School of Biosciences (Microbiology), Cardiff University, PO Box 915, Cardiff CF10 3TL, Wales, UK1
Department of Biochemistry and Molecular Genetics2 and Cellular Analysis Facility, Department of Microbiology and Immunology3, University of New South Wales, Sydney 2052, Australia
Author for correspondence: David Lloyd. Tel: +44 29 2087 4772. Fax: +44 29 2087 4305. e-mail: LloydD{at}cardiff.ac.uk
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ABSTRACT |
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Keywords: tetrazolium dye, lower eukaryotes, confocal laser scanning microscopy, rhodamine 123, mitochondria
Abbreviations: CTC, 5-cyano-2,3-ditolyl tetrazolium chloride
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INTRODUCTION |
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Alternative scenarios for the possible interactions of archaeal and bacterial precursors giving syntrophic and then symbiotic associations have been proposed (Gupta & Golding, 1996 ; Martin & Müller, 1998
; López-Garcia & Moreira, 1999
). Here we show, for the first time, that G. intestinalis has a small number of specialized plasma-membrane-associated structures that selectively partition the cationic, membrane-potential-sensitive dye rhodamine 123. The same membrane is lined at its inner face by regions that have specialized areas which act as reducing sites for a tetrazolium fluorogen.
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METHODS |
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Growth of organisms.
G. intestinalis strain Portland-1 (ATCC 30888), originally described by Meyer (1976) , was a gift from Michael R. Edwards, University of New South Wales, Sydney. Trophozoites were cultured axenically and anaerobically in screw-capped Nunclon tubes (Life Technologies) at 37 °C on Diamonds modified TYI-S-33 medium containing 2% tryptone, 1% yeast extract, 0·5% glucose, 0·106% arginine, 0·2% NaCl, 0·1% K2HPO4, 0·06% KH2PO4, 0·1% cysteine, 0·1% bovine bile, 0·02% ascorbic acid, 0·0023% ammonium ferric citrate and 3% (v/v) minimal essential medium with Earles salt (Keister, 1983
; Edwards et al., 1989
) supplemented with 10% (v/v) heat-inactivated fetal calf serum. Subculturing was performed routinely at 48 h intervals, by replacing the spent medium for fresh without detaching the cell monolayer. Cells were harvested by replacing the spent medium with fresh, chilling the tubes on ice for 20 min and then inverting them gently to detach the monolayer. Cells were counted on a FuchsRosenthal haemocytometer slide with 0·4% (w/v) Trypan blue as the viability indicator; typically this gave cell numbers of 2x106 ml-1.
Fluorescence microscopy.
Cells were harvested and washed twice in PBS (0·8 mM KH2PO4; 5 mM K2HPO4; 150 mM NaCl; pH 7·4) to remove any residual medium, then resuspended in 0·5 ml PBS containing 5·5 mM glucose. The fluorophore, rhodamine 123, was added (10 µl of 1 mg ml-1) to the cell suspensions and they were incubated for 15 min at 22 °C. The suspensions were then washed twice in PBS and resuspended in 200 µl PBS. Cells were mounted and viewed using an Olympus BH2 triocular fluorescent microscope. Images were captured on Fuji ISO 400 (daylight) 38 mm film.
Confocal laser scanning microscopy.
Cells were incubated with rhodamine 123 (as for the fluorescence microscopy) but in 0·31 M mannitol instead of PBS, or they were incubated with CTC for 1 h before analysis to allow location of electron transport components. The cells were then viewed using a Molecular Dynamics Sarastro 2000 confocal laser scanning microscope. Specimens were scanned by using a 25 mW argon laser, with the appropriate excitation and emission filters. Specimens were examined using oil emersion objectives, x60 (50 µm confocal aperture) and x100 (100 µm confocal aperture). Series sections through samples were taken at 1 µm intervals (512x512 pixels; 0·5 µm thick), and three-dimensional constructs were prepared using Molecular Dynamics Volume Workbench, running in a Silicon Graphics UNIX workstation.
X-ray microanalysis.
Cells in growth medium were fixed in 1·25% glutaraldehyde in 0·1 M sodium cacodylate buffer, pH 7·4, for 5 min at 4 °C. After washing twice in the same buffer, the cells were resuspended in 50 µl of tetrabromorhodamine 123 (1 mg ml-1 in methanol), incubated at room temperature for 30 min and then washed twice. After fixation for a further 5 min at 4 °C followed by three washes in water, cells were washed for 20 min in 70% ethanol at 4 °C. Freeze-substitution (AFS, Reichert) was programmed to give a temperature increase from -25 °C to room temperature over 52 h. Dehydration of the cells in 100% ethanol (90 min) was followed by embedding them in a graded series of Lowicryl HM20 resins (50, 70 and 95% in pure ethanol, respectively, for 30 min each), then washing them twice with 100% Lowicryl for 30 min. Pure Lowicryl was polymerized under UV light for 48 h, during which time the temperature was increased gradually from -25 °C to room temperature. Sections, 0·1 µm thick, were cut using glass knives on a Reichert Ultracut microtome; these were then mounted on pioloform-coated copper grids. The sections were analysed using a JEOL 1210 transmission electron microscope fitted with an energy-dispersive X-ray analyser (ISIS system). X-ray dot mappings of Br were collected over 2 h, and the spectra were obtained over 100 s. The characteristic X-ray emission peaks of Br K were detected at 11·92 keV.
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RESULTS |
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DISCUSSION |
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Hydrogenosomes are also now believed to have been derived from a mitochondria-like ancestral organelle (Biagini et al., 1997c ; Embley et al., 1997
) as well as evidence from their molecular biology, they share some structural characteristics (inner and outer membranes, Finlay & Fenchel, 1989
) and functions [sequestration of Ca2+ (Chapman et al., 1985
; Humphreys et al., 1998
; Biagini et al., 1997
c, d
) and transmembrane electrochemical potential (Humphreys et al., 1998
)] with mitochondria. The discovery of a ciliate hydrogenosome with DNA that shows high sequence similarity with mitochondrial SSU rRNA genes from aerobic ciliates (Akhmanova et al., 1998
) confirms the mitochondrial origin of hydrogenosomes.
Mitochondrial remnants have been described in the anaerobic parasite Entamoeba histolytica (Rodriguez et al., 1998 ). An organelle bound by two membranes has been discovered in this organism by using antibodies raised against
protein (cpn 60), which is specific to mitochondria (Tovar et al., 1999
; Mai et al., 1999
). These drastically modified remnants of the redox organelle, like hydrogenosomes, are an evolutionary consequence of attenuated function in low-O2 environments.
Rhodamine 123 is a cationic, membrane-potential-sensitive dye, which has been used extensively to locate mitochondria. It is electrophoresed through plasma membranes and then from the cytosol into the mitochondrial matrix space in energized mitochondria, the ratios of fluorophore concentrations may reach 104:102:1 between the intra- and extra-mitochondrial spaces and the extra-cellular suspending fluid, respectively (Chen, 1988 ). Rhodamine 123 has been used to stain mitochondria specifically in aerobically grown yeast (Lloyd et al., 1996
), and it also accumulates in the hydrogenosomes of Trichomonas vaginalis (Harris, 2001
). Similar specificity of uptake of the cationic cyanine dye dihexyloxacarbocyanine has been demonstrated in these hydrogenosomes (Humphreys et al., 1998
), and its uptake has also been demonstrated in the free-living, anaerobic ciliate Metopus contortus (Biagini et al., 1997b
). Our observations of structures with mitochondria-like functions in G. intestinalis further suggest the loss of fully fledged mitochondria from this parasite and the secondary nature of its amitochondrial status. The particulate nature of NAD(P)H- and pyruvate-driven electron transport chains has previously been demonstrated by subcellular fractionation after gentle mechanical disruption (Ellis et al., 1993
). Subcellular fractionation, after gentle mechanical disruption, of G. intestinalis has indicated that pyruvate:ferredoxin oxidoreductase, and NADH and NADPH oxidoreductases are mostly non-sedimentable (73%, 57% and 68%, respectively) after centrifugation at 100000 g for 1 h (Ellis et al., 1993
). The acceptors used in that study were methyl viologen for pyruvate-driven electron transport and FMN for the nicotinamide-nucleotide-linked activities. The fluorogenic tetrazolium used in the present study acts as an electron acceptor for all three donors. In organisms with mitochondria, this compound, like other tetrazolium salts with redox potential values similar to CTC (Lloyd, 1974
), has been used as a cytological marker for respiratory chain activities of the inner-mitochondrial membrane.
Extensive studies using transmission electron microscopy have not yet positively identified the nature of the structures revealed by optical methods. Multi-lamellar residues were present in some of the organisms, and their frequency of occurrence was greater after controlled exposure to low concentrations of O2 (Lloyd et al., 2000 ; Harris, 2001
). Further work is necessary to establish the precise physiological functions of the discrete membrane structures, shown by the fluorescence methods used here, and those of multi-lamellar bodies.
Electron transport and the development of membrane potential are characteristic activities of the inner-mitochondrial membrane. We might suggest that although the habitat of the trophozoite is a low-O2 environment and its energy metabolism uses characteristically anaerobic routes, not all mitochondrial functions are dispensable. The consequent structure/function relationships of the specialized membraneous elements that perform the redox-active and trans-membrane electrochemical-potential-generating activities described here may be considered as the solution that has been evolved in an organism where there are no longer fully fledged mitochondria (Lloyd et al., 1983 ).
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ACKNOWLEDGEMENTS |
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Received 31 October 2001;
revised 20 January 2002;
accepted 7 February 2002.