morphogenesis of the hydrogenosome: an ultrastructural study

Biol Cell (1996) 87, 197-205 0 Elsevier, Paris 197

Original article

Morphogenesis of the hydrogenosome: An ultrastructural study

Marlene Benchimol a, Patricia J Johnson b, Wanderley de Souza a-c

“Laboratdrio de Biologia Celular e Tecidual, Centro de Biocibncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Av Albert0 Lamego, 2000, Horto, CEP 28015620,

Campos dos Goytacazes, Rio de Janeiro, Brazil; b Department qf Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA, 90095-l 747, USA;

CLuboratdrio de Ultraestrutura Celular Hertha Meyel; Institute de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil

(Received 15 April 1996; accepted 21 November 1996)

Summary - The morphogenesis of hydrogenosomes in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming eniongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.

hydrogenosome / ultrastructure / division / Trichomonas

Introduction

Evolution from prokaryotic to eukaryotic cells was accom- panied by the formation of organelles with well defined functions such as mitochondria and peroxisomes. Eukar- yotic microorganisms living in environments poor in oxy- gen developed a special organelle first described in trichomanads as the hydrogenosome [28]. This organelle produces molecular hydrogen by oxidizing pyruvate. Later, hydrogenesomes were described in rumen ciliates [5, 32, 38-401, some free-living ciliates [36] and some fungi [41].

Previous morphological studies carried out mainly in trichomonads have shown that the hydrogenosomes are spherical or slightly elongated structures. We have shown previously that they are enveloped by two closely apposed membranes [3], with distinct distribution of intramembra- nous particles [2]. In addition, the hydrogenesomes present a special inner compartment [24, 303 designated as peripherical vesicle which accumulates calcium [4], whose membrane is labeled with wheat germ agglutinin [2]. Inter- nal invaginations of hydrogenosome membranes are also seen in its matrix [2].

There are very little data on the biogenesis of the hydrogenosome and its relationship with other cell structures [6]. A few images suggestive of a process of division of this organelle have been published [12, 22, 24, 311. However, these studies were incomplete and did not show clearly the division process.

We have been studying the fine structure of Tritrichomo- nas foetus, a parasitic protozoan of urogenital tract of cattle, as well as other related protozoa, such as T gallinae,

T augusta, T suis, T vaginalis and Monocercomonas sp. In this report we present images of the binary division of hydrogenosomes. Although they are static views of a highly dynamic event, they contribute to the elucidation of the biogenesis of this organelle.

In addition, our observations also show the relationship of the hydrogenosome to filamentous structures, glycogen granules and the endoplasmic reticulum (ER).

Materials and methods

The K strain of Tritrichomonas foetus was isolated by H Guida (EMBRAPA, Rio de Janeiro, Brazil) from the urogenital tract of a bull from the state of Rio de Janeiro, Brazil. Trichomonas vaginalis, strain 236, from ATCC, Trichomonas gallinae, strain T69, Tritrichomonas suis, strain TSM were isolated by Gerald0 DeCarli, in the state of Rio Grande do Sul, Brazil. Monocerco- monas sp was obtained from Jaroslav Kulda (Charles University, Czech Republic). All parasites were cultivated in TYM Diamond’s medium [lo], for 24 h at 36S”C, except Monocerco- monas sp which was cultivated at 28°C.

Electron microscopy

Cells were fixed overnight at room temperature in 2.5% (v/v) glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2). After fixation, the cells were washed in PBS (phosphate buffered saline) and post- fixed for 15 min in 1% 0~0, in 0.1 M cacodylate buffer plus 5 mM CaCl, and 0.8% potassium ferricyanide [17]. Cells were then washed in PBS, dehydrated in acetone and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and

198 M Benchimol et nl

observed in a Zeiss 900 or 902 electron microscope. in order to concentrated near the costa (not shown) and the axostyle localize Caz+-binding sites in some preparations 5 mM of CaCl, was added to the glutaraldehyde and washing solutions [%I.

(fig 1). Most are spherical; however, some, possibly in the process of division, were elongated (figs 14-18).

Cytochemistry

ThiLry ‘s technique, for carbohydrate detection. Periodic acid thiosemicarbazide-silver proteinate Ultrathin sections of cells fixed in glutaraldehyde and 0~0, were collected on gold grids and tretead For 20 min with 1% periodic acid. After rinsing in distilled water the sections were incubated for 12 h in aqueous solution containing 1% thiosemicarbazide, and 10% acetic acid. Thus, after rinsing sequentially with 10, 5, and 1% acetic acid and distilled water, the sections were exposed to silver proteinate (1%) for 30 min in the dark at room temperature. Sections were observed unstained [3J]. Controls were done by omission of periodic acid step.

A special compartment was observed at the periphery OZ almost all hydrogenosomes (figs 2, 10, 18, 22). It varied in size and electrondensity (figs 2, 10, l&22); in some preparations it appeared empty (fig 11) while in others it presented a certain content when the cells were fixed in a glutaraldehyde solution containing calcium and post-fixed in a reduced osmium solution (figs 18, 22). An electron-dense reaction product occupied the whole compartment (figs l&2 1).

Freezing methods

After overnight fixation in 2.5% giutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), the cells were wasbed twice in PBS and then exposed to increasing concentrations of glycerol in cacodylate buffer until a final concentration of 30% glyceral was attained. Specimens were mounted on BaIzers 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 Vat, Inc, St Louis, MO) designed by Heuser et al [lS]. A polished copper block was cooled with liquid nitrogen and the specimen was projected in the free-fall against the block (‘slarn- freezing’) [19]. 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 IO-6 TOIT or submited to deep-etching (10 min at -1OO’C) and rotatory shadowed with platinum at an angle of 15”. Replicas were recovered in distilled water, cleaned with sulfuric acid and/or sodium hypochloride, mounted on 200-mesh grids, and examined in a Zeiss 902 transmission electron microscope.

In thin sections and freeze-fracture images it was pas.. sible to see profiles of the endoplasmic retitiulum (ER) lat- erally touching the membrane of the hydrogenosome (figs 2,4,7, 10). In favorable sections, as shown in figures 2 aud 6. it was possible to see long CisEemae of the ER surroundW ing the whole hydrogenosome. The endoplasmic reticulum observed in association with the hydrogenosome could be smooth or rough (figs 2,6-8, 10). Direct continuity of these structures was observed (figs 5, 1 l-l?). This association was also seen in sections submitted to the periodic acid-thiosemicarbazide-silver proteinate technique, which reveals carbohydrate-containing structures (fig 8). This was also frequently observed in conventional freeze-fracture replicas (figs 3, 4). Concentric membranous structures, here designated as myelin-like structures, were frequently observed (figs 9, 10). In replicas of deep-etched cells, filamentous structures were seen connecting the hydrogenosome to each other (figs 29, 30). These filaments were 10 nm thick and may reach a length of 30 nm (fig 29).

Most of the hydrogenosomes were surrounded by glyco-- gen particles and rosettes as observed in freeze-etching and routine preparation of well preserved cells (figs 16. 18, &9. 22,26,27, 30).

Images ofdivision

Cell fractionation

Hydrogenosomes were isolated as previously described by La&i et al [25]. Briefly, T vaginalis ceils were harvested, washed and resuspended in SMD (0.25 M sucrose, 10 mM morpholine pro- pane sulfonic acid, pH 7.2, 10 mM dithiothreitol). Cells were lysed in a cell disruptor (Stansted) and centrifuged at 1000 g for 10 min at 4°C. Hydmgenosomes were purified by mixing an equal volume of the supematant with 90% Percoll in SMD, followed by centrifugation at 30 OW ‘pm for 1 h in a vertical VC53 rotor (Beckman). The lower band containing hydrogenosomes was collected, washed in three times with SMD and fixed in 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M cacodylate @H 7.2).

Examining a large number of sections, we occasionally found unusual hydrogenosome profiles with elongated and dumb-bell shapes (figs 14-18). Some of these were just elongated but without any constriction area. However, others contained a constriction, suggestive of a division process (figs 14, 15, 17, 18). FibriBar-lie structures were seen associated with the cleveage region (figs 14, 15). In some elongated hydrogenosomes, tubular membranous profiles were seen in the constriction area (fig 16).

Results

Association of the hydrogenmom with other structures

Examination of a large number of thin sections of Tfoetus showed hydrogenosomes scattered all around, but mainly

Spherical hydrogenosomes, presenting a straight trans- verse ‘septum’ were seen (figs 21-24). Images suggesting the gradual ‘septum’ formation are presented (figs 19-22). At higher magnification it was clear that the ‘septum’ consisted of two closely apposed unit membranes continuous with the inner membrane lining the hydragenosome (figs 21-23) thus separating the hydrogenosomal matrix into two distinct compartments. The outer membrane did not penetrate into the matrix and was not part of the ‘septum’. During the process of hydrogenosome division there was also formation of two peripherical vesicles which later were seen at opposite

Figs 1-6. 1. General view of Tritrichomonas foetus in a routine thin section. The arrow points to a hydrogenosome in division. G, Golgi k complex; H, hydrogenosomes; N, nucleus; V, vacuole. x 11 OOO. 2. Tfoetus fixed in gluta.raidehyc-Ie and post-fixed in osmium tetroxide solution containing potassium ferricyanide. Profiles of endoplasmic reticulum (arrowheads) are seen close to the hydrogenosomes (II). x 24 000. 3-5. Freeze-fracture images of Tfoetus. The association of the endoplasmic reticulum (arrows) with hydrogeriosomes (H) is demonstrated. Arrowhead in 4 points to a final step of hydrogenosome division. ER, endoplasmic reticulum; G, Golgi complex. x 66 000. In 5 a hydrogenosome (H) shows continuity (arrow) with the endoplasmic reticulum. x 50 000. 6. A close association OF a hydrogenosome (H) and the endopiasmic reticulum (ER) is observed. x 80 000.

Morphogenesis of the hydrogenosome 199


Figs 14-18. Views of the segmentation process of dividing hydrogenosomes. The organelle is elongated showing a constriction in the central region (arrow). Microfibrillar (arrow) and tubular (arrowhead) structures are associated with the division process. H, hydrogen@ some; G, glycogen; N, nucleus. 14,X Tfoetus, x 50 000.16. Monocercomonas sp, x 60 000. 17,X T vaginalis, end of segmentation process. In 17 the hydrogenosomes were obtained by cell fractionation, x 40 000. In 18, the thin arrows point to calcium deposits. x 40 000.

poles (fig 22). Figures 25 and 26 show the outer membrane enveloping two organelles. These partition figures are not frequent but could be found at any stage of the cell cycle.

find hydrogenosomes in different steps of the process of division (figs 17,25).

Carbohydrate-containing macromolecules are present in the membranous ‘septum’, as indicated by deposition of electron dense reaction product after treatment with the Thitry technique (fig 24).

Thin sections of isolated hydrogenosomes ware carefully screened by electron microscopy. It was always possible to

Discussion

Previous studies have shown that the hydrogenosomes are spherical and have no branching structures usually located close to the costa and the axostyle of trichomonads [2, 21,

4 Figs 7-13. 7. Some ribosomes are found associated with the reticulum membranes and very close to the hydrogenosomal membrane in Tfoetus. Profiles of rough endoplasmic reticulum are better visualized in this figure. ER, endoplasmic reticulum; GC, Golgi complex; G, glycogen; H, hydrogenosomes. x 36 000.8. Detection of carbohydrates in Tfoetus using the periodic acid-thiosemicarbazide-silver proteinate technique. Reaction product is found in glycogen granules (G) and in membranes of hydrogenosomes (H), vacuoles (V) and nuclear envelope. The membrane of hydrogenosomal peripheral compartment presents a more intense reaction for carbohydrates. The arrows point to membranous structures in close association to hydrogenosomes. N, nucleus. x 30 000. 9. Tfoerus presenting myelin-Iike membranes (arrows) in association with a hydrogenosome (H). x 100 000. 10, 11. These micrographs show the association of membranes (arrows) with the hydrogenosome (H) in Tfoetus. The close proximity of the endoplasmic reticulum is pointed (arrowheads). x 35 000. 12, 13. These figures show continuity of the hydrogenosome envelope with a tubular extension (arrowheads) in Tfoetus. The arrow in 13 points to the hydrogenosome double membrane. 12, x 20 000; 13, x 70 000.

202 M Benchimol et ai

Figs 19-24. Hydrogenosomes dividing via a partition process. The hydrogenosome (H) becomes larger and a invagination of the inner hydrogenosomal membrane is observed (arrow), gradually dividing the hydrogenosomal matix in twa campartements. 19. T vagiridis showing an early step of inner membrane invagination (arrow}. The arrowheads point to the peripherkcakium compartment. x 75 000. 20. Tfoetus presenting a membrane invagination as seen by freeze-fracture. x 35 000. 21. Hydrogenosome of T augusta showing a late step of the partition division. x 90 000. 22. Z’ritrichomonas suis presenting an hydrogenosome (H) with a transversal ‘septum’, wkich is formed by an invagination of the inner hydrogenosomai membrane (arrow). Two opposite periphericai cakium positive compartmenis are indicated by arrowheads. x 135 000. 23. Tritrichomonas augusta showing the same process of division. x 75 000. 24. Trichomona.~ gallinae after II&y’s technique used for demonstration of carbohydrates. A hydrogenosome (H) in the process of division by partition shows a positive reaction in the hydrogenosomal envelope and in the ‘septum’ (arrow). x 36 000.

241. This idea was recently supported by tridimensional reconstruction of the organelle [2]. As shown previously [3, 4, 91, when fixed in a glutaraldehyde-solution containing Ca2+ and post-fixed in a osmium solutiun containing Ca*+ and potassium ferricyanide, an electron-dense reaction product appears in the peripheral compartment. Using electron spectroscopic imaging these authors showed that the electron-dense reaction product seen in the hydrogenosomes

represents Ca*+-containing sites. Using this approach it was easy to recognize the hydrogenosome in g%cess of division.

Freeze-etched views after quick freezing of Iiving cells showed frlamentous strvctures projecting from and~conn&t- ing one hydrogenosome to the other. This f&mentous material also projects from the outer hydrogenosomal membme to other cytoplasmic particles, such as glycogen gram&es. These morphological data may suggest that hydrogenosomes


Figs 2530. 25-27. Progressive separation of the hydrogenosome into two compartments. 25,26. Initially the inner membrane separates the hydrogenosome (H) in two compartments, but they are still joined by the outer hydrogenosomal membrane (arrows). 25. A purified hydrogenosome isolated by Percoll-sucrose density centrifugation. 26. Glycogen rosettes (G) surrounding the hydrogenosome. 27. A final step of the division process by partition, showing two new hydrogenosomes that are still very close to each other. 25. T vaginalis, x 55 000. 26. Trichomonas gallinae, x 100 000. 27. Monocercomonas sp, x 100 000. 28. Freeze-fracture of a hydrogenosome (H) from T vaginalis showing the segmentation process of division. The furrowing regions exhibit a different pattern in the inner membrane (arrows). x 30 000. 29, 30. Freeze-etched views of T foe&s after quick freezing of living cells. The arrows point to filamentous structures projecting from and connecting one hydrogenosome (H) to the other. An asterisk indicates the peripherical compartment of the hydrogenosome. x 60 000. 30. Profiles of endoplasmic reticulum (ER) are seen very close to the hydrogenosome (H). Filamentous material (arrows) project from the hydrogenosomal membrane to other cytoplasmic particles, such as glycogen granules. x 140 000.

are kept together by these filamentous structures. It is not an drtefact since they were found in both living and fixed ceIis processed for freeze-fracture and deep-etching ES].

Using the periodic acid-thiusemicarbazide-silver proteinate technique, which reveals the presence of carbohydrates, a reaction product was seen in the membranes lining the hydrogenosomes, even in the ‘septum’. The reaction product was also seen in the membranes of the endoplasmic reticulum found close to the hydrogenosomes, suggesting that the origin of the carbohydrates found in the liydrogenosome membrane may originate from the endoplasmic reticulum.

Nielsen and Diemer [31] stated that the partition process does not exist in .hydrogenasomes, in contrast to what has been reported for mitochondria. Kulda et al [24] have presented freeze-fractured images of hydrogenosomes of Tfoe- tus in division. They were elongated and showed a furrow at the mid-region that was named as ‘septum’ by the authors. However, the images presented did not show the partition process. Our present observations show that the hydrogenosome may divide using two processes: segmentation and partition, as previouly described for mitochondria [7, 27, 341 pointing out additional similarities between the two organelles. Morpholugicd data have shown two distinct ways for mitochondria division: segmentation and partition [7]. Segmentation is defined as a replication process where there is an elongation of the organelle and a progressive attenuation of its mid-region. Membrane fusion occurs in the cleavage area. Fission then occurs and two new organelles are formed. In the case of partition, the organeile division begins by an inward furrowing of the inner organelle membrane, as occurs in cell division in many bacteria [I] and mitochondria [27]. This process invdves the formation of a membranous septum, which separates the mito- chondrion into two distinct chambers. Progr&sive constriction of the organelle at the level of partition ultimately resulted in formation of two daughter mitochondria [34]. In the present study using parasitic trichomanads (Tfoe&s, T vaginalis, T suis, T augusta, T galliwe and Monocerco- monad sp) we have seen images very similar to those described for mitochrondria division in all of these cells. Few differences were found among the different species here studied. In Monocercumonas sp the hydrogenosomes in process of division were often elongated, much larger then those found in the other groups.

It was possible to find tubular and fibrillar-like structures at the constricted mid-region suggesting involvement of cytoskeletal structures and/or profiles of the endoplasmic reticulum in the division process. Further studies are necessary to clarify the role played and the nature of these structures in the constriction zone. The process of division by segmentation was only observed in elongated hydrogeno-, somes. In contrast, the partition process took place in spherical hydrogenosomes. Observations of hydrogenosomes isolated following cell disruption and centrifugation frequently showed organelles in the process of partition. Elongated hydrogenosomes were uncommon in this preparation.

Our electron microscopic studies clearly demonstrate that new hydrogenosomes are generated by division of pre- existing organelles and do not support budding of new organelles from the endoplasmic reticulum. We and others [2* 1.5, 241 have observed an intimate association of the hydrogenosome with the endoplasmic reticulum (ER). As the hydrogenosome enlarges before its division, new membranes have to be formed. The ER could participate by pro- viding the membrane lipids. Previous studies on the peroxisome morphogenesis in hepatocytes revelead continuity between ER and peroxisomes [201, leading to the sugges-

tion that the loop- or hook-shaped smooth endoplasmic reticuhim could play an important +ole in the initial stage of peroxisoma formation. The images we observed show& an association of the ER with the hydrogenosomes are very similar to those reported for the peroxisomes.

Mitochondria and peroxisomes require special mecha- nisms for the import of proteins and lipids for their growth, Most mitochondria proteins and all of peroxisome proteins are imported from the cytosol, where they are synthesized by free ribosomes. These organelles do not synthesize lipids de mvo; instead, their lipids have to be importe’d from the ER, either directly or indirectly via other cellular niem- branes. It is well known that the extensive ER Retwork serves as a factory for the prod@ction of almost all the cell’s lipids. Exchange proteins act to distribute phos@olipids~at random among all membranes present in a cell Ii ]. The double-membrane loops and myelin-like structures frequently observed in the hydrogenosome surface 127 may represent ;> site of insertion of lipids into hydrogenosomes. Similar structures have been observed either in direct contact with. or in the vicinity of liver peroxisomes of rats treated with hypolipidaemic drugs which induce peroxiscime prolif’era- tion [ 11,371. Lahti and Johnson [26) have shown- that hydra-. genosomal proteins in T vagina& are synthesized on free polyrihosomes, released in the cytoplasm and subsequenfy translocated into the organelle. Proteins that are sorted into organelles which multiply by fission, invariably.are niade on free rihosomes, while organelles that multiply by budding from endoplasmic reticulum are synthesized on membrane-,~ associated ribosomes [ 13, 14, 16,231. .lohson and colleztgues have demonstrated that hydrogenosomaf proteins -contain amino-terminal, cleavable sequences and have hypothesized that these sequences are the signals that direct proltens to the organelle [23]. Interestingly, hydrogenosomal amino. terminal sequences are similar to mitochondrial targeting sequences. They both have an amino-terminal location, are cleaved from the mature protein found in the organ&e ami have similar amino acid composition. The major difference between these sequences is in length. The amino tefminal extensions on hydrogenosornal proteins are typically 8-12 amino acids, whereas presequences of yeast as&mamma&m mitochondrial proteins are typically 20-80 amino acids.

The morphologicai observations reported here contiibute to our understanding on the structural organization of the hydrogenosomes which resembles that observed for mitochondria. Other characteristics that are shared by hydrogenosomes and mitochandria include: a) presence of two membranes, with internal specializations~ [2]; b) Ca2+ coti-. centration [4]; and c) ATP production [29]. Similarities between hydrogenosomes and peroxisomes also exist, such as: a) the general shape and electron density of the matrix; and b) the association with the endoplasmic reticulum. Simi - larities between hydrogenosomes and mitochondria have led to the hypothesis that hydrogenosomes are converter mitochondria [81. Alternatively, it has also hn propdsed that hydrogenosomes arose by symbiosis of an anaerobic bacteria with a primitive host cell [29]. Additional studies will be necessary to clarify whether hydrogenosomes origi- nated as an independent or have a common origin with mitochondria.

Acknowledgments

This work has been supported by Financiadora de Estudos e Pm- jetos (FINJZP), Conselho National de Desenvolvimento Cietitif- ice e TecnoMgico (CNPq) and Funda@o Estadual Norte Flumi-


nense (FENORTE). The authors thank Mrs Marcia Adriana Car- valho for help in the preparation of the photographs.

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morphogenesis of the hydrogenosome: an ultrastructural study

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