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Ultrastructural characterization of theisolated hydrogenosome in Tritrichomonas foetus
Tissue & Cell, 2000 32 (6) 518-526 ¢~ 2000 Harcourt Publishers Ltd ® DOI: 10.1054/tice.2000.0142. available online at http:l/www.idealibrary.com I Illlg ~ l
Ultrastructural characterization of the i•s o l a t e d h y d r o g e n o s o m e in "
Tritrichomonas foetus M. Benchimol
Abstract.
In the present study we show new aspects of the hydrogenosome ultrastructure as well alterations induced by the fractionation technique. The morphology of freshly isolated hydrogenosomes as well those found in whole cells of Tritrichomonas foetus were examined in thin-sections, in replicas of fast-freezing, and conventional freeze-fracture, freeze-etching, and by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). The true surface as well the concave and convex fracture faces of the inner and outer membranes are shown. We showed that after fractionation procedures the hydrogenosome ultrastructure can be changed, since isolated hydrogenosomes present patchwork-like structures, rosettes and the inner hydrogenosomal membrane is displaced. The peripheral vesicle is seen as a distinct compartment, since its content and morphological appearance is quite different from the rest of the organelle. The peripheral vesicle shows a smooth surface but presenting pores with 20 nm in diameter with a density of 7/gm 2 when observed after freeze-etching. We report the existence of characteristic intramembrane particles distribution and density on hydrogenosome membranes of isolated and whole T. foetus, suggesting that this organelle can have its morphology changed as consequence of technical modifications or as expression of its metabolic state. © 2000 Harcourt Publishers Ltd
Introduction The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments, such as certain freeliving ciliates (Finlay & Fenchel, 1989), rumen ciliates (Yarlett et al., 1981) and also in some fungi (Yarlett et al., 1986). The organisms presenting hydrogenosomes live in anaerobic habitats and they all lack mitochondria.
Laboratbrio de Ultraestrutura Celular, Universidade Santa Ursula and Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Received 28 March 2000
Accepted 22 August 2000 Correspondence to: Marlene Benchimol, Universidade Santa Ursula, Rua Jornalista Orlando Dantas, 59, CEP 222 31010. Botafogo, Rio de Janeiro, Brazil. TeL:/Fax: 55 21 553 1615; E-mail: [email protected]
The hydrogenosome contains enzymes that participate in the metabolism of pyruvate and is the site of formation of ATP and molecular hydrogen (Reviewed in Mtiller, 1990, 1993). The origin of the hydrogenosome has been the subject of intense discussion, since like mitochondria, it is enveloped by two membranes (Benchimol & DeSouza, 1983), divides autonomously by fission (Benchimol et al., 1996), imports proteins post-translationally (Johnson et al., 1993), and produces ATP (Lindmark & Mfiller, 1973). However, it differs from mitochondria in that it seems to lack a genome, a respiratory chain, cytochromes, the F0-Fi ATPase, the tricarboxylic acid cycle, oxidative phosphorylation, and cardiolipin (Mfiller, 1990). The hydrogenosome uses pyruvate:ferredoxin oxidoreductase, a counterpart to the mitochondrial pyrurate dehydrogenase complex, and presents hydrogenase, an enzyme typically restricted to anaerobes (for review, see Miiller, 1993).
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Hypotheses have been proposed for the origin of the trichomonad hydrogenosome: an independent endosymbiosis of an anaerobic eubacterium with a eukaryotic host or the conversion of an established mitochondrion adapted to an anaerobic lifestyle. The common origin hypothesis for mitochondria and hydrogenosome was refined to state that both organelles evolved from a common progenitor structure present in eukaryotes before the advent of true mitochondria or hydrogenosome (Embley et al., 1997). Hydrogenosome is a spherical or slightly elongated structure (when in process of division), 0.5 ~tm diameter, usually associated with cytoskeletal structures such as the axostyle and costa in trichomonads. The matrix of the hydrogenosome is homogeneously granular. Crista-like invaginations of inner-membrane were described in hydrogenosome of some rumen ciliates (Finlay & Fenchel, 1989). The hydrogenosome of trichomonads contains a peripheral, flattened, membrane-bounded compartment which contains high levels of magnesium, calcium and phosphorus and, possibly functions in intracellular calcium regulation (Benchimol et al., 1982; Chapman et al., 1985; De Souza and Benchimol, 1988). Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosome was observed (Benchimol et al., 1996). Although the hydrogenosomes were discovered more then 25 years ago (Lindmark & Miiller, 1973), information concerning their ultrastructure is limited (for review, see Benchimol, 1999). Many studies were performed using isolated hydrogenosomes in order to characterize this organelle biochemically (Lindmark & Miiller, 1973; Miiller, 1988; Miiller, 1990, Miiller, 1993; Morgado & DeSouza, 1997; Johnson et al., 1990, 1993). In the present work an ultrastructural analysis was undertaken using complementary techniques that allow the observation of membrane details of freshly isolated hydrogenosome or in whole cells of THtrichomonasfoetus, a parasitic protist of urogenita! tract in cattle.
Materials and Methods Cells Trichomonads The strains of Trichomonas vaginalis used in this study were obtained from ATCC (strain 30236) while the K strain of Tritrichomonas foetus was isolated by Dr H. Guida (Embrapa, Rio de Janeiro, Brazil) from the urogenital tract of a bull from the state of Rio de Janeiro, Brazil and has been maintained in TYM Diamond's medium (Diamond, 1957). The cells were cultivated for 24h at 36.5°C, which corresponds to the end of the logarithmic phase of growth. They were collected by low speed centrifugation, washed three times in phosphate-buffered saline (PBS, pH 7.2) and processed using one of the following procedures.
High resolution scanning electron microscopy The sample of the highly enriched hydrogenosome fraction was fixed overnight in a solution containing 2.5% glutaraldehyde. Small drops of the sample were placed on a specimen holder, post-fixed with 1% osmium tetroxide, washed several times in PBS, dehydrated in ethanol, critical point-dried in CO2, coated with chromium using high resolution sputtering equipment and examined in a Jeol JSM-6340F Field Emission Scanning Electron Microscope. Transmission electron microscopy (TEM) The cells were fixed overnight at room temperature in 2.5% glutaraldehyde, 4% paraformaldehyde, 5raM CaCI2 in PHEM or cacodylate buffer, pH 7.2. Postfixation was performed in 1% OsO4 in the same buffer, plus 5 mM CaCI2 and 0.8% potassium ferricyanide, for 30 min (Hepler, 1980). Thereafter, the cells were washed in PBS, dehydrated in acetone and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed either in a Zeiss 900 or Zeiss 902 electron microscope. When necessary some thin-sections were shifted and the image rotated up to l0 ° using the Zeiss 902 goniometer accessory. Freeze-fracture After overnight fixation in 2.5% glutaraldehyde in 0. I M Na-cacodylate buffer, pH 7.2, whole cells or isolated hydrogenosomes were washed twice in PBS and then infiltrated to increasing concentrations of glycerol in Na-cacodylate buffer until a final concentration of 30% glycerol was attained. Specimens were mounted on Balzers support disks and rapidly frozen in the liquid phase of Freon 22 cooled by liquid nitrogen and immediately transferred to liquid nitrogen. Also, previously fixed or unfixed specimens were deposited on a gelatin square and then mounted to the holder of a freezing device (Cryopress Med. Vac, Inc., St Louis, MO, USA) designed by Heuser et al. (1976). A polished copper block was cooled with liquid nitrogen and the specimen was projected in the free-fall against the block ('slam-freezing'). Subsequently, the specimens were freeze-fractured at -115°C in a Balzers BAF 300 freeze-etching machine and either immediately shadowed with platinum/carbon at 2 x 10-6 Torr or submitted to deep-etching (10min at -100 ° C) and rotatory shadowed with platinum at an angle of 15°. Replicas were recovered in distilled water, cleaned with sulfuric acid and/or sodium hyp.ochlodde, mounted on 200-mesh grids, and examined in a Zeiss 902 transmission electron microscope. Isolation of the hydrogenosomes Isolated hydrogenosomes were obtained by differential and Percoll gradient centrifugation following the procedure reported by Morgado and De Souza (1997) and fixed for electron microscopy as above described. Briefly, one liter of the culture was harvested by centrifugation,
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washed three times in a isotonic buffer containing 0.25 M sucrose, 2mM MgCI2, and 10mM Tris-HCI, pH 7.2 (H buffer). The pellet was disrupted in a Potter-type homogenizer with Teflon pestle. The resulting homogenate was submitted to differential centrifugation in Percoll gradient solutions of 53, 45, 24 and 18%, prepared by diluting a stock solution of Percoll in H buffer (0.25 M sucrose, 2mM MgCI_~, and 10mM Tris-HCI, pH 7.2). Aliquots (I ml) of the Po fraction were layered on top of the gradient, and centrifuged in a Beckman (Palo Alto, CA/USA) ultracentrifuge using a swinging bucket rotor (Beckman SW. 50. I ) at 36 000 g for 30 rain, without using brake.
Results and Discussion The multiplicity ofconfigurational states in which hydrogenosomes can exist when in whole cell or when in isolated state, was not previously documented. However. several studies were performed using isolated hydrogenosomes (for review, see Miiller, 1988. 1993). In the present study, after extensive analyses of many frozen-fractured cells, using different ways of freezing (fast- freezing, slow freezing of previously fixed and cryoprotected cells, conventional freeze-fracture, deepetching) we could recognize all four membranes in the hydrogenosomes. However. different configurational states were observed, as direct result of: 1. if the cell was in perfect conditions, i.e. intact, motile, and in log phase of growth; 2. if we were working with isolated hydrogenosomes, which were under stress during several hours, before be frozen, and 3. intermediate states, in which rosettes or particle clusters were observed, both in intact or fractionated cells. We analyzed the morphology of whole T..foetus cells (Figs 1-2) and freshly isolated hydrogenosomes using conventional freeze-fracture (Figs 3-9), freeze-etching (Figs I 0. 12-15), high resoltuion scanning electron microscopy (Figs 16-20) and thin-sections (Figs II, 21-22). Conventional freeze-fracture replicas revealed the presence of two membranes enveloping the hydrogenosome. Four fracture faces could be identified: two concave faces representing the P faces of the outer and the inner membranes and two convex faces representing the E faces of the two membranes (Figs 3-7). The frequency and size of these particles was characteristic of different fracture faces. The exoplasmic fracture face of the outer membrane (OEF) contained fewer particles than the inner hydrogenosomal membrane. The particle size ranged from 10 to 50 nm on the external faces (outer membrane) and around 15 nm, and 50 nm at exoplasmic and protoplasmic fracture face of the inner membrane, respectively (Figs 3-6). The particles found in the matrix measured around 13 nm. By freeze-etching using hydrogenosomes in whole cells, it was observed a special array of particles, both in the outer and inner hydrogenosomal membranes.
Hydrogenosomal inner membrane and matrix are composed of a high percentage of proteins, as seen by freezefracture and freeze-etching (Figs 10, 12-15) probably corresponding to respiratory and metabolic enzymes. Pore-like structures could be present to transport of material in and out the hydrogenosome (Figs 10, 12 & 13). However, we did not find projections corresponding to FoFt complexes as observed in freeze-etched mitochondria (Allen et al., 1989), confirming the study of Lloyd et al (I 979), in which trichomonads do not appear to contain FoFt ATPase. Patchwork-like structures (Fig. 7) were frequently found in outer membranes (OEF) of isolated hydrogenosomes submitted to freeze-fracture, although very rarely, we were able to find them also in hydrogenosomes of whole cells, and sometimes a special arrangement of intramembrane particles forming rosette-like pattern after freeze-fracture (Figs 8 & 9). These rosette-like formations consisting of eight to 13 intramembranous particles were found in both PF and EF of the inner hydrogenosomal membranes (Figs 8 & 9). Different number of these rosettes was observed in each isolated hydrogenosomes: in some hydrogenosomes only two rosettes were seen whereas in others the entire surface was covered with these structures (Figs 8 & 9). This structure was previously described in mitochondria and interpreted as being the result of the fracture plane, jumping from the outer to the inner limiting membrane and vice versa at sites of contact (Veneti~ & Verkleij, 1982). These authors proposed a semi-fusion model, in which non-bilayer lipids are involved in these contact sites. It is important to note that these patchwork-like structures were seen only in isolated mitochondria and thus could be an artifact. The hydrogenosome presenting patchworklike structures seen in our studies are very similar to those described in mitochondria. Concerning the rosettes, at first we thought that it could correspond to portion of flagellar membrane contaminating the fraction, since similar specialization was described in the anterior flagella of T../betus (Benchimol et al., 1981). However, several differences were observed: 1) the structures present a rounded shape and are quite different from those of flagella, 2) the particle number found in each rosette was different from that found in the flagella replicas, 3) the frequency and size of the particles were different, and 4) hydrogenosomes present different number and distribution of these particle clusters. Studies performed by Wrigglesworth et al. (1970), showed alterations of mitochondria membranes induced by poor fixation, such as particles in linear and circular arrays after freeze-etching. We suggest that the stress provoked by the isolation procedure, in which cells are broken by mechanical strength, centrifuged in very high speed without any fixation, are maintained in contact with Percoll during the gradient separation, could induce to membrane alterations. By freeze-etching of hydrogenosome in whole cells it was possible to see the hydrogenosomal membrane in
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General view of Trilrichm,onu.vj'oem.~" in a routine thin section. H, hydrogenosomes: N. nucleus: V. vacuole. G, glycogen granules, x l3 000.
Fig. 2 Trilricll,mcmu.~J'oelu.~"hydrogenosome fixed in glutaraldehyde and post-fixed in osmium tetroxide solution containing potassium ferricyanide. The hydrogenosome (H) presents a closely apposed double membrane, and a flat peripheral compartment (star). also bounded by a double membrane, x]00000. Figs 3-"/ Frecze-fract ure images of Tritricla,monu.tfuvl,.~ hydrogenosome after fractionation procedure (Figs 3 & 4), and in whole cells (Figs 5-7). Different aspects of intramembranous particles distribution is demonstrated. The flat vesicle presents a smooth surface (P). as seen in Fig. 4, which presents IEF (inner membrane. E face). The patchwork-like image is seen in Fig. 7 (OEF. outer membrane. E face). Fig. 3 presents the face P of the outer hydrogenosoma] (OPF)
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membrane and Fig. 6 demonstrate the E face of the outer membrane (OEF), whereas Figs 4 & 5 show the E face of the hydrogenosomal inner membrane (I EF). Note the higher number of particles in the inner membrane. Fig. 4 x l00 000: Fig. 5:x0 000; Fig. 6:x82 000; Fig. 7:x55 000. Figs 8-9 Hydrogenosomes from isolated fraction observed by freeze-fracture. Note the clusters of intramcmbranous particles forming rosettes (arrowheads), in the inner hydrogenosomal membrane, E face (IEF). Fig. 8:x83 000; Fig. 9: xl00000. Fig. 10
Inner membrane. E face (IEF) of hydrogenosome from cryofixed whole cell seen after freeze-etching. Few rosettes formation are seen. x 100000.
Fig. I I Electron micrograph of a thin-section showing a general view of the purified hydrogenosomal fraction obtained using a Percoll gradient. The hydrogenosome matrix (H) is homogeneous. The peripheral vesicles present an electron-dense content (arrows).x30 000. Figs. 12-15 Frecze-etching views ofhydrogenosomes in whole T.foetuseellsfrozenbyquick-freezing(slam-freezing). Figs 12& 13 show the outer (OEF) and inner hydrogsnosomal (IEF) membranes, respectively. Fig. 12: few particles are distributed over the hydrogenosomal membrane surface. A small pore is depicted at the middle of each protein (arrow). xl00000. Fig. 13 The hydrogenosome presents a smooth surface in its peripheral vesicle and large pore-like structures (arrow). The inner hydrogenosomal membrane is seen presenting densely packed particles, and also particles in a circular array. This micrograph is ,'1 higher magnification of Fig. 15. x l00 000. Figs 14 & 15: Hydrogenosomal matrix. Fig. 14: An array of proteins (arrowheads), and a large peripheral vesicle (P) with heterogeneous content are seen. x45 000. Fig. 15: an overall view of several hydrogenosomes (H) in different fracture planes. The hydrogenosomal matrix is seen in all hydrogenosomes, except one which presents a smooth peripheral vesicle (P) as demonstrated at higher magnification in Fig. 13. The axostyle microtubules (A) and glycogen particles (G) are seen in close proximity of hydrogenosomes (H 1. x35 000.
cross-section, and also the E face of the fractured inner or outer faces and also the matrix (Figs 10, 12-15). The hydrogenosomal matrix was rich in proteins, some of which were disposed in a special arrangement (Fig. 14). A freeze-fracture study of T. vaginalis and T. foetus was performed by Honigberg et al. (1984) and the authors presented figures of whole cells with hydrogenosomes without alterations. No description of the distribution of the intramembranous particles was presented. The hydrogenosomal membranes present special arrangement of particles as seen by freeze-etching, and at higher magnification small holes are seen at the middle of the membranous particles (Figs 12 & 13). The particle density was quite different when the E face of the outer membrane was seen: few particles arranged with a central pore (Fig. 12). Occasionally were observed membrane particles forming rosettes (Fig. 10) and in linear arrangement (Fig. 14). We were able to observe that the membrane lining the peripheral vesicle is morphologically different from the hydrogenosomal membrane envelope: it presents very few intramembranous particles, being almost smooth. It was seen in different views, depending of the fracture plane, such as a) an empty space (Fig. 2), b) a space containing material (Fig. 14), c) a membrane fractured image with particles (Fig. 5) and d) its true external surface (Fig. 13). In this last observation, it was possible to see its smooth surface with pores with uniform diameter (about 20 nm), which could represent a region of high permeability. These pores are in low density, only 7/lam2. The peripheral vesicle occupies 10 to 25% of the whole hydrogenosome area in fast-frozen cells submitted to freeze-etching. Morgado and DeSouza (1997) showed that the peripheral vesicle membrane has different properties from the enveloping hydrogenosomal membrane, although previous freeze-fracture studies did not show a special organization of their membrane proteins (Honigberg et al., 1984; Benchimol et al., 1996). The isolated peripheral vesicles maintained their flattened morphology, suggesting that each individual
vesicle could have its own inherent structural framework. Morgado and DeSouza (1997) also demonstrated by SDS-PAGE analysis of the vesicle fraction very few protein bands, especially one of 45 kDa. Previous studies performed with cryosections of T..[betus incubated in the presence of gold-labeled WGA showed positive reaction in the membrane lining the peripheral vesicle, only in the luminal portion (Benchimol et al., 1996). The disturbance of the density and distribution of intramembranous particles found in hydrogenosomes membranes after isolation procedure could be due to the hypertonic medium used during the isolation steps or an alteration of the hydrogenosome metabolic state. In the former case, the outer membrane could allow the entry of the external medium and the intermembranar space acquired the same osmotic tension, while the selective permeability of the inner membrane could lead to shrink the organellar matrix. Other studies have shown the influence of chemicals in the disorganization of protein-lipid association at specific regions of the plasma membrane (Ghosh & Nanninga, 1976) and in transformed cells (Furcht & Scott, 1975). We also examined the true external surface of isolated hydrogenosomes using field emission in-lens scanning electron microscopy (FEISEM) (Figs 16-20), which has a resolution comparable to that of the transmission electron microscope (reviewed by Allen and Goldberg, 1993). The images produced in this way are rather different to conventional SEM: the lower accelerating voltage, thinner metal coating, increased brightness, allow surface details observation and higher resolution. Therefore, details of the hydrogenosome surface were seen revealing pits and membrane invaginations (Figs 16-20), which could represent a process of material entry in the organelle. Previous study performed by Tanaka (1980), using SEM for visualization of intracellular structures, provided evidences that mitochondria present a smooth contoured outer membrane, whereas the inner mitochondrial membrane has a granular surface (for review, see Tanaka,
HYDROGENOSOME ULTRASTRUCTURE
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Figs. 16-20 Isolated hydrogenosomes seen by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). Different views of the true hydrogenosomal surface is presented: invaginations (Figs 16-18), rugosities (Figs 16-18, 19), pits (arrowheads in Fig. 19) and, peripheral vesicles (Figs 19--20) are seen. Fig. 16: xlS0000; Fig. 17: xl00,000; Fig. 18000: Fig. 19-20: x85000. Figs. 21-22 Thin-sections of isolated hydrogenosomes. The inner hydrogenosomal membrane is seen detached from the outer membrane (arrow) at some points (arrowheads)• The volume of the inner comparlment is decreased while the volume of the ouLer compartment is increased. P. peripheral vesicle. Fig. 21: x52 000; Fig. 22: x110000.
HYDROGENOSOME ULTRASTRUCTURE 525
1980). Both findings are similar to those here described for hydrogenosomes. Thin sections o f routine T E M o f isolated h y d r o g e n o somes revealed that the inner m e m b r a n e could be found separated from the outer m e m b r a n e (Figs 21 & 22), leaving only some regions o f the inner and outer m e m b r a n e s closely apposed to each other. Kulda et al. (1987) published micrographs o f dividing h y d r o g e n o s o m e s seen by freeze-fracture after glutaraldehyde fixation. They proposed that both single and double m e m b r a n e h y d r o g e n o somes could be found. In view o f o u r findings, we believe that these authors found some a b n o r m a l hydrogenosomes, in which the outer m e m b r a n e was detached as seen in the present study in isolated hydrogenosomes. Allen et al., (1989) also revealed the outer and inner m e m b r a n e s o f mitochondria to be separated after the isolation procedure. We established what is a characteristic o r t h o d o x h y d r o g e n o s o m e configuration: two closely apposed membranes, with no space between them. In isolated hydrogenosomes, by freeze-fracture, a patchwork-like structure or rosettes was frequently found, and by thinsection, a condensed configuration, where a large interm e m b r a n a r space is seen as result o f displacement o f the inner h y d r o g e n o s o m a l membrane. The two m e m b r a n e s are still connected to some extent, on the places were peripheral vesicles are located. This observation could be explained by: I. an artifact resulting from the stress o f the isolation, since it was not seen in intact cells: 2. this could be a condensed configuration, similar to that found in m i t o c h o n d r i a after being isolated (Hertsens & Jacob, 1987), where large intracristal spaces and a small matrix c o m p a r t m e n t are found: 3. this could be a real configuration, occasionally fourtd by some authors w h o described the h y d r o g e n o s o m e as an organelle enveloped by a single m e m b r a n e (Kulda et al., 1987). Studies carried out on m i t o c h o n d r i a considered the influence o f the metabolic or the energetic state on the ultrastructure o f the mitochondrial inner m e m b r a n e on the basis o f results from standardized freeze-fracture experiments (Hertsens & Jacob, 1987). In conclusion, h y d r o g e n o s o m e s studied after isolation procedures must be carefully examined since the conditions o f preparation play a crucial role in determining the final ultrastructure. It would be important to analyze if there is loss o f membrane-associated enzyme activities a c c o m p a n i e d by a corresponding alteration o f the structural components.
ACKNOWLEDGMENTS The authors wish to thank D r Wanderley de Souza for critical reading the manuscript. This investigation received financial support f r o m C N P q (Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico), P R O N E X ( P r o g r a m a de Nficleos de Excel6ncia), F A P E R J
( F u n d a g i o de A m p a r o ~ Pesquisa do E s t a d o do Rio de Janeiro), and A U S U ( A s s o c i a ~ o Universit~ria Santa 0rsula).
REFERENCES Allen, R.D. Schroeder, C.C. and Fok, A.K. 1989. An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques. J. Cell Biol., 108, 2233-2240. Allen, D. and Goldbcrg, M.W. 1993. High resolution SEM in cell biology. Trends Cell Biol., 3, 205-208. Benchimol. M. 1999.The Hydrogenosome. Acta Microscop., 8, 1-22. Benchimol, M.. Almeida, J.C.A. and De Souza, W. 1996.Further studies on the organization of the hydrogenosomein Tritrichomoaas foetus. Tiss Cell, 28, 287-299.
Benchimol, M. and De Souza, W. 1983. Fine structure and cytochemistry of the hydrogenosome of Tritrichomonaafoetus. J. Protozool., 30, 422-425. Benchimol, M., Elias C.A. and De Souza, W. 1982. Tritrichomonas foetus: U hrastructural localization of calcium in the plasma membrane and in the hydrogenosome. Exp. Parasitol., 54, 277-284. BenchimoL M., Elias, C.A. and DeSouza, W. 1981. Specializations in the flagellar membrane of Tritrichomonasfoelus. J. Parasitol., 67, 174-178. Benchimol, M., Johnson, P.J. and De Souza, W. 1996. Morphogenesis of the Hydrogenosome: An Ultrastructural Study. Biol. Cell., 87, 197-205. Chapman, A., Hann, A.O.C., Lindstead, D. and Lloyd, D. 1985. Energy dispersive X-ray microanalysis of membrane-associated inclusion in hydrogenosomes isolated from Trichomonas vaginalis. J. Gen. Microbiol., 131,2933-2939. De Souza, W. and Benchimol, M. 1988. Electron spectroscopicimaging ofcalcium in the hydrogenosomes of Tritrichomonasfoetus. J. Submicrose. Cytol. Palhol.. 20, 619-621. Diamond, L.S. 1957.The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol., 43, 488-490. Embley, T.M., Homer, D.A. and Hirt, R.P. 1997.Anaerobic eukaryote evolution: hydrogenosomes as biochemically modified mitochondria? TREE., 12, 437-441. Finlay, B.J. and Fenchel, T. 1989. Hydrogenosomes in some anaerobic protozoa resemble mitochondria. FEMS Microb. Lett., 65, 31 I-314. Furcht, L.T. and Scott, R.S. 1975. Modulation of the distribution of plasma membrane intramembranous particles in contactinhibited and transformed cells. Biochem. Biophys. Acta, 401, 213-220. Ghosh, B.K. and Nanninga, N. 1976. Polymorphism of the mesosome in Bacillus licheniformis. J. Ultrastruct. Res., 56, 107-120. Hepler, P.K. 1980. Membranes in the mitotic apparatus of barley cells. J. Cell Biol.. 86, 490--499. Hertsens, R.C. and Jacob, W.A. 1987. Freeze-fracture study of heart mitochondria in the condensed or orthodox state. Biochem. Biophys. Acta, 894. 507-514. Heuser, J.E., Reese, T.S. and Landis, D.M.D. 1976. Preservation of synaptic structure by rapid freezing. Cold Spring Harbor Symposium Quant. Biol., 40, 17-2421. Honigberg, B.M.. Volkmann, D., Entzeroth. R. and Scholtyseek, E. 1984. A freeze-fracture electron microscopy study of Trichomonas vaginalis Donne and Tritri£.homonasfoetus (Riedmiiller). J. Protozool., 31, 116-131. Johnson, P.J., d'Oliveira, C.E., Gorrel, T.E. and Mfiller, M. 1990. Molecular analysis of the hydrogenosomal ferrodoxin of the anerobic protist Trichomonas vaginalia. Proc. Natl. Acad. Sci. USA., 87, 6097-6101. Johnson, P.J., Lahti, C.J. and Bradley, P.J. 1993. Biogenesisof the hydrogenosome: an unusual organelle found in the anaerobic protist Trichomwzaa vaginalis. J. Parasitol., 79, 664-670. Kulda J., Noh~,nkovd, E. and Ludvik, J. 1987. Basic structure and function of the trichomonal cell. Acta Universitatis Carolinae., 30, 181-198. Lindmark, D.G. and Milller, M. 1973. Hydrogenosome, a cytoplasmic organeUeof the anaerobic flagellate, Tritrichomonaa foetus, and its role in pyruvate metabolism. J. Biol. Chem., 248, 7724-7728.
526
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Lloyd, D., Lindmark, D.G. and M011er,M. 1979.Adenosine triphosphatasc activity of Tritrichomona.~foelus. J. Gen. Microbiol., 115, 301-307. Morgado Diaz, J.A. and DeSouza, W. 1997.Purification and biochemical characterization of the hydrogenosomesof l,he flagellate protist Tritrichonmnas.[oetu.~. Eur. J. Cell Biol., 74. 85-9 I. M01ler. M. 1988. Energy metabolism of protozoa withoul, mil.ochondria. Ann. Rev. Microbiol.. 42, 465-488. MQIler, M. 1990.Sl,ructure. In: Trichomonads Parasil,icin Humans, ed. B.M. Honigberg., Springer-Verlag, New-York, 5-35. M011er, M., 1993.The hydrogenosome.J. Gen. Microb., 139, 2879-2889. MLiller, M. and Moor, H. 1984.Cryofixation ofthick specimensby higll pressure freezing. In: Scienceof Biological SpecimenPreparal,ion. vol. 2 Revel, J.P., Barnard T. and Haggis. G.H. eds. SEM. Inc., AMF O'Hare, Chicago, 131-138.
Tanaka, K. 1980.Scanning elecl,ronmicroscopy of i.ll,racellular strucl,ures. Int. Rev. Cyl,ol., 68, 97-] 25. Van VcnetiE, R. and Vcrkleij, A.J. 1982.Possiblerole of non-bilaycr ]ipids in the structure o£ mil,ochondria. A freeze-fractureelecl,ronmicroscopy study. Biochem. Biophys. Acl.a, 692, 397--405. Yarlel,l,.N.. Harm, A.C., Lloyd, D. and Williams, A.G. 1981. Hydrogenosomes in l,herumen protozoon Da3j,lricharuminanltlm Scuberg. Biochem. J., 200, 365-372. Yarlel,l,,N., Orpin, C.G., Munn, E.A.. Yarlel,t.N.C. and Greenwood. C.A. 1986. Hydrogenosomes in l,herumen fungus Neocallimaslix p
Ultrastructural characterization of the i•s o l a t e d h y d r o g e n o s o m e in "
Tritrichomonas foetus M. Benchimol
Abstract.
In the present study we show new aspects of the hydrogenosome ultrastructure as well alterations induced by the fractionation technique. The morphology of freshly isolated hydrogenosomes as well those found in whole cells of Tritrichomonas foetus were examined in thin-sections, in replicas of fast-freezing, and conventional freeze-fracture, freeze-etching, and by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). The true surface as well the concave and convex fracture faces of the inner and outer membranes are shown. We showed that after fractionation procedures the hydrogenosome ultrastructure can be changed, since isolated hydrogenosomes present patchwork-like structures, rosettes and the inner hydrogenosomal membrane is displaced. The peripheral vesicle is seen as a distinct compartment, since its content and morphological appearance is quite different from the rest of the organelle. The peripheral vesicle shows a smooth surface but presenting pores with 20 nm in diameter with a density of 7/gm 2 when observed after freeze-etching. We report the existence of characteristic intramembrane particles distribution and density on hydrogenosome membranes of isolated and whole T. foetus, suggesting that this organelle can have its morphology changed as consequence of technical modifications or as expression of its metabolic state. © 2000 Harcourt Publishers Ltd
Introduction The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments, such as certain freeliving ciliates (Finlay & Fenchel, 1989), rumen ciliates (Yarlett et al., 1981) and also in some fungi (Yarlett et al., 1986). The organisms presenting hydrogenosomes live in anaerobic habitats and they all lack mitochondria.
Laboratbrio de Ultraestrutura Celular, Universidade Santa Ursula and Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Received 28 March 2000
Accepted 22 August 2000 Correspondence to: Marlene Benchimol, Universidade Santa Ursula, Rua Jornalista Orlando Dantas, 59, CEP 222 31010. Botafogo, Rio de Janeiro, Brazil. TeL:/Fax: 55 21 553 1615; E-mail: [email protected]
The hydrogenosome contains enzymes that participate in the metabolism of pyruvate and is the site of formation of ATP and molecular hydrogen (Reviewed in Mtiller, 1990, 1993). The origin of the hydrogenosome has been the subject of intense discussion, since like mitochondria, it is enveloped by two membranes (Benchimol & DeSouza, 1983), divides autonomously by fission (Benchimol et al., 1996), imports proteins post-translationally (Johnson et al., 1993), and produces ATP (Lindmark & Mfiller, 1973). However, it differs from mitochondria in that it seems to lack a genome, a respiratory chain, cytochromes, the F0-Fi ATPase, the tricarboxylic acid cycle, oxidative phosphorylation, and cardiolipin (Mfiller, 1990). The hydrogenosome uses pyruvate:ferredoxin oxidoreductase, a counterpart to the mitochondrial pyrurate dehydrogenase complex, and presents hydrogenase, an enzyme typically restricted to anaerobes (for review, see Miiller, 1993).
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Hypotheses have been proposed for the origin of the trichomonad hydrogenosome: an independent endosymbiosis of an anaerobic eubacterium with a eukaryotic host or the conversion of an established mitochondrion adapted to an anaerobic lifestyle. The common origin hypothesis for mitochondria and hydrogenosome was refined to state that both organelles evolved from a common progenitor structure present in eukaryotes before the advent of true mitochondria or hydrogenosome (Embley et al., 1997). Hydrogenosome is a spherical or slightly elongated structure (when in process of division), 0.5 ~tm diameter, usually associated with cytoskeletal structures such as the axostyle and costa in trichomonads. The matrix of the hydrogenosome is homogeneously granular. Crista-like invaginations of inner-membrane were described in hydrogenosome of some rumen ciliates (Finlay & Fenchel, 1989). The hydrogenosome of trichomonads contains a peripheral, flattened, membrane-bounded compartment which contains high levels of magnesium, calcium and phosphorus and, possibly functions in intracellular calcium regulation (Benchimol et al., 1982; Chapman et al., 1985; De Souza and Benchimol, 1988). Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosome was observed (Benchimol et al., 1996). Although the hydrogenosomes were discovered more then 25 years ago (Lindmark & Miiller, 1973), information concerning their ultrastructure is limited (for review, see Benchimol, 1999). Many studies were performed using isolated hydrogenosomes in order to characterize this organelle biochemically (Lindmark & Miiller, 1973; Miiller, 1988; Miiller, 1990, Miiller, 1993; Morgado & DeSouza, 1997; Johnson et al., 1990, 1993). In the present work an ultrastructural analysis was undertaken using complementary techniques that allow the observation of membrane details of freshly isolated hydrogenosome or in whole cells of THtrichomonasfoetus, a parasitic protist of urogenita! tract in cattle.
Materials and Methods Cells Trichomonads The strains of Trichomonas vaginalis used in this study were obtained from ATCC (strain 30236) while the K strain of Tritrichomonas foetus was isolated by Dr H. Guida (Embrapa, Rio de Janeiro, Brazil) from the urogenital tract of a bull from the state of Rio de Janeiro, Brazil and has been maintained in TYM Diamond's medium (Diamond, 1957). The cells were cultivated for 24h at 36.5°C, which corresponds to the end of the logarithmic phase of growth. They were collected by low speed centrifugation, washed three times in phosphate-buffered saline (PBS, pH 7.2) and processed using one of the following procedures.
High resolution scanning electron microscopy The sample of the highly enriched hydrogenosome fraction was fixed overnight in a solution containing 2.5% glutaraldehyde. Small drops of the sample were placed on a specimen holder, post-fixed with 1% osmium tetroxide, washed several times in PBS, dehydrated in ethanol, critical point-dried in CO2, coated with chromium using high resolution sputtering equipment and examined in a Jeol JSM-6340F Field Emission Scanning Electron Microscope. Transmission electron microscopy (TEM) The cells were fixed overnight at room temperature in 2.5% glutaraldehyde, 4% paraformaldehyde, 5raM CaCI2 in PHEM or cacodylate buffer, pH 7.2. Postfixation was performed in 1% OsO4 in the same buffer, plus 5 mM CaCI2 and 0.8% potassium ferricyanide, for 30 min (Hepler, 1980). Thereafter, the cells were washed in PBS, dehydrated in acetone and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed either in a Zeiss 900 or Zeiss 902 electron microscope. When necessary some thin-sections were shifted and the image rotated up to l0 ° using the Zeiss 902 goniometer accessory. Freeze-fracture After overnight fixation in 2.5% glutaraldehyde in 0. I M Na-cacodylate buffer, pH 7.2, whole cells or isolated hydrogenosomes were washed twice in PBS and then infiltrated to increasing concentrations of glycerol in Na-cacodylate buffer until a final concentration of 30% glycerol was attained. Specimens were mounted on Balzers support disks and rapidly frozen in the liquid phase of Freon 22 cooled by liquid nitrogen and immediately transferred to liquid nitrogen. Also, previously fixed or unfixed specimens were deposited on a gelatin square and then mounted to the holder of a freezing device (Cryopress Med. Vac, Inc., St Louis, MO, USA) designed by Heuser et al. (1976). A polished copper block was cooled with liquid nitrogen and the specimen was projected in the free-fall against the block ('slam-freezing'). Subsequently, the specimens were freeze-fractured at -115°C in a Balzers BAF 300 freeze-etching machine and either immediately shadowed with platinum/carbon at 2 x 10-6 Torr or submitted to deep-etching (10min at -100 ° C) and rotatory shadowed with platinum at an angle of 15°. Replicas were recovered in distilled water, cleaned with sulfuric acid and/or sodium hyp.ochlodde, mounted on 200-mesh grids, and examined in a Zeiss 902 transmission electron microscope. Isolation of the hydrogenosomes Isolated hydrogenosomes were obtained by differential and Percoll gradient centrifugation following the procedure reported by Morgado and De Souza (1997) and fixed for electron microscopy as above described. Briefly, one liter of the culture was harvested by centrifugation,
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washed three times in a isotonic buffer containing 0.25 M sucrose, 2mM MgCI2, and 10mM Tris-HCI, pH 7.2 (H buffer). The pellet was disrupted in a Potter-type homogenizer with Teflon pestle. The resulting homogenate was submitted to differential centrifugation in Percoll gradient solutions of 53, 45, 24 and 18%, prepared by diluting a stock solution of Percoll in H buffer (0.25 M sucrose, 2mM MgCI_~, and 10mM Tris-HCI, pH 7.2). Aliquots (I ml) of the Po fraction were layered on top of the gradient, and centrifuged in a Beckman (Palo Alto, CA/USA) ultracentrifuge using a swinging bucket rotor (Beckman SW. 50. I ) at 36 000 g for 30 rain, without using brake.
Results and Discussion The multiplicity ofconfigurational states in which hydrogenosomes can exist when in whole cell or when in isolated state, was not previously documented. However. several studies were performed using isolated hydrogenosomes (for review, see Miiller, 1988. 1993). In the present study, after extensive analyses of many frozen-fractured cells, using different ways of freezing (fast- freezing, slow freezing of previously fixed and cryoprotected cells, conventional freeze-fracture, deepetching) we could recognize all four membranes in the hydrogenosomes. However. different configurational states were observed, as direct result of: 1. if the cell was in perfect conditions, i.e. intact, motile, and in log phase of growth; 2. if we were working with isolated hydrogenosomes, which were under stress during several hours, before be frozen, and 3. intermediate states, in which rosettes or particle clusters were observed, both in intact or fractionated cells. We analyzed the morphology of whole T..foetus cells (Figs 1-2) and freshly isolated hydrogenosomes using conventional freeze-fracture (Figs 3-9), freeze-etching (Figs I 0. 12-15), high resoltuion scanning electron microscopy (Figs 16-20) and thin-sections (Figs II, 21-22). Conventional freeze-fracture replicas revealed the presence of two membranes enveloping the hydrogenosome. Four fracture faces could be identified: two concave faces representing the P faces of the outer and the inner membranes and two convex faces representing the E faces of the two membranes (Figs 3-7). The frequency and size of these particles was characteristic of different fracture faces. The exoplasmic fracture face of the outer membrane (OEF) contained fewer particles than the inner hydrogenosomal membrane. The particle size ranged from 10 to 50 nm on the external faces (outer membrane) and around 15 nm, and 50 nm at exoplasmic and protoplasmic fracture face of the inner membrane, respectively (Figs 3-6). The particles found in the matrix measured around 13 nm. By freeze-etching using hydrogenosomes in whole cells, it was observed a special array of particles, both in the outer and inner hydrogenosomal membranes.
Hydrogenosomal inner membrane and matrix are composed of a high percentage of proteins, as seen by freezefracture and freeze-etching (Figs 10, 12-15) probably corresponding to respiratory and metabolic enzymes. Pore-like structures could be present to transport of material in and out the hydrogenosome (Figs 10, 12 & 13). However, we did not find projections corresponding to FoFt complexes as observed in freeze-etched mitochondria (Allen et al., 1989), confirming the study of Lloyd et al (I 979), in which trichomonads do not appear to contain FoFt ATPase. Patchwork-like structures (Fig. 7) were frequently found in outer membranes (OEF) of isolated hydrogenosomes submitted to freeze-fracture, although very rarely, we were able to find them also in hydrogenosomes of whole cells, and sometimes a special arrangement of intramembrane particles forming rosette-like pattern after freeze-fracture (Figs 8 & 9). These rosette-like formations consisting of eight to 13 intramembranous particles were found in both PF and EF of the inner hydrogenosomal membranes (Figs 8 & 9). Different number of these rosettes was observed in each isolated hydrogenosomes: in some hydrogenosomes only two rosettes were seen whereas in others the entire surface was covered with these structures (Figs 8 & 9). This structure was previously described in mitochondria and interpreted as being the result of the fracture plane, jumping from the outer to the inner limiting membrane and vice versa at sites of contact (Veneti~ & Verkleij, 1982). These authors proposed a semi-fusion model, in which non-bilayer lipids are involved in these contact sites. It is important to note that these patchwork-like structures were seen only in isolated mitochondria and thus could be an artifact. The hydrogenosome presenting patchworklike structures seen in our studies are very similar to those described in mitochondria. Concerning the rosettes, at first we thought that it could correspond to portion of flagellar membrane contaminating the fraction, since similar specialization was described in the anterior flagella of T../betus (Benchimol et al., 1981). However, several differences were observed: 1) the structures present a rounded shape and are quite different from those of flagella, 2) the particle number found in each rosette was different from that found in the flagella replicas, 3) the frequency and size of the particles were different, and 4) hydrogenosomes present different number and distribution of these particle clusters. Studies performed by Wrigglesworth et al. (1970), showed alterations of mitochondria membranes induced by poor fixation, such as particles in linear and circular arrays after freeze-etching. We suggest that the stress provoked by the isolation procedure, in which cells are broken by mechanical strength, centrifuged in very high speed without any fixation, are maintained in contact with Percoll during the gradient separation, could induce to membrane alterations. By freeze-etching of hydrogenosome in whole cells it was possible to see the hydrogenosomal membrane in
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General view of Trilrichm,onu.vj'oem.~" in a routine thin section. H, hydrogenosomes: N. nucleus: V. vacuole. G, glycogen granules, x l3 000.
Fig. 2 Trilricll,mcmu.~J'oelu.~"hydrogenosome fixed in glutaraldehyde and post-fixed in osmium tetroxide solution containing potassium ferricyanide. The hydrogenosome (H) presents a closely apposed double membrane, and a flat peripheral compartment (star). also bounded by a double membrane, x]00000. Figs 3-"/ Frecze-fract ure images of Tritricla,monu.tfuvl,.~ hydrogenosome after fractionation procedure (Figs 3 & 4), and in whole cells (Figs 5-7). Different aspects of intramembranous particles distribution is demonstrated. The flat vesicle presents a smooth surface (P). as seen in Fig. 4, which presents IEF (inner membrane. E face). The patchwork-like image is seen in Fig. 7 (OEF. outer membrane. E face). Fig. 3 presents the face P of the outer hydrogenosoma] (OPF)
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membrane and Fig. 6 demonstrate the E face of the outer membrane (OEF), whereas Figs 4 & 5 show the E face of the hydrogenosomal inner membrane (I EF). Note the higher number of particles in the inner membrane. Fig. 4 x l00 000: Fig. 5:x0 000; Fig. 6:x82 000; Fig. 7:x55 000. Figs 8-9 Hydrogenosomes from isolated fraction observed by freeze-fracture. Note the clusters of intramcmbranous particles forming rosettes (arrowheads), in the inner hydrogenosomal membrane, E face (IEF). Fig. 8:x83 000; Fig. 9: xl00000. Fig. 10
Inner membrane. E face (IEF) of hydrogenosome from cryofixed whole cell seen after freeze-etching. Few rosettes formation are seen. x 100000.
Fig. I I Electron micrograph of a thin-section showing a general view of the purified hydrogenosomal fraction obtained using a Percoll gradient. The hydrogenosome matrix (H) is homogeneous. The peripheral vesicles present an electron-dense content (arrows).x30 000. Figs. 12-15 Frecze-etching views ofhydrogenosomes in whole T.foetuseellsfrozenbyquick-freezing(slam-freezing). Figs 12& 13 show the outer (OEF) and inner hydrogsnosomal (IEF) membranes, respectively. Fig. 12: few particles are distributed over the hydrogenosomal membrane surface. A small pore is depicted at the middle of each protein (arrow). xl00000. Fig. 13 The hydrogenosome presents a smooth surface in its peripheral vesicle and large pore-like structures (arrow). The inner hydrogenosomal membrane is seen presenting densely packed particles, and also particles in a circular array. This micrograph is ,'1 higher magnification of Fig. 15. x l00 000. Figs 14 & 15: Hydrogenosomal matrix. Fig. 14: An array of proteins (arrowheads), and a large peripheral vesicle (P) with heterogeneous content are seen. x45 000. Fig. 15: an overall view of several hydrogenosomes (H) in different fracture planes. The hydrogenosomal matrix is seen in all hydrogenosomes, except one which presents a smooth peripheral vesicle (P) as demonstrated at higher magnification in Fig. 13. The axostyle microtubules (A) and glycogen particles (G) are seen in close proximity of hydrogenosomes (H 1. x35 000.
cross-section, and also the E face of the fractured inner or outer faces and also the matrix (Figs 10, 12-15). The hydrogenosomal matrix was rich in proteins, some of which were disposed in a special arrangement (Fig. 14). A freeze-fracture study of T. vaginalis and T. foetus was performed by Honigberg et al. (1984) and the authors presented figures of whole cells with hydrogenosomes without alterations. No description of the distribution of the intramembranous particles was presented. The hydrogenosomal membranes present special arrangement of particles as seen by freeze-etching, and at higher magnification small holes are seen at the middle of the membranous particles (Figs 12 & 13). The particle density was quite different when the E face of the outer membrane was seen: few particles arranged with a central pore (Fig. 12). Occasionally were observed membrane particles forming rosettes (Fig. 10) and in linear arrangement (Fig. 14). We were able to observe that the membrane lining the peripheral vesicle is morphologically different from the hydrogenosomal membrane envelope: it presents very few intramembranous particles, being almost smooth. It was seen in different views, depending of the fracture plane, such as a) an empty space (Fig. 2), b) a space containing material (Fig. 14), c) a membrane fractured image with particles (Fig. 5) and d) its true external surface (Fig. 13). In this last observation, it was possible to see its smooth surface with pores with uniform diameter (about 20 nm), which could represent a region of high permeability. These pores are in low density, only 7/lam2. The peripheral vesicle occupies 10 to 25% of the whole hydrogenosome area in fast-frozen cells submitted to freeze-etching. Morgado and DeSouza (1997) showed that the peripheral vesicle membrane has different properties from the enveloping hydrogenosomal membrane, although previous freeze-fracture studies did not show a special organization of their membrane proteins (Honigberg et al., 1984; Benchimol et al., 1996). The isolated peripheral vesicles maintained their flattened morphology, suggesting that each individual
vesicle could have its own inherent structural framework. Morgado and DeSouza (1997) also demonstrated by SDS-PAGE analysis of the vesicle fraction very few protein bands, especially one of 45 kDa. Previous studies performed with cryosections of T..[betus incubated in the presence of gold-labeled WGA showed positive reaction in the membrane lining the peripheral vesicle, only in the luminal portion (Benchimol et al., 1996). The disturbance of the density and distribution of intramembranous particles found in hydrogenosomes membranes after isolation procedure could be due to the hypertonic medium used during the isolation steps or an alteration of the hydrogenosome metabolic state. In the former case, the outer membrane could allow the entry of the external medium and the intermembranar space acquired the same osmotic tension, while the selective permeability of the inner membrane could lead to shrink the organellar matrix. Other studies have shown the influence of chemicals in the disorganization of protein-lipid association at specific regions of the plasma membrane (Ghosh & Nanninga, 1976) and in transformed cells (Furcht & Scott, 1975). We also examined the true external surface of isolated hydrogenosomes using field emission in-lens scanning electron microscopy (FEISEM) (Figs 16-20), which has a resolution comparable to that of the transmission electron microscope (reviewed by Allen and Goldberg, 1993). The images produced in this way are rather different to conventional SEM: the lower accelerating voltage, thinner metal coating, increased brightness, allow surface details observation and higher resolution. Therefore, details of the hydrogenosome surface were seen revealing pits and membrane invaginations (Figs 16-20), which could represent a process of material entry in the organelle. Previous study performed by Tanaka (1980), using SEM for visualization of intracellular structures, provided evidences that mitochondria present a smooth contoured outer membrane, whereas the inner mitochondrial membrane has a granular surface (for review, see Tanaka,
HYDROGENOSOME ULTRASTRUCTURE
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Figs. 16-20 Isolated hydrogenosomes seen by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). Different views of the true hydrogenosomal surface is presented: invaginations (Figs 16-18), rugosities (Figs 16-18, 19), pits (arrowheads in Fig. 19) and, peripheral vesicles (Figs 19--20) are seen. Fig. 16: xlS0000; Fig. 17: xl00,000; Fig. 18000: Fig. 19-20: x85000. Figs. 21-22 Thin-sections of isolated hydrogenosomes. The inner hydrogenosomal membrane is seen detached from the outer membrane (arrow) at some points (arrowheads)• The volume of the inner comparlment is decreased while the volume of the ouLer compartment is increased. P. peripheral vesicle. Fig. 21: x52 000; Fig. 22: x110000.
HYDROGENOSOME ULTRASTRUCTURE 525
1980). Both findings are similar to those here described for hydrogenosomes. Thin sections o f routine T E M o f isolated h y d r o g e n o somes revealed that the inner m e m b r a n e could be found separated from the outer m e m b r a n e (Figs 21 & 22), leaving only some regions o f the inner and outer m e m b r a n e s closely apposed to each other. Kulda et al. (1987) published micrographs o f dividing h y d r o g e n o s o m e s seen by freeze-fracture after glutaraldehyde fixation. They proposed that both single and double m e m b r a n e h y d r o g e n o somes could be found. In view o f o u r findings, we believe that these authors found some a b n o r m a l hydrogenosomes, in which the outer m e m b r a n e was detached as seen in the present study in isolated hydrogenosomes. Allen et al., (1989) also revealed the outer and inner m e m b r a n e s o f mitochondria to be separated after the isolation procedure. We established what is a characteristic o r t h o d o x h y d r o g e n o s o m e configuration: two closely apposed membranes, with no space between them. In isolated hydrogenosomes, by freeze-fracture, a patchwork-like structure or rosettes was frequently found, and by thinsection, a condensed configuration, where a large interm e m b r a n a r space is seen as result o f displacement o f the inner h y d r o g e n o s o m a l membrane. The two m e m b r a n e s are still connected to some extent, on the places were peripheral vesicles are located. This observation could be explained by: I. an artifact resulting from the stress o f the isolation, since it was not seen in intact cells: 2. this could be a condensed configuration, similar to that found in m i t o c h o n d r i a after being isolated (Hertsens & Jacob, 1987), where large intracristal spaces and a small matrix c o m p a r t m e n t are found: 3. this could be a real configuration, occasionally fourtd by some authors w h o described the h y d r o g e n o s o m e as an organelle enveloped by a single m e m b r a n e (Kulda et al., 1987). Studies carried out on m i t o c h o n d r i a considered the influence o f the metabolic or the energetic state on the ultrastructure o f the mitochondrial inner m e m b r a n e on the basis o f results from standardized freeze-fracture experiments (Hertsens & Jacob, 1987). In conclusion, h y d r o g e n o s o m e s studied after isolation procedures must be carefully examined since the conditions o f preparation play a crucial role in determining the final ultrastructure. It would be important to analyze if there is loss o f membrane-associated enzyme activities a c c o m p a n i e d by a corresponding alteration o f the structural components.
ACKNOWLEDGMENTS The authors wish to thank D r Wanderley de Souza for critical reading the manuscript. This investigation received financial support f r o m C N P q (Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico), P R O N E X ( P r o g r a m a de Nficleos de Excel6ncia), F A P E R J
( F u n d a g i o de A m p a r o ~ Pesquisa do E s t a d o do Rio de Janeiro), and A U S U ( A s s o c i a ~ o Universit~ria Santa 0rsula).
REFERENCES Allen, R.D. Schroeder, C.C. and Fok, A.K. 1989. An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques. J. Cell Biol., 108, 2233-2240. Allen, D. and Goldbcrg, M.W. 1993. High resolution SEM in cell biology. Trends Cell Biol., 3, 205-208. Benchimol. M. 1999.The Hydrogenosome. Acta Microscop., 8, 1-22. Benchimol, M.. Almeida, J.C.A. and De Souza, W. 1996.Further studies on the organization of the hydrogenosomein Tritrichomoaas foetus. Tiss Cell, 28, 287-299.
Benchimol, M. and De Souza, W. 1983. Fine structure and cytochemistry of the hydrogenosome of Tritrichomonaafoetus. J. Protozool., 30, 422-425. Benchimol, M., Elias C.A. and De Souza, W. 1982. Tritrichomonas foetus: U hrastructural localization of calcium in the plasma membrane and in the hydrogenosome. Exp. Parasitol., 54, 277-284. BenchimoL M., Elias, C.A. and DeSouza, W. 1981. Specializations in the flagellar membrane of Tritrichomonasfoelus. J. Parasitol., 67, 174-178. Benchimol, M., Johnson, P.J. and De Souza, W. 1996. Morphogenesis of the Hydrogenosome: An Ultrastructural Study. Biol. Cell., 87, 197-205. Chapman, A., Hann, A.O.C., Lindstead, D. and Lloyd, D. 1985. Energy dispersive X-ray microanalysis of membrane-associated inclusion in hydrogenosomes isolated from Trichomonas vaginalis. J. Gen. Microbiol., 131,2933-2939. De Souza, W. and Benchimol, M. 1988. Electron spectroscopicimaging ofcalcium in the hydrogenosomes of Tritrichomonasfoetus. J. Submicrose. Cytol. Palhol.. 20, 619-621. Diamond, L.S. 1957.The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol., 43, 488-490. Embley, T.M., Homer, D.A. and Hirt, R.P. 1997.Anaerobic eukaryote evolution: hydrogenosomes as biochemically modified mitochondria? TREE., 12, 437-441. Finlay, B.J. and Fenchel, T. 1989. Hydrogenosomes in some anaerobic protozoa resemble mitochondria. FEMS Microb. Lett., 65, 31 I-314. Furcht, L.T. and Scott, R.S. 1975. Modulation of the distribution of plasma membrane intramembranous particles in contactinhibited and transformed cells. Biochem. Biophys. Acta, 401, 213-220. Ghosh, B.K. and Nanninga, N. 1976. Polymorphism of the mesosome in Bacillus licheniformis. J. Ultrastruct. Res., 56, 107-120. Hepler, P.K. 1980. Membranes in the mitotic apparatus of barley cells. J. Cell Biol.. 86, 490--499. Hertsens, R.C. and Jacob, W.A. 1987. Freeze-fracture study of heart mitochondria in the condensed or orthodox state. Biochem. Biophys. Acta, 894. 507-514. Heuser, J.E., Reese, T.S. and Landis, D.M.D. 1976. Preservation of synaptic structure by rapid freezing. Cold Spring Harbor Symposium Quant. Biol., 40, 17-2421. Honigberg, B.M.. Volkmann, D., Entzeroth. R. and Scholtyseek, E. 1984. A freeze-fracture electron microscopy study of Trichomonas vaginalis Donne and Tritri£.homonasfoetus (Riedmiiller). J. Protozool., 31, 116-131. Johnson, P.J., d'Oliveira, C.E., Gorrel, T.E. and Mfiller, M. 1990. Molecular analysis of the hydrogenosomal ferrodoxin of the anerobic protist Trichomonas vaginalia. Proc. Natl. Acad. Sci. USA., 87, 6097-6101. Johnson, P.J., Lahti, C.J. and Bradley, P.J. 1993. Biogenesisof the hydrogenosome: an unusual organelle found in the anaerobic protist Trichomwzaa vaginalis. J. Parasitol., 79, 664-670. Kulda J., Noh~,nkovd, E. and Ludvik, J. 1987. Basic structure and function of the trichomonal cell. Acta Universitatis Carolinae., 30, 181-198. Lindmark, D.G. and Milller, M. 1973. Hydrogenosome, a cytoplasmic organeUeof the anaerobic flagellate, Tritrichomonaa foetus, and its role in pyruvate metabolism. J. Biol. Chem., 248, 7724-7728.
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Lloyd, D., Lindmark, D.G. and M011er,M. 1979.Adenosine triphosphatasc activity of Tritrichomona.~foelus. J. Gen. Microbiol., 115, 301-307. Morgado Diaz, J.A. and DeSouza, W. 1997.Purification and biochemical characterization of the hydrogenosomesof l,he flagellate protist Tritrichonmnas.[oetu.~. Eur. J. Cell Biol., 74. 85-9 I. M01ler. M. 1988. Energy metabolism of protozoa withoul, mil.ochondria. Ann. Rev. Microbiol.. 42, 465-488. MQIler, M. 1990.Sl,ructure. In: Trichomonads Parasil,icin Humans, ed. B.M. Honigberg., Springer-Verlag, New-York, 5-35. M011er, M., 1993.The hydrogenosome.J. Gen. Microb., 139, 2879-2889. MLiller, M. and Moor, H. 1984.Cryofixation ofthick specimensby higll pressure freezing. In: Scienceof Biological SpecimenPreparal,ion. vol. 2 Revel, J.P., Barnard T. and Haggis. G.H. eds. SEM. Inc., AMF O'Hare, Chicago, 131-138.
Tanaka, K. 1980.Scanning elecl,ronmicroscopy of i.ll,racellular strucl,ures. Int. Rev. Cyl,ol., 68, 97-] 25. Van VcnetiE, R. and Vcrkleij, A.J. 1982.Possiblerole of non-bilaycr ]ipids in the structure o£ mil,ochondria. A freeze-fractureelecl,ronmicroscopy study. Biochem. Biophys. Acl.a, 692, 397--405. Yarlel,l,.N.. Harm, A.C., Lloyd, D. and Williams, A.G. 1981. Hydrogenosomes in l,herumen protozoon Da3j,lricharuminanltlm Scuberg. Biochem. J., 200, 365-372. Yarlel,l,,N., Orpin, C.G., Munn, E.A.. Yarlel,t.N.C. and Greenwood. C.A. 1986. Hydrogenosomes in l,herumen fungus Neocallimaslix p