Tritrichomonas foetus: Ultrastructural localization of calcium in the plasma membrane and in the hydrogenosome

EXPERIMENTAL PARASITOLOGY 54,211-284(1982)

Tritrichomonas foetus: Ultrastructural Localization of Calcium Plasma Membrane and in the Hydrogenosome MARLENE

BENCHIMOL,

Znstituto de Biofisica.

CEZAR ANTONIO

ELIAS,

AND WANDERLEY

Universidnde Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil (Accepted

for publication

20 March

in the

DE SOUZA’

Z/ha do Fundrio, 21910,

1981)

BENCHIMOL, M., ELIAS, C. A., AND DE SOUZA, W. 1982. Tritrichomonas foetus: Ultrastructural localization of calcium in the plasma membrane and in the hydrogenosome. Experimental Parasitology 54, 277-284. The osmium tetroxide-potassium pyroantimonate technique was used to localize Ca2+ -containing sites in the protozoan Tritrichomonas foetus. Reaction product was seen in association with the plasma membrane and with a membranebound organelle, the hydrogenosome. Reaction product was also seen in some cytoplasmic vesicles and in lysosomes. Treatment of the ultrathin sections with EGTA resulted in removal of the pyroantimonate precipitate. These results suggest that the hydrogenosome may be involved in the control of the intracellular concentration of CaZ+ in T. foetus. INDEX DESCRIPTORS: Tritrichomonas foetus; Protozoa, parasitic; Hydrogenosome; Plasma membrane; Ultrastructural cytochemistry; Intracellular calcium.

of the formation of molecular hydrogen (Lindmark and Muller 1973; Muller and Calcium ions (Ca”‘) play an essential role Lindmark 1978; Cerkasov et al. 1978). Miin many biological processes such as tochondria and hydrogenosomes, however, flagellar or ciliary activity (Plattner 197.5), have some common biochemical properties muscular contraction (Katz and Miledi such as high content of cardiolipin, circular 1968), depolarization-secretion coupling DNA, and a cyanide-insensitive superoxide (Baker er al. 1971), etc. The concentration dismutase (Cerkasov et al. 1978). of free Ca2+ in the cytoplasm regulates a In the study being reported we tried to large number of biological phenomena. determine, by using ultrastructural cytoTherefore, the cell has some mechanisms to chemistry, the sites of accumulation of calcontrol the concentration of free Ca’+. cium in Tritrichomonus foetus. Among these mechanisms, the most important is the uptake and/or binding of Ca*+ MATERIALS AND METHODS by the mitochondria, the endoplasmic reMicroorganism. Tritrichomonus foetus was ticulum, and the plasma membrane (De isolated by Dr. H. Guida (EMBRAPA, Rio de Aralijo Jorge et al. 1979; Lehninger et ul. Janeiro, Brazil) from the urogenital tract of 1978; Carafoli and Crompton 1978). a bull in Rio de Janeiro, and has been Protozoans of the family Trichomonadi- maintained in a medium of the following dae are facultative anaerobic parasites in composition (g/liter): liver hydrolisate, which mitochondria are absent. However, 2.5.O; D-(+)-glucose, 5 .O; sodium chloride, they have a membrane-bound organelle, the 6.5; agar-agar, 0.5; and 10% of inactivated hydrogenosome, which has some enzymes and filter-sterilized fresh bovine serum. The that participate in the metabolism of pyru- cells were cultivated for 24 hr at 36.5 C vate formed in glycolysis and is the site which corresponds to the end of the logarithmic growth phase. INTRODUCTION

1 To whom all correspondence

Electron

should be addressed.

microscopy

and cytochemistry.

211 0014-4X94/82/060277-08$02.00/0 Copyright All rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

278

BENCHIMOL,ELIAS,

The cells were collected by centrifugation (1OOOgfor 10 mm), washed with 0.1 M Nacacodylate buffer, pH 7.2, and fixed in 2.5% glutaraldehyde in cacodylate buffer for 2 hr at room temperature. After fixation, they were washed in cacodylate buffer, postfixed with 1% Os04, dehydrated in acetone, and embedded in Epon. For localization of Ca’+-containing sites, the cells were fixed in glutaraldehyde as described above, washed twice with 0.1 M cacodylate buffer and twice with 0.1 M potassium phosphate buffer, and postfixed with 1% osmium tetroxide and 2% potassium pyroantimonate for 1 hr at 4 C. The latter solution was prepared as follows: potassium pyroantimonate (Merck) was dissolved in distilled water in order to get a 5% solution which was heated to boiling and then cooled. The original volume was restored and the solution was then filtered to remove pyroantimonate which might have reprecipitated (Komnick and Komnick 1963; Herman et al. 1973; Weakely 1979; Simson and Spicer 1975; Appleton and Morris 1979; Debas et al. 1975). By adding 1 ml of 2% aqueous osmium tetroxide, 1 ml of 5% potassium pyroantimonate, and 0.5 ml of 0.1 M potassium phosphate, the final concentrations were 1 and 2% for the 0~0~ and pyroantimonate, respectively. The pH of this mixture was adjusted to 7.5-7.8 with 0.1 N acetic acid in order to achieve efficient precipitation and to maintain a physiologic pH during fixation. After the OsO,-pyroantimonate fixation, the cells were washed twice in potassium phosphate buffer, dehydrated in acetone, and embedded in Epon. Ultrathin sections were obtained with a LKB Ultratome III, collected on copper grids, stained with many1 acetate and lead citrate, and observed in an AEI EMdB electron microscope. In order to control the specificity of the technique, thin sections of cells treated with the Os04pyroantimonate solution were exposed for 20-30 min at 60 C in 5 mM ethylene glycol

AND

DE

SOUZA

bis(P-aminoethyl ether)N,N’-tetraacetic acid (EGTA), washed with distilled water, stained with uranyl acetate and lead citrate, and observed with the electron microscope. RESULTS

The general structure of Tritrichomonas foe&s has been described in detail by Honigberg et al. (1971). A membrane-bound organelle has been observed throughout the cell. It has a circular profile, with a mean diameter of 0.67 pm (Fig. 1). Its matrix appears to be homogeneous and more electron dense than that of the cytoplasm. This organelle corresponds to that called hydrogenosome, chromatic granule, paraxostylar, or paracostal granule (Lindmark and Muller 1973; Nielson and Diemer 1976; Honigberg et al. 1971). We did not observe a preferential localization of the hydrogenosomes relative to the costa. However, in many sections we could observe that they were linearly arranged following the microtubules of the axostyle (Fig. 2). In certain regions of the hydrogenosome membrane, electron-dense areas were observed (Figs. 1, 3). T. foetus exposed to the osmium tetroxide-potassium pyroantimonate solution appeared well preserved. The reaction product appeared as round, oval, or irregularly shaped electron-dense particles in association with specific cellular structures. The particles were seen in all the hydrogenosomes of all the cells examined (Figs. 4-6). They were not associated with a particular region of the organelle, but distributed throughout it. Some unidentified membrane-bound structures resembling the hydrogenosomes did not show a reaction product (Fig. 6). The reaction product was also seen to be associated with the plasma membrane of many, but not all, cells (Fig. 4). It is interesting to note that the reaction product was seen only in the plasma membrane which envelopes the cell body. Few or no particles were seen in the flagellar membrane.

Tritrichomonasfoetus:

LOCALIZATION

OF CALCIUM

FIG. 1. General aspect of the anterior region of Tritrichomonas foe&s showing several hydrogenosomes (H) near the costa (C). Electron-dense areas are seen in some regions of the membrane of hydrogenosomes (arrows). ~22,500. FIG. 2. Hydrogenosomes of Tritrichomonas foetus localized near the microtubules which form the axostyle. In some regions (arrow) profiles of membrane are seen in the interior of the organelle. x 90,000.

FIG. 3. Electron-dense areas are seen in some regions of the membrane of the Tritrichomonas foetus hydrogenosomes (arrow). ~60,000.

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ELIAS,

AND

DE SOUZA

pyroantimonate techFIG. 4. Tritrichomonas foetus submitted to the osmium tetroxide-potassium nique. Reaction products are seen in association with the hydrogenosomes (H) and the plasma membrane which surrounds the cell body. No reaction product is seen in the flagellar membrane (F) or in the costa (C). x 22,500.

Tritrichomonasfoetus:

LOCALIZATION OF CALCIUM

In the cytoplasm, a few particles were found associated with small vesicles located near the cell surface (Fig. 5). Deposit of a fine granular electron-dense precipitate was seen in the interior of some vacuoles (Fig. 5). No reaction product was seen in other cellular structures such as the nucleus, the endoplasmic reticulum, the microtubules which form the pelta and the axostyle, the glycogen particles, and the costa. In order to control the specificity of the osmium tetroxide - potassium pyroantimonate technique and determine if the electron-dense particles contained calcium, ultrathin sections of treated cells were incubated in the presence of 5 mM EGTA. It was observed that the electron-dense deposits, associated mainly with the hydrogenosome and the plasma membrane, were dissolved by such treatment, suggesting that they had been composed by calcium salts. As shown in Fig. 7, after solubilization of the reaction product by EGTA treatment, electron-transparent points appear in areas in which pyroantimonate had been precipitated. DISCUSSION

Protozoa of the family Trichomonadidae, which include agents of important human and animal diseases, have a typical cytoplasmic organelle, the hydrogenosome. It is now established that the most important metabolic function of this organelle is the anaerobic conversion of pyruvate to acetate via acetyl-CoA, accompanied by substrate phosphorylation and by production of molecular hydrogen. As certain peroxisomes, hydrogenosomes oxidize malate but do not have catalase, the enzyme typical of peroxisomes. Several characteristics such as synthesis of ATP from ADP and inorganic phosphate, the presence of superoxide dismutase, adenylate kinase, the high content of cardiolipin, and the presence of 3 FM circular DNA bring the hydrogenosomes close to mitochondria (Lindmark

281

and Muller 1973; Muller 1975; Cerkasov et al. 1978; Muller and Lindmark 1978). The hydrogenosomes also contain basic proteins as cytochemically detected with the ethanolic phosphotungstic acid and the ammoniacal silver techniques (Benchimol et al. 1982). It is well known that the Ca2+ concentration within the cells is maintained at levels lower than those in the environment. Although it is allowed to fluctuate widely below these levels, Ca2+ is regulated in the intracellular medium since a large number of cellular reactions are sensitive to Ca2+ in the micromolar concentration range. The regulation of intracellular Ca2+ concentration depends on the operation of specific Ca’+-pumping systems located mainly in the plasma membrane, in the endoplasmic reticulum, and in the mitochondria (De Araujo Jorge et al. 1979; Lehninger et al. 1978; Carafoli and Crompton 1978). Based on morphometric determinations of the total area of Ca2+-transporting membranes, it has been suggested that mitochondria are, quantitatively, the most important Ca2+transporting system in eucaryotic cells (Carafoli and Crompton 1978). The results obtained by us in Tritrichomonas foetus suggest that the hydrogenosomes, together with the plasma membrane, are the main sites of Ca2+ uptake in this pathogenic protozoan. Although profiles of the endoplasmic reticula were seen, mainly around the nucleus, no reaction product was observed associated with them. A few dense granules were seen in association with small vesicles which seem to derive from the plasma membrane during pinocytosis, and with some vacuoles which may represent lysosomes. It should be stressed that all hydrogenosomes show a large number of electron-dense granules indicative of calcium. These results suggest for this organelle a function which is known to be carried out in other eucaryotic cells by the mitochondria and the endoplasmic reticulum. It is interesting to note that few or no

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DE SOUZA

FIGS. 5 AND 6. 7’ritrichomonas foetus subjected to osmium tetroxide-potassium pyroantimonate technique. Reaction products are seen in association with the hydrogenosomes (H), in some vesicles localized near the cell surface (arrow) and in some vacuoles which may represent lysosomes (L). Some membrane-bound structures which resemble the hydrogenosomes (* in Fig. 6), profiles of the endoplasmic reticulum (ER), and the nuclei (N) do not include reaction products. Fig. 5, x 15,000; Fig. 6, x 22,500. FIG. 7. Thin section of Trirrichomonas foerus subjected to the osmium tetroxide-potassium pyroantimonate technique and treated with EGTA. The electron-dense deposits associated with the hydrogenosomes were dissolved by the treatment, leaving electron-transparent areas (arrow). x20,000

Tritrichomonas

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LOCALIZATION

OF CALCIUM

283

narticles were seen in association with the thank Mr. A. L. de Oliveira for help with photography. flagellar membrane of the T. foetus. It has This work has been supported by the Conselho Nade Desenvolvimento Cientifico e Tecnol6gico been reported that little or no reaction cional (CNPq), Conselho de Ensino para Graduados da product was seen in the ciliary membrane UFRJ ~_~ (CEPG). and Financiadora de Estudos e Proof the gill ciliated epithelium of freshwater jetos(FIN&’ mussels (Satir and Gilula 1970), thus sugREFERENCES gesting the existence of differences between the membrane which surrounds the AMOS, W. B., GRIMSTONE, A. V., ROTHSCHILD, L. J., AND ALLEN, R. D. 1979. Structure, protein compocell body and that of the cilia and flagella. sition and birefrigence of the costa: A motile flagelPrevious observations of the flagellar memlar root fibre in the flagellate Trichomonas. Journal brane of T. foe&s with the freeze-fracture of Cell Science 35, 139- 164. technique showed that the flagellar mem- APPLETON, J., AND MORRIS, D. C. 1979. The use of the potassium pyroantinomate-osmium method as brane has a lower density of intramembraa means of identifying and localizing calcium at the nous particles than the plasma membrane ultrastructural level in the cells of calcifying sys(Benchimol et al. 1982). tems. Journal of Biological Chemistry 27, 676-680. The pyroantimonate technique has been BAKER, P. F., HODGIKIN, A. L., AND RIDSWAY, E. B. widely used to detect several ions including 1971. Depolarization and calcium entry in squid giant axons. Journal of Physiology 218, 709-755. Ca’+ (Komnick and Komnick 1963; Herman et al. 1973; Weackley 1979; Simson BENCHIMOL, M., ELIAS, C. A., AND DE SOUZA, W. 1982. Tritrichomonas foetus: Ultrastructural loand Spicer 1975; Appleton and Morris 1979; calization of basic proteins and carbohydrates. ExDebas et al. 1975). In our study we did not perimental Parasitology 54, 135- 144. find a random deposition of electron-dense CARAFOLI, E., AND CROMPTON, M. 1978. The regulation of intracellular calcium by mitochondria. particles, indicative of antimonate-reacting Annals of the New York Academy of Sciences 307, sites. The particles were always associated 269-284. with specific cellular structures. Exposure CERKASOV, J., CERKASOVO.~, A., KULDA, J., AND of the section to EGTA resulted in comVILHELMOVA, D. 1978. Respiration of hydrogenoplete removal of the pyroantimonate presomes of Tritrichomonas foetus. I. ADP-dependent oxidation of malate and pyruvate. Journal of Biocipitate, a result which indicates that callogical Chemistry 25, 1207-1214. cium was involved in the formation of the DE ARA~JO JORGE, T. C., DE SOUZA, W., AND electron-dense product. MACHADO, R. D. 1979. Ultrastructural localization Cations, particularly Ca2+, appear to play of calcium-binding sites in the electrocyte of Electrophorus electricus. Journal of Cell Science 38, an important role in cell secretion, activa97- 104. tion of the adenylcyclase system, function of microtubules and microfilaments, etc. In DEBAS, G., HOFFMAN, L., LANDON, E. J., AND HORWITZ, L. 1975. Electron microscopic localizathe cytoplasm of Trichomonas spp. at least tion of calcium in vascular smooth muscle. two structures may be considered as possibly Anatomic Record 182, 447-451. influenced by Ca’+: a complex system of HERMAN, L., SATO, T., AND HALES, C. N. 1973. The electron microscopic localization of cation to panmicrotubules which form the axostylecreatic islets of Langehans and their possible role pelta complex and the costa, a structure in insulin secretion. Journal of Ultrastructure Rewhich appears to be related to cell movesearch 42, 298-311. ment (Amos et al. 1979). However, further HONIGBERG, B. M., MATTERN, C. F. T., AND studies are necessary in order to determine DANIEL, W. A. 1971. Fine structure of the mastigont system in Tritrichomonas foetus. Journal of Prothe influence of Ca2+ in the cell physiology tozoology 18, 183- 198. of Trichomonas spp. ACKNOWLEDGMENTS The authors thank Dr. H. Meyer for advice suggestions in the preparation of this manuscript.

and We

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