DE SOUZA, W. & MEYER, H. (1975). An electron microscopic and cytochemical study of the cell coat of Trypanosoma cruzi in tissue cultures. Z. Parasitenk. 46, DE SOUZA, W., ROSSI, M. A., KITAJIMA, E. W., SANTOS, R. R. & ROITMAN, I. (1976). An electron microscopic study of Herpetomonas sp. (Leptomonas pessoai). Can. J. Microbiol. 22, ^ EASTERBROOK, K. B. (1971). The ultrastructure of Crithidia fasciculata. A freeze-etching study. Can. J. Microbiol. 17, GILULA, N. & SATIR, P. (1972). The ciliary necklace. A ciliary membrane specialization, jf. Cell Biol. S3, 494-5O9- HOGAN, J. C. & PATTON, C. L. (1976). Variation in intramembrane components of Trypanosoma brucei from intact and X-irradiated rats. A freeze-cleave study. J. Protozool. 23, LANGRETH, S. G. & BALBER, A. E. (1975). Protein uptake and digestion in bloodstream and culture forms of Trypanosoma brucei. J. Protozool. 22, LINDER, J. C. & STAEHELIN, L. A. (1977). Plasma membrane specializations in a trypanosomatid flagellate. J. Ultrastruct. Res. 60,

15 Cell surface of T. cruzi 299 LUFT, J. H. (1971). Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Artat. Rec. 171, MARIA, T. A., TAFURI, W. & BRENER, Z. (1972). The fine structure of different bloodstream forms of Trypanosoma cruzi. Ann. trop. Med. Parasit. 66, MARTINEZ-PALOMO, A. (1978). Ultrastructural characterization of the cuticule of Onchocerca volvus microfilaria. J. Parasit. 64, (in Press). MARTINEZ-PALOMO, A., PINTO DA SILVA, P. & CHAVEZ, B. (1976). Membrane structure of Entamoeba histolytica: Fine structure of freeze-fractured membranes. J. Ultrastruct. Res. 54, MARTINEZ-PALOMO, A., DE SOUZA, W. & GONZALEZ-ROBLES, A. (1976). 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. 69, 5O MEYER, H. & DE SOUZA, W. (1973). On the fine structure of Trypanosoma cruzi in tissue cultures of pigment epithelium from the chick embryo. Uptake of melanin granules by the parasite..7. Protozool. 20, MILDER, R. & DEANE, M. P. (1969). The cytostome of Trypanosoma cruzi and T. conorhini. J. Protozool. 16, 730^737. NOGUEIRA, N., BIANCO, C. & COHN, Z. (1975). Studies on the selective lysis and purification of Trypanosoma cruzi. jf. exp. Med. 142, PINTO DA SILVA, P. & BRANTON, D. (1970). Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker. J. Cell Biol. 45, PINTO DA SILVA, P., MARTINEZ-PALOMO, A. & GONZALEZ-ROBLES, A. (1975). Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate. J. Cell Biol. 64, SMITH, D. S., NJOGU, A. R., CAYER, M. & JARLFORS, U. (1974). Observations of freezefractured membranes of a trypanosome. Tissue & Cell 6, STEINERT, M. & NOVIKOFF, A. B. (i960). The existence of a cytostome and the occurrence of pinocytosis in the trypanosome, Trypanosoma mega. J. Cell Biol. 8, ' VICKERMAN, K. (1969a). The fine structure of Trypanosoma congolense in its bloodstream phase. jf. Protozool. 16, VICKERMAN, K. (19696). On the surface coat and flagellar adhesion in trypanosomes. J. Cell Sci. 5, {Received 30 January 1978)

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