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Effects of total anaerobiosis on the mitochondrial system of Rhodotorula gracilis
Ceil Differentiation, 7 (1978) 223--233 © Elsevier[North-Holland Scientific Publishers Ltd.
223
E F F E C T S OF T O T A L ANAEROBIOSIS ON T H E M I T O C H O N D R I A L SYSTEM O F R H O D O T O R U L A G R A C I L I S G.R. ROSSI and M. COCUCCI Istituto di Scienze Botaniche. Universitd di Milano, Centro di studio C.N.R. per la Biologia Cellulare e Moleeolare delle Piante, via G. Colombo 60, 20133 Milano, Italy
Accepted 12 March, 1978 Absence of oxygen induced marked changes in the mitochondrial system of the obligate aerobic yeast Rhodotorula graeilis. The number of mitochondrial profiles diminished progressively in proportion to increase in time of anaerobic growth. After 8 h of anaerobiosis, when the cell survival rate was still high (95%) almost al[cells showed a single broad mitochondrial profile. Protein and RNA synthesis were blocked, and cytochrome levels a~, a and c remained unchanged. When oxygen was again introduced, growth continued and the cell promptly recovered from all the changes observed under anaerobic conditions. The morphological changes in the mitochondrial system were interpreted as a change of form in what was probably a single mitochondrion.
R h o d o t o r u l a gracilis, an aerobic yeast, unlike facultative aerobic yeasts, u n d e r w e n t a complete block o f biosynthesis and metabolism under anaerobic conditions, and appeared t o have no energy-producing fermentative mechanism although its survival rate was total for 8 h (Cocucci, 1972). When R h . gracilis was cultivated under low-oxygen conditions sufficient t o permit 30% o f m a x i m u m growth rate, t he levels o f certain mitochondrial enzymes and the mitochondrial system as a whole u n d e r w e n t a substantial increase; accordingly there was no fall in availability o f % P; t he increase o f the mitochondrial system seemed to constitute an adaptation of t he organism t o low-oxygen conditions (Cocucci et al., 1975). This paper is based on the supposition t h a t if t h e mitochondriaI'increase observed in low-oxygen conditions is t o be seen as an adaptation, no such d e v e l o p m e n t will be observed if t ot al anaerobiosis com pl et el y blocks the metabolism. It was thus felt t hat it would be interesting t o investigate t he effects o f anaerobic conditions on t he mitochondrial system o f R h . gracilis.
MATERIALS AND METHODS Cultures o f R h . gracilis were set up as described previously (Beffagna et al., 1972). Exponentially growing cells were harvested by filtration on cellulose acetate filters (Sartorious, I, 2 p diameter) at 4°C and resuspended to a density corresponding t o 1 O.D.660 nm, in a minimal m edi um containing 2% glucose and 2% a m m o n i u m sulphate. These cultures were transferred t o a glass-stainless steel f e r m e n t at 30°C fitted out with an 02 pressure cont rol
224 system consisting of a Clark polarographic electrode (Radiometer, Copenhagen) (Cocucci et al., 1975). Anaerobic conditions were obtained by a flow of highly-purified nitrogen.
Biochemical determinations Cells were collected by filtration on Sartorious membrane filters and thoroughly washed free of traces of the medium by repeated washing in ice-cold incubation medium. Cells were fractionated according to the Nieman and Poulsen method (1963). Incorporation of L-[1-'4C]leucine (5 X 10 -4 M, spec. act. 40 pCi/mmol) into proteins was determined by measuring radioactivity into the NaOH soluble, PCA insoluble fraction. The acid-soluble fraction was assayed for intracellular level of free leucine. Incorporation of [32p] orthophosphate {5.73" 10 -3 M, spec. act. 0.35 ~Ci/ pmol) into RNA was determined by measuring the radioactivity present in the acid-soluble fraction obtained after alkaline hydrolysis of PCAinsoluble fraction. Intracellular level of inorganic orthophosphate was evalued according to Lindberg and Ernester (1956), as the radioactivity of phosphomolybdic complex of acid-soluble fraction. Radioactivity was determined in a Packard TriCarb liquid-scintillation counter. Cytochromes were assayed in intact cells with a Dual Wavelength Aminco-Chance spectrophotometer according to Chance (1957) as previously described (Cocucci et al., 1975). ATP level: cells were collected at 4°C by rapid filtration (approx. 6") under the same oxygen pressure on cellulose acetate filters and extracted in cold perchloric acid. ATP level was determined by enzymatic method according to Lamprecht and Trautschold {1974).
Electron microscopy Cells were harvested as described previously, washed twice with cold distilled water and resuspended in fixating solutions. Fixation and inclusion were performed as reported in a previous paper (Cocucci and Rossi, 1972). In this case we chiefly used permanganate fixation because it was more convenient for evaluating mitochondrial membranes.
Analysis of micrographs For the evaluation of the number of mitochondria, sections were cut a constant thickness and sampled at intervals sufficiently long to ensure that every cell was photographed once. Mitochondrial profiles were counted on the micrographic prints. Three-dimensional mitochondrial profiles were analysed by ultrathin serial section technique, transferring ribbons of 30--40 consecutive sections, about 70 nm thick, onto single-hole copper grids covered with Formvar and carbon layers. The mitochondrial and cell area were determined according to Hawley and Wagner (1967).
225 RESULTS
Morphological observations Fig. 2 shows Rhodotorula gracilis cells grown in total anaerobiosis for 8 h. Comparison with control cells cultivated under normal aerobic conditions (Fig. 1} shows that the mitochondrial profiles o f the anaerobic cells become larger, whilst they simultaneously become less numerous until there is a single profile for each cell. The fall in the number of mitochondrial profiles per cell after progressively longer periods of anaerobiosis is gradual, as can be seen in Table I. Under normal conditions of growth only 19% of cell sections examined have a single mitochondrial profile, whilst under strict anaerobiosis this percentage rises gradually to 60% after 2 h and to 90'% after 8 h. To ascertain whether this reduction in the number of mitochondrial profiles represented an effective reduction in the number of mitochondria/ cell, we used conventional ultrathin serial sectioning techniques to analyze the shape of the mitochondria of Rh. gracilis. Fig. 3 presents an approximate diagrammatic reconstruction of the mitochondria of part of a Rh. gracilis cell cultivated under normal aerobic conditions: the various mitochondrial profiles present in a given section of the cell do not correspond to so many different organelles, but represent sections of a single mitochondrion with long thin ramifications. This indicates that the mitochondrial system of Rh. gracilis consist of a single mitochondrion or o f at most a few mitochondria. The reduction in the number of mitochondrial profiles observed in sections of cells grown under strict anaerobiosis is quite conceivably the result of changes in the form of the organelle. Only the mitochondrial system is apparently affected during the first 4 h of anaerobic treatment. No change can be detected in the nucleus and in the density of ribosomes (this has been observed in electron micrographs of osmium-fixed samples that are n o t reported here). If anaerobic conditions are maintained for a longer time (up to 8 h) the mitochondria often show clear s y m p t o m s of degradation: the matrix becomes progressively less electron-dense and the cristae change s in form and distribution (Fig. 4). Moreover Table II shows that under anaerobiosis mitochondrial area increases in comparison with the area of whole cell. Since the percentage of area occupied b y any cellular inclusion in a random cell section is indicative o f the percentage v o l u m e in the whole cell, the data in Table II suggest a significant increase in the volume of the mitochondrial system. After 8 h of total anaerobiosis the nucleus also shows alterations: the nuclear envelope becomes discontinuous with large gaps and swelling of the perinuclear space (Fig. 5).
Reversibility of the effects o f anaerobiosis When non-growing anaerobic cells are brought back to normal aerobic
226
Fig. 1. Logarithmic phase cells o f Rh. gracilis grown under aerobic conditions. N, nucleus; M, mitochondria; Va, vacuole. Fig. 2. Rh. gracilis cells grown for 8 h in total anaerobiosis.
227 TABLE I Variations in the number of mitochondrial profiles in cells of Rh. gracilis after strict anaerobiosis and return to normal conditions for 2 h. Growth conditions
% Cell sections with
Aerobiosis icontrol) Anaerobiosis 2 h Anaerobiosis 8 h Recovery from 2 h anaerobiosis Recovery from 8 h anaerobiosis
1 mitochondria
2 mitochondria
3--4--5 mitochondria
19 60 90 29 56
28 31 10 39 38
53 9 0 32 6
No. of cell sections examined
66 95 92 75 70
71 35-
"~ 70-
7
~
10
o
~ lo5-
15-~ !
140--
20"
175--
25: Mitochondrial
profiles
in cell section
Fig. 3. Schematic profile o f mitochondrial apparatus o f a portion of Rh. gracilis cell grown under normal aerobic conditions, obtained from an intact ribbon of consecutive ultrathin sections, starting from the periphery o f the cell. Thickness o f sections: 70 rim. The continuous lines represent, approximately, the position and long axis of sectioned mitochondria; the dotted lines their tangential sections.
228
Fig. 4. Anaerobic Rh. gracilis cell showing a single broad mitochondrial profile whose internal membrane system is deeply altered. Fig. 5. Rh. gracilis cells grown for 8 h or longer in anaerobiosis. Note the gaps of the nuclear membrane.
229 T A B L E II C h a n g e s in m i t o c h o n d r i a l a n d cell area o f Rh. gracilis cells a f t e r 8 h o f anaerobiosis. Growth conditions
Mitochondrial area (~t2 )a
Cell w h o l e area (~t2 )a
Aerobiosis ( c o n t r o l ) Anaerobiosis 8 h
0.718 1.15,0
5.22 5.47
a Average o f 109 d i f f e r e n t a n d r a n d o m cell sections.
conditions growth is resumed; the morphology also becomes normal again. In almost all cells the nucleus appears to be normal 2 h after restoring aerobic conditions (Fig. 6); where the nucleus is still damaged repair processes are seen to be under way (Fig. 6a). The mitochondria also resume the aspect typical for aerobic conditions both as regards the number/cell and length and distribution of the cristae {Fig. 6a).
Fig. 6 and 6a. Rh. gracilis cells g r o w n f o r 2 h u n d e r aerobic c o n d i t i o n s a f t e r 8 h o f total anaerobiosis. T h e n u c l e u s a n d m i t o c h o n d r i a resume the normal aspect.
230
Quantitative data about variations in number of mitochondrial profiles in cells brought back to normal oxygen tension after 2 and 8 h of anaerobiosis are given in Table I. The percentage of the cells with a single mitochondrion decreases from 60% to 29% and from 90% to 56% respectively and cells with more I mitochondrion increase in number. Biochemical observations
Under anaerobic conditions protein and RNA synthesis in Rh. gracilis cells appear to be totally blocked (Cocucci, 1972). Return to normal oxygen tension in Rh. gracilis cultures kept under strict anaerobiosis for 2 h, leads to a rapid return to normal activity of incorporation of [t4C]leucine into proteins and [32p] orthophosphate in RNA; Fig. 7a shows how, after a brief period (7 min) incorporation once again begins in a linear manner and at a rate equal to that of cells which have not undergone anaerobiosis treatment. Under total anaerobiosis the absence of incorporation cannot be put down
(a)
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Fig. 7. Effects o f anaerobiosis and r e s t o r a t i o n o f pO~ availability after 120 rain o f
anaerobiois on protein and RNA synthesis, a) Incorporation of [1,~4C]leucine into proteins and the intracellular level of free leucine (see methods). L-Leucine (5 × 10 -4 M, spec. act. 40 ttCi/mmol) was added at zero time. The values are expressed as tzmol/100 ml of culture. The plot in the inset shows [14C]leucine incorporation into proteins of control cells (100 mg Hg O~). The initial amounts of protein were: 36 mg/100 ml of culture and, in the inset, 37.2 mg/100 ml. Continuous line: leucine incorporation into proteins; broken line: intraceUular level of free leucine. Open symbols: pO 2 saturating; closed symbols: anaerobiosis, b) Incorporation of [3:P]orthophosphate into RNA, intracellular level of inorganic orthophosphate and acid soluble organic phosphate compounds (see methods). ~:P-orthophosphate (5.73 × 10 -3 M, spec. act. 0.35 uCi/ttmol) was added at zero time. The values are expressed as ttmol Pi/100 ml of culture. The plot in the inset shows [32P]orthophosphate into RNA of control cells (100 mm Hg O 2). The initial amounts of protein were: 22 mg/100 ml of culture and, in the inset, 25.1 mg/100 ml. Continuous line: orthophosphate incorporation into RNA • o and acid soluble phosphate compounds • a ; broken line : intracellular level of inorganic orthophosphate. Open symbols: pO~ saturating; closed symbols: anaerobiosis.
231 I lOOmm Hgl~ 0 mm Hg 02 021 10-
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Fig. 8. Effects of anaerobiosis and restoration of saturating pO 2 availability after 2 and 8 h of anaerobiosis on cytochromes a, a 3 and c levels in Rh. gracilis cells. Cytochromes were assayed spectrophotometrically in intact cells. The values are expressed as ~mol/100 ml of culture. Broken lines refer to aerobic conditions after 2 and 8 h of anaerobiosis. Open symbols: pO 2 saturating; closed symbols: anaerobiosis.
to a blockage of the uptake since the labelling levels of the acid-soluble fraction are of the same entity as those found in aerobic controls. A similar pattern can be observed in Fig. 7b for RNA synthesis: under anaerobic conditions practically no [32P]orthophosphate incorporation can be observed, whilst a return to normal oxygen level leads to a rapid accumulation of labelling. Analysis of inorganic orthophosphate level variations in the acid-soluble fraction would appear to exclude a blockage of assumption during anaerobiosis. Variations in the levels of certain cytochromes induced b y anaerobiosis were studied to obtain information on the variations affecting the mitochondrial system. Fig. 8 shows that under aerobiosis the levels o f 3 cytochromes (a3, a, c) increase in step with cell growth. Anaerobiosis leads not only to no further growth b u t even to a slight fall in levels. Return to oxygen saturation induces a rapid increase of the level o f the cytochromes studied both after 2 and 8 h of anaerobiosis. The increase observed is in agreement with the data described earlier showing a rapid reactivation of the metabolism, induced b y oxygenation o f the medium. CONCLUSION AND DISCUSSION
Under strict anaerobiosis the infoldings and projections of the mitochondrial profile decrease in number and mitochondrial volume significantly increases. Only after 8 h do the mitochondria begin to show signs of degeneration. These changes do n o t seem to involve de novo protein synthesis given that b o t h incorporation of [32p] orthophosphate in RNA and that of [14C] leucine in proteins under anaerobiosis are totally blocked. Furthermore, there would
232
not seem to be any important degenerative phenomena given the slight variation of cytochromes a3, a and c. The fact that the cytochromes examined do not increase whilst the mitochondrial volume undergoes a considerable increase is interpreted as a swelling of the structure. This phenomenon can be connected with the swelling which is known to take place "in vitro" under certain conditions (Briefly, 1976). Reduction of mitochondrial profiles with the appearance of giant mitochondria was observed in the alga Chlamydomonas reinhardi under normal physiological conditions during a brief intermediate phase of the cell cycle. At this moment a considerable reduction of the cell' oxygen uptake activity was observed (Osafune et al., 1972). Giant mitochondria can also be produced experimentally by various treatments inhibiting cellular respiration (riboflavine deficiency, antimycine, cuprizone) (Tandler et al., 1969; Calvayrac et al., 1971; Suzuki, 1969; Tandler et al., 1973) is as widely differing species as rat and Euglena. In all such cases these changes are reversible and are interpreted as phenomena of fragmentation and fusion of the mitochondrial apparatus of the cell. In Rh. gracilis recovery also appears to be very rapid both after 2 h and 8 h of.,~aerobiosis, and the morphological observation is confirmed by biochemical analysis. The appearance of giant mitochondria in Rh. gracilis cultivated under anaerobiosis cannot correspond to a real increase of mitochondrial mass given the observed blockage of syntheses. Nor can it be due to the merging of pre-existent smaller separate mitochondria. Observations made during this study showed, in fact, that the mitochondrial system of this obligate yeast is, under normal conditions of growth, of the type proposed by Hoffmann and Avers (1973) for Saccharomyces cerevisiae: one, or at most a few, widely-ramified mitochondria. The data presented showing a blockage of cellular syntheses may be interpreted as a consequence of the reduction of % P availability produced by strict anaerobiosis: in fact, ATP level assays show a level falling to about 10% of that in controls after 90 min of anaerobiosis (from 8.4 nMoli/mg proteins in controls falls to 0.73 nMoli/mg proteins under anaerobiosis). The morphological changes in the mitochondrial system can be interpreted by the interesting hypothesis that abolition of A pH (due to the absence of oxygen) in the mitochondrial membrane leads to swelling of the mitochondrion. This hypothesis would agree with "in vitro" observations under particular experimental conditions (Brierly, 1976). REFERENCES Beffagna, N., S. Coeueci, M. Coeucci and M.C. Cocucei: Ann. Microbiol. 22,119--130 (1972). Brierley, G.P.: In: Mitochondria Bioenergetics, Biogenesis and Membrane Structure, eds. L. Packer and A. Gomez-Puyou pp. 3--20 (1976). Calvayrac, R., F. Van Lente and R.A. Butow, Scienc~ 173,252--254 (1971).
233
Chance, B.: In: Methods in Enzymology, eds. S.P. Colowick and N.O. Kaplan (Academic Press, New York and London) Vol. 4, pp. 273--329 (1957). Cocucci, M. : Ann. Microbiol. 22, 63--70 (1972). Cocucci, M.C. and G. Rossi: Arch. Mikrobiol. 85,267--279 (1972). Cocucci, M., G. Rossi and T. Vandoni: Cell Differentiation 4,155--165 (1975). Hawley, E.S. and R.P. Wagner: J. Cell Biol. 35,489---499 (1967). Hoffmann, H.P. and C.J. Avers: Science 181,749--751 (1973). Lamprecht, W. and I. Trautschold: In: Methoden der enzymatischen Analyse, ed. H.U. Bergmeyer, Band II, (Verlag Chemie Weinheim/Berg str.) pp. 2151--2162 1974. Lindberg, O. and L. Ernester: In: Methods of Biochemical analysis, ed. D. Glick, Vol. III, 1--22 (Interscience Publishers, New York-London) pp. 1--22 1956. Nieman, R.H. and L.L. Poulsen: Plant Physiol. 38, 31--35 (1963). Osafune, T., S. Mihara, E. Hase and I. Ohkuro: Plant Cell Physiol. 13,211--227 (1972). Suzuki, K. : Science 163, 81--82 (1969). Tandler, B., R.A. Erlandson, A.L. Smith and E.L. Winder: J. Cell Biol. 41, 477--493 (1969). Tandler, B. and C.L. Hoppel: J. Cell Biol. 56,266--272 (1973).
223
E F F E C T S OF T O T A L ANAEROBIOSIS ON T H E M I T O C H O N D R I A L SYSTEM O F R H O D O T O R U L A G R A C I L I S G.R. ROSSI and M. COCUCCI Istituto di Scienze Botaniche. Universitd di Milano, Centro di studio C.N.R. per la Biologia Cellulare e Moleeolare delle Piante, via G. Colombo 60, 20133 Milano, Italy
Accepted 12 March, 1978 Absence of oxygen induced marked changes in the mitochondrial system of the obligate aerobic yeast Rhodotorula graeilis. The number of mitochondrial profiles diminished progressively in proportion to increase in time of anaerobic growth. After 8 h of anaerobiosis, when the cell survival rate was still high (95%) almost al[cells showed a single broad mitochondrial profile. Protein and RNA synthesis were blocked, and cytochrome levels a~, a and c remained unchanged. When oxygen was again introduced, growth continued and the cell promptly recovered from all the changes observed under anaerobic conditions. The morphological changes in the mitochondrial system were interpreted as a change of form in what was probably a single mitochondrion.
R h o d o t o r u l a gracilis, an aerobic yeast, unlike facultative aerobic yeasts, u n d e r w e n t a complete block o f biosynthesis and metabolism under anaerobic conditions, and appeared t o have no energy-producing fermentative mechanism although its survival rate was total for 8 h (Cocucci, 1972). When R h . gracilis was cultivated under low-oxygen conditions sufficient t o permit 30% o f m a x i m u m growth rate, t he levels o f certain mitochondrial enzymes and the mitochondrial system as a whole u n d e r w e n t a substantial increase; accordingly there was no fall in availability o f % P; t he increase o f the mitochondrial system seemed to constitute an adaptation of t he organism t o low-oxygen conditions (Cocucci et al., 1975). This paper is based on the supposition t h a t if t h e mitochondriaI'increase observed in low-oxygen conditions is t o be seen as an adaptation, no such d e v e l o p m e n t will be observed if t ot al anaerobiosis com pl et el y blocks the metabolism. It was thus felt t hat it would be interesting t o investigate t he effects o f anaerobic conditions on t he mitochondrial system o f R h . gracilis.
MATERIALS AND METHODS Cultures o f R h . gracilis were set up as described previously (Beffagna et al., 1972). Exponentially growing cells were harvested by filtration on cellulose acetate filters (Sartorious, I, 2 p diameter) at 4°C and resuspended to a density corresponding t o 1 O.D.660 nm, in a minimal m edi um containing 2% glucose and 2% a m m o n i u m sulphate. These cultures were transferred t o a glass-stainless steel f e r m e n t at 30°C fitted out with an 02 pressure cont rol
224 system consisting of a Clark polarographic electrode (Radiometer, Copenhagen) (Cocucci et al., 1975). Anaerobic conditions were obtained by a flow of highly-purified nitrogen.
Biochemical determinations Cells were collected by filtration on Sartorious membrane filters and thoroughly washed free of traces of the medium by repeated washing in ice-cold incubation medium. Cells were fractionated according to the Nieman and Poulsen method (1963). Incorporation of L-[1-'4C]leucine (5 X 10 -4 M, spec. act. 40 pCi/mmol) into proteins was determined by measuring radioactivity into the NaOH soluble, PCA insoluble fraction. The acid-soluble fraction was assayed for intracellular level of free leucine. Incorporation of [32p] orthophosphate {5.73" 10 -3 M, spec. act. 0.35 ~Ci/ pmol) into RNA was determined by measuring the radioactivity present in the acid-soluble fraction obtained after alkaline hydrolysis of PCAinsoluble fraction. Intracellular level of inorganic orthophosphate was evalued according to Lindberg and Ernester (1956), as the radioactivity of phosphomolybdic complex of acid-soluble fraction. Radioactivity was determined in a Packard TriCarb liquid-scintillation counter. Cytochromes were assayed in intact cells with a Dual Wavelength Aminco-Chance spectrophotometer according to Chance (1957) as previously described (Cocucci et al., 1975). ATP level: cells were collected at 4°C by rapid filtration (approx. 6") under the same oxygen pressure on cellulose acetate filters and extracted in cold perchloric acid. ATP level was determined by enzymatic method according to Lamprecht and Trautschold {1974).
Electron microscopy Cells were harvested as described previously, washed twice with cold distilled water and resuspended in fixating solutions. Fixation and inclusion were performed as reported in a previous paper (Cocucci and Rossi, 1972). In this case we chiefly used permanganate fixation because it was more convenient for evaluating mitochondrial membranes.
Analysis of micrographs For the evaluation of the number of mitochondria, sections were cut a constant thickness and sampled at intervals sufficiently long to ensure that every cell was photographed once. Mitochondrial profiles were counted on the micrographic prints. Three-dimensional mitochondrial profiles were analysed by ultrathin serial section technique, transferring ribbons of 30--40 consecutive sections, about 70 nm thick, onto single-hole copper grids covered with Formvar and carbon layers. The mitochondrial and cell area were determined according to Hawley and Wagner (1967).
225 RESULTS
Morphological observations Fig. 2 shows Rhodotorula gracilis cells grown in total anaerobiosis for 8 h. Comparison with control cells cultivated under normal aerobic conditions (Fig. 1} shows that the mitochondrial profiles o f the anaerobic cells become larger, whilst they simultaneously become less numerous until there is a single profile for each cell. The fall in the number of mitochondrial profiles per cell after progressively longer periods of anaerobiosis is gradual, as can be seen in Table I. Under normal conditions of growth only 19% of cell sections examined have a single mitochondrial profile, whilst under strict anaerobiosis this percentage rises gradually to 60% after 2 h and to 90'% after 8 h. To ascertain whether this reduction in the number of mitochondrial profiles represented an effective reduction in the number of mitochondria/ cell, we used conventional ultrathin serial sectioning techniques to analyze the shape of the mitochondria of Rh. gracilis. Fig. 3 presents an approximate diagrammatic reconstruction of the mitochondria of part of a Rh. gracilis cell cultivated under normal aerobic conditions: the various mitochondrial profiles present in a given section of the cell do not correspond to so many different organelles, but represent sections of a single mitochondrion with long thin ramifications. This indicates that the mitochondrial system of Rh. gracilis consist of a single mitochondrion or o f at most a few mitochondria. The reduction in the number of mitochondrial profiles observed in sections of cells grown under strict anaerobiosis is quite conceivably the result of changes in the form of the organelle. Only the mitochondrial system is apparently affected during the first 4 h of anaerobic treatment. No change can be detected in the nucleus and in the density of ribosomes (this has been observed in electron micrographs of osmium-fixed samples that are n o t reported here). If anaerobic conditions are maintained for a longer time (up to 8 h) the mitochondria often show clear s y m p t o m s of degradation: the matrix becomes progressively less electron-dense and the cristae change s in form and distribution (Fig. 4). Moreover Table II shows that under anaerobiosis mitochondrial area increases in comparison with the area of whole cell. Since the percentage of area occupied b y any cellular inclusion in a random cell section is indicative o f the percentage v o l u m e in the whole cell, the data in Table II suggest a significant increase in the volume of the mitochondrial system. After 8 h of total anaerobiosis the nucleus also shows alterations: the nuclear envelope becomes discontinuous with large gaps and swelling of the perinuclear space (Fig. 5).
Reversibility of the effects o f anaerobiosis When non-growing anaerobic cells are brought back to normal aerobic
226
Fig. 1. Logarithmic phase cells o f Rh. gracilis grown under aerobic conditions. N, nucleus; M, mitochondria; Va, vacuole. Fig. 2. Rh. gracilis cells grown for 8 h in total anaerobiosis.
227 TABLE I Variations in the number of mitochondrial profiles in cells of Rh. gracilis after strict anaerobiosis and return to normal conditions for 2 h. Growth conditions
% Cell sections with
Aerobiosis icontrol) Anaerobiosis 2 h Anaerobiosis 8 h Recovery from 2 h anaerobiosis Recovery from 8 h anaerobiosis
1 mitochondria
2 mitochondria
3--4--5 mitochondria
19 60 90 29 56
28 31 10 39 38
53 9 0 32 6
No. of cell sections examined
66 95 92 75 70
71 35-
"~ 70-
7
~
10
o
~ lo5-
15-~ !
140--
20"
175--
25: Mitochondrial
profiles
in cell section
Fig. 3. Schematic profile o f mitochondrial apparatus o f a portion of Rh. gracilis cell grown under normal aerobic conditions, obtained from an intact ribbon of consecutive ultrathin sections, starting from the periphery o f the cell. Thickness o f sections: 70 rim. The continuous lines represent, approximately, the position and long axis of sectioned mitochondria; the dotted lines their tangential sections.
228
Fig. 4. Anaerobic Rh. gracilis cell showing a single broad mitochondrial profile whose internal membrane system is deeply altered. Fig. 5. Rh. gracilis cells grown for 8 h or longer in anaerobiosis. Note the gaps of the nuclear membrane.
229 T A B L E II C h a n g e s in m i t o c h o n d r i a l a n d cell area o f Rh. gracilis cells a f t e r 8 h o f anaerobiosis. Growth conditions
Mitochondrial area (~t2 )a
Cell w h o l e area (~t2 )a
Aerobiosis ( c o n t r o l ) Anaerobiosis 8 h
0.718 1.15,0
5.22 5.47
a Average o f 109 d i f f e r e n t a n d r a n d o m cell sections.
conditions growth is resumed; the morphology also becomes normal again. In almost all cells the nucleus appears to be normal 2 h after restoring aerobic conditions (Fig. 6); where the nucleus is still damaged repair processes are seen to be under way (Fig. 6a). The mitochondria also resume the aspect typical for aerobic conditions both as regards the number/cell and length and distribution of the cristae {Fig. 6a).
Fig. 6 and 6a. Rh. gracilis cells g r o w n f o r 2 h u n d e r aerobic c o n d i t i o n s a f t e r 8 h o f total anaerobiosis. T h e n u c l e u s a n d m i t o c h o n d r i a resume the normal aspect.
230
Quantitative data about variations in number of mitochondrial profiles in cells brought back to normal oxygen tension after 2 and 8 h of anaerobiosis are given in Table I. The percentage of the cells with a single mitochondrion decreases from 60% to 29% and from 90% to 56% respectively and cells with more I mitochondrion increase in number. Biochemical observations
Under anaerobic conditions protein and RNA synthesis in Rh. gracilis cells appear to be totally blocked (Cocucci, 1972). Return to normal oxygen tension in Rh. gracilis cultures kept under strict anaerobiosis for 2 h, leads to a rapid return to normal activity of incorporation of [t4C]leucine into proteins and [32p] orthophosphate in RNA; Fig. 7a shows how, after a brief period (7 min) incorporation once again begins in a linear manner and at a rate equal to that of cells which have not undergone anaerobiosis treatment. Under total anaerobiosis the absence of incorporation cannot be put down
(a)
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70 min.
Fig. 7. Effects o f anaerobiosis and r e s t o r a t i o n o f pO~ availability after 120 rain o f
anaerobiois on protein and RNA synthesis, a) Incorporation of [1,~4C]leucine into proteins and the intracellular level of free leucine (see methods). L-Leucine (5 × 10 -4 M, spec. act. 40 ttCi/mmol) was added at zero time. The values are expressed as tzmol/100 ml of culture. The plot in the inset shows [14C]leucine incorporation into proteins of control cells (100 mg Hg O~). The initial amounts of protein were: 36 mg/100 ml of culture and, in the inset, 37.2 mg/100 ml. Continuous line: leucine incorporation into proteins; broken line: intraceUular level of free leucine. Open symbols: pO 2 saturating; closed symbols: anaerobiosis, b) Incorporation of [3:P]orthophosphate into RNA, intracellular level of inorganic orthophosphate and acid soluble organic phosphate compounds (see methods). ~:P-orthophosphate (5.73 × 10 -3 M, spec. act. 0.35 uCi/ttmol) was added at zero time. The values are expressed as ttmol Pi/100 ml of culture. The plot in the inset shows [32P]orthophosphate into RNA of control cells (100 mm Hg O 2). The initial amounts of protein were: 22 mg/100 ml of culture and, in the inset, 25.1 mg/100 ml. Continuous line: orthophosphate incorporation into RNA • o and acid soluble phosphate compounds • a ; broken line : intracellular level of inorganic orthophosphate. Open symbols: pO~ saturating; closed symbols: anaerobiosis.
231 I lOOmm Hgl~ 0 mm Hg 02 021 10-
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Fig. 8. Effects of anaerobiosis and restoration of saturating pO 2 availability after 2 and 8 h of anaerobiosis on cytochromes a, a 3 and c levels in Rh. gracilis cells. Cytochromes were assayed spectrophotometrically in intact cells. The values are expressed as ~mol/100 ml of culture. Broken lines refer to aerobic conditions after 2 and 8 h of anaerobiosis. Open symbols: pO 2 saturating; closed symbols: anaerobiosis.
to a blockage of the uptake since the labelling levels of the acid-soluble fraction are of the same entity as those found in aerobic controls. A similar pattern can be observed in Fig. 7b for RNA synthesis: under anaerobic conditions practically no [32P]orthophosphate incorporation can be observed, whilst a return to normal oxygen level leads to a rapid accumulation of labelling. Analysis of inorganic orthophosphate level variations in the acid-soluble fraction would appear to exclude a blockage of assumption during anaerobiosis. Variations in the levels of certain cytochromes induced b y anaerobiosis were studied to obtain information on the variations affecting the mitochondrial system. Fig. 8 shows that under aerobiosis the levels o f 3 cytochromes (a3, a, c) increase in step with cell growth. Anaerobiosis leads not only to no further growth b u t even to a slight fall in levels. Return to oxygen saturation induces a rapid increase of the level o f the cytochromes studied both after 2 and 8 h of anaerobiosis. The increase observed is in agreement with the data described earlier showing a rapid reactivation of the metabolism, induced b y oxygenation o f the medium. CONCLUSION AND DISCUSSION
Under strict anaerobiosis the infoldings and projections of the mitochondrial profile decrease in number and mitochondrial volume significantly increases. Only after 8 h do the mitochondria begin to show signs of degeneration. These changes do n o t seem to involve de novo protein synthesis given that b o t h incorporation of [32p] orthophosphate in RNA and that of [14C] leucine in proteins under anaerobiosis are totally blocked. Furthermore, there would
232
not seem to be any important degenerative phenomena given the slight variation of cytochromes a3, a and c. The fact that the cytochromes examined do not increase whilst the mitochondrial volume undergoes a considerable increase is interpreted as a swelling of the structure. This phenomenon can be connected with the swelling which is known to take place "in vitro" under certain conditions (Briefly, 1976). Reduction of mitochondrial profiles with the appearance of giant mitochondria was observed in the alga Chlamydomonas reinhardi under normal physiological conditions during a brief intermediate phase of the cell cycle. At this moment a considerable reduction of the cell' oxygen uptake activity was observed (Osafune et al., 1972). Giant mitochondria can also be produced experimentally by various treatments inhibiting cellular respiration (riboflavine deficiency, antimycine, cuprizone) (Tandler et al., 1969; Calvayrac et al., 1971; Suzuki, 1969; Tandler et al., 1973) is as widely differing species as rat and Euglena. In all such cases these changes are reversible and are interpreted as phenomena of fragmentation and fusion of the mitochondrial apparatus of the cell. In Rh. gracilis recovery also appears to be very rapid both after 2 h and 8 h of.,~aerobiosis, and the morphological observation is confirmed by biochemical analysis. The appearance of giant mitochondria in Rh. gracilis cultivated under anaerobiosis cannot correspond to a real increase of mitochondrial mass given the observed blockage of syntheses. Nor can it be due to the merging of pre-existent smaller separate mitochondria. Observations made during this study showed, in fact, that the mitochondrial system of this obligate yeast is, under normal conditions of growth, of the type proposed by Hoffmann and Avers (1973) for Saccharomyces cerevisiae: one, or at most a few, widely-ramified mitochondria. The data presented showing a blockage of cellular syntheses may be interpreted as a consequence of the reduction of % P availability produced by strict anaerobiosis: in fact, ATP level assays show a level falling to about 10% of that in controls after 90 min of anaerobiosis (from 8.4 nMoli/mg proteins in controls falls to 0.73 nMoli/mg proteins under anaerobiosis). The morphological changes in the mitochondrial system can be interpreted by the interesting hypothesis that abolition of A pH (due to the absence of oxygen) in the mitochondrial membrane leads to swelling of the mitochondrion. This hypothesis would agree with "in vitro" observations under particular experimental conditions (Brierly, 1976). REFERENCES Beffagna, N., S. Coeueci, M. Coeucci and M.C. Cocucei: Ann. Microbiol. 22,119--130 (1972). Brierley, G.P.: In: Mitochondria Bioenergetics, Biogenesis and Membrane Structure, eds. L. Packer and A. Gomez-Puyou pp. 3--20 (1976). Calvayrac, R., F. Van Lente and R.A. Butow, Scienc~ 173,252--254 (1971).
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Chance, B.: In: Methods in Enzymology, eds. S.P. Colowick and N.O. Kaplan (Academic Press, New York and London) Vol. 4, pp. 273--329 (1957). Cocucci, M. : Ann. Microbiol. 22, 63--70 (1972). Cocucci, M.C. and G. Rossi: Arch. Mikrobiol. 85,267--279 (1972). Cocucci, M., G. Rossi and T. Vandoni: Cell Differentiation 4,155--165 (1975). Hawley, E.S. and R.P. Wagner: J. Cell Biol. 35,489---499 (1967). Hoffmann, H.P. and C.J. Avers: Science 181,749--751 (1973). Lamprecht, W. and I. Trautschold: In: Methoden der enzymatischen Analyse, ed. H.U. Bergmeyer, Band II, (Verlag Chemie Weinheim/Berg str.) pp. 2151--2162 1974. Lindberg, O. and L. Ernester: In: Methods of Biochemical analysis, ed. D. Glick, Vol. III, 1--22 (Interscience Publishers, New York-London) pp. 1--22 1956. Nieman, R.H. and L.L. Poulsen: Plant Physiol. 38, 31--35 (1963). Osafune, T., S. Mihara, E. Hase and I. Ohkuro: Plant Cell Physiol. 13,211--227 (1972). Suzuki, K. : Science 163, 81--82 (1969). Tandler, B., R.A. Erlandson, A.L. Smith and E.L. Winder: J. Cell Biol. 41, 477--493 (1969). Tandler, B. and C.L. Hoppel: J. Cell Biol. 56,266--272 (1973).