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Living unicellular eukaryote Tetrahymena pyriformis as a model for study of mitochondrial energetics in mammalian cells under conditions of

reduced oxidative metabolism. E.N.Mokhova, A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992,Moscow, Russia, E-mail: [email protected]

Abstract Some “animal-like” protozoa are used instead of mammalian cells in diverse investigations. Tetrahymena pyriformis cells in stationary phase of growth and washed from oxidative substrates (T.pyriformis) function under conditions of reduced oxidative metabolism. To elucidate if T.pyriformis can be applied as a model for mitochondrial energetics study in mammalian cells during hibernation, the features of T.pyriformis mitochondria were compared with that of liver mitochondria isolated from hibernating animals; the published earlier data were used. Based on the respiration recording and observation of Mito Tracker Red fluorescence in living T.pyriformis we tentatively concluded that the mitochondrial electrical membrane potential, Δψ, is low, and that the most part of the proton motive force is stored as the difference of pH between the sides of inner mitochondrial membrane. A sharp decrease of the maximal uncoupler-stimulated respiration rates was observed in the liver mitochondria isolated from hibernating animals. These and other data are explained by hypothesis that remodeling the liver mitochondria to a condense configuration is prerequisite for reduction of the oxidative metabolism. The rearrangement possibly resembles the apoptosis with the initial Δψ decrease induced by the respiratory activity suppression; the process has been earlier

Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 studied by E. Gotlieb and coauthors. The data suggest that T.pyriformis are suitable to study of some aspects of mitochondrial role in cellular adaptation to metabolic depression; that the evolutionary ancient mechanisms are conserved in a modified form in the ciliate and mammalian cells. Key words: Tetrahymena pyriformis; hibernation; metabolic depression, mitochondria, condensed configuration; mitochondrial electrical potential; ATP/ADP-antiporter. Running title: mitochondria during metabolic depression. Abbreviation: ΔP- proton motive force, or, difference in the electrochemical potential of hydrogen ions across the inner mitochondrial membrane; Δψ-electrical potential difference across the inner mitochondrial membrane; ΔpH-difference in hydrogen ion concentrations between the sides of the inner mitochondrial membrane; FCCP-p- trifluoromethoxycarbonylcyanide phenylhydrazone; DNP-2,4-dinitrophenol; T.pyriformis- 2

Tetrahymena pyriformis cells, taken during stationary growth phase and washed from oxidative substrates; ATP/ADP-antiporter- adenine nucleotide translocase; fatty acids-long- chain free fatty acids; Mito Tracker Red, Mito Tracker Red CMXRo.

Introductory part Some unicellular eukaryotes are widely used instead of mammalian cells for detection of various environmental toxic factors and, less frequently, in fundamental research. However, a number of publications demonstrate that experiments with Tetrahymena pyriformis and some other unicellular eukaryotes provide valuable information for solving fundamental problems in biology and medicine. Tetrahymena pyriformis cells are similar to the mammalian cells in many aspects, including functioning of the essential metabolic systems. Comprehensive characterization of these unicellular eukaryotes can be found in the monograph of A.M. Eliott (Ed) Biology of Tetrahymena (1973). These ciliates resemble mammalian cells even in a high sensitivity to very low hormone concentrations and in a response to stress at the hormonal level [1]. The present paper provides an analysis of the published data to compare (1) the mitochondrial energetics of living Tetrahymena pyriformis cells in the stationary phase of growth and washed from oxidative substrates (T.pyriformis) and (2) that of liver mitochondria isolated from the hibernating (winter slipping) animals. In both cases the cells survive under conditions of the oxidative metabolism reduction. During hibernation the animal oxidative metabolism drops much greater compared to that explained by the body temperature decrease [2]. A great decrease of the maximal uncoupler-stimulated respiration rate was observed in the liver mitochondria isolated from hibernating animal compared to those from active animals ([2-4] Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 and references within). Mitochondria from T.pyriformis and liver are very alike in their structural and functional characteristics: they have similar content of the cytochrome c type components of the respiratory chain, and comparable values of the P/O ratio showing the effectiveness of the oxidative phosphorylation, and etc [5]. They contain even similar number of mitochondria per cell [6]. During hibernation liver mitochondria have to support the cell viability and fulfill their functions under low level of oxidative substrates, strong decrease in body temperature, hypoxia, and some other unfavorable conditions. It is remarkable that the same is true for T.pyriformis. These cells enter the stationary growth phase due to the deficit in food, oxygen, and other environmental changes resulting from long living inside of closed space. Under these conditions both the cell types use lipid droplets as a fuel source [3, 7, 8]. Close 3

contacts between lipid droplets and mitochondria were clearly detected in hepatocytes of the hibernating animals (see Fig 1B in [3]) and in T.pyriformis cells (see Fig. 17 in [7]). Both the liver mitochondria and T.pyriformis cells did not utilize the long-chain fatty acids as oxidative substrate. In the liver mitochondria long-chain fatty acids, and in particular, essential fatty acids do not undergo the beta - oxidation cycle during hibernation. They can be used for the membrane phospholipids rearrangement for adaptation to low temperature, and/or for synthesis of endogenous regulators of the cellular metabolism and other functions ([9-11) and references within). Mitochondria from T.pyriformis can oxidize the short-chain fatty acids. The beta-oxidation of long-chain free fatty acids occurs mainly in peroxisomes [12].

Unexpected difference in DNP- and FCCP-induced uncoupling in experiments with T.pyriformis The integral characteristic of mitochondrial energetics, protonmotive force (ΔP) is composed from the electrical component, Δψ, and the concentration component, ΔpH. In mitochondria of the eukaryotic cells most of ΔP is stored in form of Δψ ([13 - 15] and references within). Synthesized in mitochondria ATP should be rapidly transported to cytosol for supporting the energy-dependent processes, for normal functioning the cells. The electrophoretic exchange of intramitochondrial ATP4– for extramitochondrial ADP3– is rapidly performed by the ATP/ADP- antiporter only at high Δψ [16]. In addition to the main function, the ATP/ADP-antiporter is involved in the uncoupling effect of low concentrations of fatty acids and DNP ([14, 17-20]). This uncoupling, which is often called the protonophopric effect, can occur only at high Δψ as well. However, some powerful Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 uncouplers, such as FCCP, increase the proton permeability of the inner mitochondrial membrane via different mechanism [21-22]. During experiments with T.pyriformis we found that maximal stimulation of endogenous cellular respiration by DNP (or oleic acid) was close to two while that achieved by FCCP was close to six (Fig.1), it was quite unexpected result. The difference in uncoupling activity of FCCP and DNP (or oleic acid) remained near the same in the presence of 20-30 mM pyruvate (for details, see [23]). Such strong stimulation of the respiration rate by FCCP indicated to a high ΔP in T.pyriformis mitochondria. The result was tentatively explained by low value of Δψ. This means that (i) the most part of mitochondrial ΔP should be stored in the form of ΔpH and, (ii) that the ATP/ADP-antiporter activity should be low.

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Fig. 1. Effects of DNP (1) and FCCP (2) on respiration of T. pyriformis cells. Before recording respiration, the cells were incubated for 1 day in medium containing 30 mM

KCl, 10 mM NaCl, 6 mM KH2PO4, and 5 mM Hepes, pH 7.4. The respiration rates are mean values (from 10 to 15 experiments) ± S. E. The figure is given according to the permission of “Pleiades Publishing, Ltd. in the RF”. It was firstly published in Biochemistry (Moscow) –see details in [23].

Additional evidence consistent with our assumption of low Δψ in T.pyriformis mitochondria were obtained in experiments with the cells stained with Mito Tracker Red, a Δψ-sensitive probe [23, 24]. In contrast to experiments with other eukaryotic cells, we did not observed mitochondria with bright fluorescence in T.pyriformis. The weak intensity of Mito Tracker Red

Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 fluorescence in T.pyriformis mitochondria was also reported by other authors [25]. It is unlikely, that (i) low DNP – induced uncoupling (Fig.1) and (ii) the unexpected low fluorescence of Mito Tracker Red in the intact T.pyriformis mitochondria were due to their pumping out the cells by the multidrug efflux pump. DNP at high concentrations noticeably suppresses respiration of T.pyriformis (Fig.1), as it does in isolated mitochondria. Mito Tracker Red in contrast with another Δψ-sensitive probe rhodamin 123, did not usually clear out from mitochondria during fixation. The bright fluorescence of Mito Tracker Red was clearly seen in mitochondria-like organelles inside the fixed cells [23, 24], where this Δψ-sensitive probe is concentrated within the phospholipid area It needs to be emphasized that ability to store the most part of ∆P in the form of ΔpH is essential for surviving of free-living T. pyriformis under unfavorable conditions such as hypoxia, fasting, and other. ΔP stored in the form of ΔpH has greater buffer capacity as 5

compared with that stored in Δψ form [15]. Conversion of Δψ to ΔpH results in enhanced transport of some oxidative substrates into mitochondria. On the other hand, if the Δψ value is low the Δψ – dependent functions should be sharply reduced; possibly, from time to time the energy stored in ΔpH converted to Δψ form for supporting the functions important for survival the cells. In addition, many energy dependent processes had been reduced in the liver mitochondria during hibernation and in infusorians during the stationary phase of growth as well. In short series of experiments with mitochondria, isolated from T.pyriformis suspension, Δψ, was recorded by safranin method. In our hands the mitochondria were unstable and had Δψ, close to that of liver mitochondria, only during the several first consecutive recordings, and when mitochondrial suspension was incubated in the presence of EGTA, oxidative substrates, oligomycin and nigericin [23]. The last two reagents produced no any effect on the respiration rates when being added to the polarographic well after or before T.pyriformis. Possibly it was due to low permeability of the cell membranes to these substances.

Insight into the nature of liver mitochondrial energetics during switching into and living during hibernation Possibly, the liver mitochondrial energetics during transition from an active to hibernating state have a resemblance with initial stages of apoptosis or autophagy induced by food deprivation. . Recently it was found that at the beginning of an apoptosis, induced by the respiratory activity suppression, an initial Δψ decrease led to remodeling the mitochondrial structure from an orthodox to a condensed configuration. Relationships between the mitochondrial ultrastructure and Δψ were studied during modification of Δψ in experiments Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 with cell cultures and isolated mitochondria ([26] and references within). The obtained results confirmed the supposition. The described data are understandable as Δψ decrease induces efflux of a number of cations (such as e.g. K+ or Ca2+) from the matrix; and this leads to the matrix shrinkage. It is desirable to reproduce these very important experiments with another Δψ-sensitive fluorescent probe; preferentially to use сarbocyanine diS-C3-(5) instead of rhodamin 123. Our experiments with lymphocyte suspension show that just diS-C3-(5) is suitable for recording the Δψ kinetics [27]. It could not be excluded, that an initial Δψ decrease during the described above apoptosis [26] is due to a partial conversion of ΔP from Δψ to ΔpH form, that this conversion gives to cells the last possibility for reparation to avoid an apoptosis. I speculate that the evolutionary ancient mechanisms of reparation on the cellular level may be activated in our organism, e.g., during 6

deep relaxation in the time of a suitable meditation when the nervous system is in a “dormant state”. Similar rearrangements of the mitochondrial energetics may be reasonable and for liver mitochondria during transition from active to hibernating state. The mitochondria should diminish their energy-consuming processes, and it could be performed by remodeling their structure to a condense configuration. The Δψ decrease may occur due to reduction of the respiratory activity resulting from a reduction of oxidative substrates or hypoxia, or a decrease in the temperature or, etc. In the condense configuration the respiration activity may be reduced due to changing the mitochondrial morphology, including an essential increase of the intermembrane space and intercristal space with parallel decrease of the matrix volume. The similar effects are observed during incubation of mitochondria in a hypertonic medium. . In a condense configuration the number of contact sites between the inner and outer mitochondrial membranes are much lower compared with that in an orthodox state. The activity of the ATP/ADP-antiporter and associated with this anion carrier processes, and Ca2+ fluxes in mitochondria should be significantly reduced (for illustration, see scheme in Fig. 5 in [28] and references within). After remodeling of the mitochondrial structure to a condense configuration great changes occur in the structure and function of the supramolecular protein complexes located in the contact sites between inner and outer mitochondrial membrane. They include the ATP/ADP- antiporter and other proteins involved in regulation of cellular energetics ([29] and references within). The weighty arguments were represented by authors that these complexes govern not only energetics but the fate of cells [29]. Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 Mitochondria of various configurations were observed in T.pyriformis during the stationary phase of growth [7]. Possibly the autophagy of the mitochondria was activated in Tetrahymena pyriformis during later growth phase; some mitochondria were incorporated into vacuoles containing acid phosphatase [7]. The moderate autophagy facilitates surviving of the cells under the metabolic depression; it defends mitochondria from apoptosis ([30] and references within). During imitation the hibernation state in experiments with isolated mitochondria EGTA was present in the incubation medium to avoid diverse ways of the oxidative metabolism intensification by Ca2+, especially in the presence of fatty acids. Reagents and water may content impurities of Ca2+. In our experiments with isolated liver mitochondria a very low Ca2+ concentration, from 3 μM, produced a great effect on the mitochondrial energetics being added in the presence of 3 μM 7

myristic or palmitic acid. Depending on the experimental conditions, they either decreased Δψ without stimulation of the respiration, or induced the non-specific pore opening in the inner mitochondrial membrane [28, 31]. Probably calcium was bound during stationary growth phase of Tetrahymena pyriformis cells; inclusions were found in these cells, which were tentatively considered as complex of Ca2+ with other substances [7]. During incubation of a liver mitochondria in the EGTA containing medium, carboxyatractylate, the specific inhibitor of the ATP/ADP-antiporter, decreased the succinate oxidation rate. The rate of the maximal uncoupled by DNP respiration was in 5-6 times lower in hibernating animal as compares to that of arousing (body temperature 25-30 o C) animals. The rate of succinate oxidation were about 2 – folds lower in hibernating animal as compared to that of similar arousing animals [32]. Unexpectedly, the degree of the suppression of succinate oxidation by carboxyatractylate was about similar in these groups. This led to a suggestion that during hibernation only part of mitochondria gives essential input to respiration in the presence of DNP. The similar puzzling mechanism of the oxygen consumption regulation was observed in our experiments with T.pyriformis, when FCCP produced strong stimulation of the respiration. Addition of pyruvate to these cells mostly resulted in about similar stimulation of the initial and uncoupled respiration rates. We had never observed such relationships in the experiments with well coupled isolated liver mitochondria: an oxidation substrate addition stimulated the uncoupling respiration stronger compared with that of the initial respiration rate. All these data have a reasonable explanation in the assumption that a part of mitochondrial population gives only a small input to the respiration due to being in the condense Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 configuration. Possibly, a number of factors, e.g., pyruvate addition, may activate them, may induce their transformation to the ortodox configuration Important results were obtained during parallel recording of the oxygen consumption and analysis of the mitochondrial ultrastructure [33]. It was found that mitochondria from both animal states, active and hibernating, had the condensed configuration after their isolation and storing in the cold. However if their fixation was performed after 4 min of incubation at 27o C, mitochondria from active animal had the orthodox configuration, and mitochondria from hibernating animal remained the condensed configuration with shrunken matrix, their respiration was still reduced. Incubation of the mitochondria from hibernated animal in a hypotonic medium resulted in remodeling of their structure into the orthodox configuration as well; however some structural differences between the samples of mitochondria from active and hibernating animals were still preserved (see Fig.3 in [33]). 8

Some methodological aspects of experiments with mitochondria in living T.pyriformis and isolated from liver of hibernating animals. Isolated mitochondria and mitochondria in intact cells usually differ in a number of important properties of their energetics; especially it concerns the energy coupling characteristics. Recently a new method was created: the experimental conditions were elaborated in order to preserve the mitochondrial reticulum during homogenate preparation and following incubation [34]. Mitochondrial respiration rates were calculated from the homogenate oxygen consumption. Under such conditions the mitochondrial metabolic state in vivo was close the state in vitro. Validity of the method was demonstrated during study of a number of physiological models. It is reasonable to utilize this method [34] at least during tentative experiments with mitochondria isolated from liver of active and hibernating animals. Before beginning the experiments with liver mitochondria from hibernating animals, the suitable characteristics of mitochondrial metabolism should be chosen. It should be underlined that the metabolic state of the mitochondria with the condense configuration should be characterized by other parameters compared to that in the orthodox one. For example, in the first case the relation between rates of succinate oxidation in the presence and absence of an uncoupler may be dependent not only on the proton conductance of the inner mitochondrial membrane but and on the uncoupler- induced shrinking of the matrix due to an uncoupler – induced inhibition of cations influx. The parameters should be previously chosen in specials experiments. Probably, the first step is a selection of a suitable hypertonic EGTA-containing incubation medium. Its osmolarity should be increased until the maximal uncoupled respiration of the Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 mitochondria from active animal becomes as low as that of mitochondria from the hibernating group during incubation in the isotonic medium. Under the same conditions an analysis of the mitochondrial ultractructure should be performed either by light scattering recording, or, preferentially, by the same method, as described above during consideration of the apoptosis [26]. As to Δψ estimation, the application of a TPP+-sensitive electrode for Δψ comparison of liver mitochondria isolated from hibernating and active animals is questionable. One of the problems is the matrix volume estimation. Hepatocyte mitochondria have very complicated matrix structure and barriers for some substances movement between intermembrane and intracristal spaces [35 - 36]. Ultrastructural features of liver mitochondria isolated from hibernating animals were not investigated by similar 9

methods. In addition, the energy-independent binding of TPP+ with the isolated from hibernating animal mitochondria was not estimated. The other difficulty is heterogeneity of mitochondria. TPP+ and similar Δψ -sensitive probes are accumulated mainly in mitochondria with highest Δψ value (according to the Nernst equation); so obtained with TPP+ - electrode results refer to the mitochondrial fraction with the highest Δψ. Probably, the measured with TPP+-sensitive electrode Δψ value referred firstly to mitochondria with the orthodox configuration. Due to mitochondrial heterogeneity the calculation of proton conductance by usual method is difficult. In many studies, the proton conductance in isolated mitochondria was estimated from the ratio of the respiration rate and Δψ during succinate oxidation without and with of the consecutive additions of malonate [2]. While TPP+ or other Δψ –sensitive probes are accumulated mainly in mitochondria with the highest Δψ, the main contribution to the respiration rates comes from the mitochondria with the lowest energy coupling, with the highest respiration rates. Thus, different fractions of mitochondria may contribute to the ratio [37]. In some cases it is useful to utilize suitable Δψ-sensitive fluorescent probes for recording Δψ kinetics, we used, diS-C(3)-5 [27]. For visual observation with the help of a microscope we used rhodamin 123 or its derivatives. To dissipate Δψ in the control experiments, it is reasonable to add an inhibitor of the cytochtrome c oxidase, e.g., cyanide [38], but not FCCP. Experiments with T.pyriformis have some features. The great advantage of working with T.pyriformis is that they respond to some unfavorable environmental factors by change in their movement and shape ([23-24] and references within). Just by this method T.pyriformis demonstrated that digitonin even at very low concentrations, using for the membrane Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 permeabilization, is toxic for these ciliates. So, we did not use digitonin in the main experiments with T.pyriformis. On the other hand, in the case of suitable treatment of T.pyriformis during experiment (see details in [23]) shortly after recording of the oxygen consumption the cells restore their normal motion, the shape was not changed. So measurement of the cellular respiration rates by the polarographic method is not only informative but and noninvasive for T.pyriformis. The respiration rate is sharply dependent on the cell concentration. Usually the concentration was chosen to decrease the data variability. It should be added that the experiments with Tetrahymena thermophila showed that the siliate can not survive for long period if the suspension is too diluted [39]. Due to large size, T.pyriformis can be easily observed under a regular microscope after staining or with side lightening .The only complication for microscopic observation is their high 10

sensitivity to the bright light. Such sensitivity is common for a number of unicellular eukaryotes [40]. Prolong illumination even with a tungsten lamp changes their behavior. In experiments with T.pyriformis the optimal Δψ-sensitive fluorescent probes appears to be Mito Tracker Red. [23]. For proper interpretation of the data it is important to perform the similar experiments with Tetrahymena pyriformis cells loaded with Mito Tracker Red and taken from different stages of the stationary and exponential growth phases. The results will be useful and for elucidation of the question concerning Δψ value in these ciliates. Two following methods did not used in the discussed studies; however the methods are very important for study of mitochondrial features during metabolic depression. One of them is recording the cytochrome spectra of biological objects with high light scattering, e.g., of suspension of mitochondria, cells, etc, with the help of a special high sensitive differential Aminco-Chance type spectrophotometer. The recording of the steady states of the respiratory chain carrier reduction gives data about location of the limiting steps of the respiratory chain [41]. The most important, this method is able to estimate a portion of the respiratory chains which does not participate in the respiration. Addition of high concentration of T.pyriformis to spectropjotometric cell did not give any signal: the cells escaped the illuminated by light beam areas. In our previous experiments with low quantity of mitochondria we used a special device for support the close to liquid nitrogen temperature of the frozen samples and for concentration of the scattered light on a light detector [42]. Such installation was constructed by V. N. Larionov; their prototype was created by W. Estabrook [43]. A plastic thin cell with mitochondrial suspension was immersed several times to liquid nitrogen. Its light scattering was strongly increased. So, the optical path was increased as well, and very low concentrations of mitochondria or cells may be used for Nature Precedings : hdl:10101/npre.2010.4521.2 Posted 22 Sep 2010 recording of cytochrome spectra. The most appropriate method for T.pyriformis oxidative metabolism study under the natural physiological condition is using an infrared imaging camera with the corresponding accessories. The measurements are absolutely noninvasive; only the far infrared irradiation from the object is collected. The mitochondrial oxidative metabolism can be selected, as T.pyriformis respiration is more than 90 per cent suppressed by such inhibitor as cyanide [24]. It should be added that this method has been successfully applied to study of isolated liver mitochondria energetics [44].

Conclusion The paper demonstrates that mitochondria in the living T.pyriformis are similar to liver mitochondria in regard to a number of properties, and that mitochondria of both the objects are 11

able to function under conditions of an essential decrease of the oxidative metabolism. According to my conjecture, the mitochondrial rearrangement to the state with oxidative metabolism reduction may be induced by Δψ decrease with suitable ΔpH increase, ΔP being unchanged. .As it was shown earlier, a Δψ decrease leads to remodeling of the mitochondrial configuration to a condense structure with low oxidative metabolism at least in a portion of mitochondria. The similar approach might be useful for study of the mitochondrial energetics in lymphocytes from the capable to hibernation animals. They may be easily isolated in the intact state. Such experiments are especially interesting during yearly winter involution of the thymus and the subsequent reparation. On the other hand, T.pyriformis and some other “animal-like” free living in water unicellular eukaryotes give a unique possibility to research diverse effects of water structure and low concentrations of dissolved in water substances on the mitochondrial energetics in living cells.

Acknowledgments I wish to express my deep gratitude to academician V. P. Skulachev for the consistent support of research on the uncoupling effect of long-chain free fatty acids and related fields. I am heartfelt thanks to Galina P. Kirillova, whose help and wise advices have been invaluable throughout the preparation of the whole manuscript. I am very grateful to my highly educated colleagues Michael L. Altshuler and Emil E. Khavkin, who always were ready to share their vast encyclopedic knowledge.

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