Morphological variations of isolated liver mitochondria during spontaneous deterioration

Summary The effects of mitochondrial concentration, incubation temperature and composition of suspension medium on spontaneous swelling of rat liver mitochondria were investigated. The swelling was measured simultaneously by four different methods — optical density changes at 520 m, mitochondrial diameter measurements and differential counts made on phase contrast micrographs of fixed suspensions, and electron microscopical examination of the fine structural alterations.The findings indicate that, in isosmotic sucrose and KCl media, both mitochondrial concentration and incubation temperature influence the course of spontaneous swelling. It was also found that the ratio of Tris (hydroxymethyl) aminomethane to mitochondria concentration profoundly affects the morphological transformations.Correlation of the results obtained with the four methods shows that measurement of optical density changes alone is insufficient for full assessment of mitochondrial swelling and that this method therefore is best used in conjunction with electron microscopical studies.The authors wish to thank Mr. George O'Shea for valuable technical assistance.     

Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH

Fluorescentdeterminations of NADH in porcine heart mitochondria were subject tosignificant errors caused by alterations in inner filter effects duringnumerous metabolic perturbations. These inner filter effects wereprimarily associated with changes in mitochondrial volume andaccompanying light scattering. The observed effects were detected in astandard commercial fluorometer with emission orthogonal to theexcitation light path and, to a lesser extent, in a light path geometrydetecting only the surface fluorescence. A method was developed todetect and correct for inner filter effects on mitochondrial NADHfluorescence measurements that were independent of the optical pathgeometry using an internal fluorescent standard and linearleast-squares spectral analysis. A simple linear correction with theinner fluorescence reference was found to adequately correct for innerfilter effects. This approach may be useful for other fluorescenceprobes in isolated mitochondria or other light-scattering media.

     

Effect of ozone on swelling of tobacco mitochondria

The swelling of mitochondria isolated from leaves, roots, and callus tissues of Nicotiana tabacum, L, var. White Gold, was measured by following changes in optical density at 520 mμ in buffered 0.25 m sucrose or 0.125 m KCl. Ozone induced rapid swelling of the isolated mitochondria and increased the permeability of mitochondrial membranes. The extent of mitochondrial swelling and the amount of soluble proteins and other substances absorbing at 260 and 280 mμ released from mitochondria into the suspending medium were positively correlated with the length of exposure to O₃. The correlation between the extent of mitochondrial swelling and the loss of intramitochondrial materials was also highly significant.     

Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria

The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the α, β, and [ill] mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

Mitochondria optical parameters are dependent on their energy state: a new electrooptical effect?

The membrane potential of mitochondria determines, to a large extent, their functional state and the response to the changing conditions of the environment. A correlation was found between the changes in the optical characteristics of isolated mitochondria and the composition of the incubation medium, which determines the potential of the inner mitochondrial membrane. The measurements performed by the method of coherent phase microscopy made it possible to establish a linear dependence of the phase height on the value of the membrane potential and to calculate the electrooptical constant, K(3–4)×10^–7 m/V.Abbreviations CPM coherent phase microscopy - MEOE membrane electrooptical effect - MP membrane potential - OP optical path - PH phase height     

Optical isolation of individual mitochondria ofPhysarum polycephalum for PCR analysis

Summary We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria fromPhysarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EDTA ethylenediaminetetraacetic acid - mtDNA mitochondrial DNA - PCR polymerase chain reaction     

Variation of mitochondrial size during the cell cycle: A multiparameter flow cytometric and microscopic study.

BACKGROUND: Changes in mitochondrial structure and size are observed in response to alterations in cell physiology. Flow cytometry provides a useful tool to study these changes in intact cells. We have used flow cytometry and digital fluorescence microscopy to analyze the variations in mitochondrial size in relation to specific phases of the cell cycle. METHODS: Supravital staining of rat fibroblasts was done with Hoechst 33342 and rhodamine 123, and cells were analyzed in a dual-laser flow cytometer. Synchronized cells at various stages of the cell cycle were analyzed for changes in mitochondrial size. These cells were also examined by electron microscopy, digital fluorescence microscopy and computerized image analysis to compare the lengths of the mitochondria. RESULTS: By using fluorescence pulse width analysis, we observed two populations of mitochondria in intact cells. The percentage of cells with small and large mitochondria at specific stages of the cell cycle indicated that mitochondrial size increases during the cell cycle; early G1 phase cells had the smallest mitochondria and the mitotic phase cells had the largest mitochondria. These results were confirmed by microscopic analysis of cells. CONCLUSIONS: Flow cytometry can distinguish the relative mitochondrial size in intact cells, and in combination with digital microscopy it can be used to study mitochondrial variation during the cell cycle.     

The role of the ATP-sensitive potassium channel in the activation of the K+ cycle in rat liver mitochondria

It is known that the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) plays a key role in protecting myocardium during ischemia. We have suggested that the mechanism of this protection is associated with the potassium cycle in mitochondria. In this paper, for the first time, a direct proof was obtained of the existence of a cycle of potassium ions in rat liver mitochondria that are associated with the functioning of mitoK_ATP. Activation of the cycle was recorded by optical density changes of mitochondrial suspension in the form of two or three swelling-contraction waves of the organelles. Using activators and inhibitors of mitoK_ATP we showed that a significant role in the potassium cycle belongs to the channel. It was found that in vitro sildenafil has a direct effect on mitoK_ATP, being its activator. The results obtained indicate that the cardioprotective effect of sildenafil observed previously is associated with the activation of mitoK_ATP. In order to study the structure and volume changes of mitochondria in various stages of the cycle in the presence of potassium channel modulators, the electron microscopy studies of mitochondria preparations were carried out. A correlation between the optical density decrease of mitochondrial suspension and the swelling of mitochondria was revealed. The data obtained in this study suggest participation of mitoK_ATP in the protection of tissues from hypoxic damage.     

The responses of mitochondrial proteome in rat liver to the consumption of moderate ethanol: the possible roles of aldo-keto reductases

A large body of evidence supports the view that mitochondria are a primary target of alcohol stress. Changes in mitochondrial proteins due to moderate ethanol intake, however, have not been broadly and accurately estimated. For this study, rats were fed low doses of ethanol and the mitochondria were isolated from heart, kidney, and liver, using ultracentrifugation with Nycodenz density gradient. The mitochondrial proteins were well resolved upon two-dimensional electrophoresis (2DE), and the alcohol-responsive 2DE spots were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Compared with the control group, the proteins extracted from liver mitochondria of ethanol-fed rats exhibited the significant changes on 2DE images, whereas the 2DE images obtained from the kidney and the heart mitochondria remained almost unchanged by ethanol feeding. Significantly, over 50% of the alcohol-responsive proteins in liver mitochondria were members of aldo-keto reductase family (AKR), which were usually present in cytoplasm. The organelle distributions of AKR proteins in liver mitochondria were further confirmed by Western blot analysis as well as by confocal microscopy. In addition, translocations of AKR were examined in the CHANG cell line, which was cultured with and without ethanol. The results of Western blot strongly suggested that the abundances of AKR proteins in the mitochondria were greatly reduced by the presence of ethanol in culture medium. The results of this study show that, even with moderate ethanol feeding, the mitochondrial proteome in rat liver was more sensitive to alcohol stress than that of either the kidney or the heart. The translocation of AKR proteins may be involved in the detoxification of liver cells.     

Physicochemical differences between fragments of plasma membrane and endoplasmic reticulum

Specific turbidities, densities, and refractive indices of fragments of plasma membrane (PM) and endoplasmic reticulum (ER) from Ehrlich ascites carcinoma have been measured. A spherical shell model of specified dimensions and refractive index was established for PM fragments. The ionic composition of the dispersion medium was varied systematically. Increases in Γ/2 caused increases in the turbidity of both PM and ER suspensions, the greatest effects being observed with Ca²⁺ and Mg²⁺. In the case of PM this effect is attributable mainly to aggregation, whereas "structural" changes account for most of the turbidity increase with ER. The pH was also varied systematically to obtain pH- density and turbidity profiles and to establish the isoelectric pH of the two membrane types (PM—3.6; ER—4.35). Turbidity was maximum at "isoelectric" pH, which corresponds in each case to the region of minimum charge on the particle surfaces. Both PM and ER show large increases of density at the "isoelectric" pH, but only ER shows substantial structurally based turbidity increase under these conditions. Both PM and ER show operation of electrostatic attractions near "isoelectric" pH. PM has been shown to have ionically distinctive inner and outer surfaces while ER shows no such dissymmetry. The necessary theoretical background for interpretation of turbidity and density measurements is included, as well as a discussion of the limitations of our conclusions and the biological importance of our results.     

冷冻电子断层扫描研究原代培养神经元中的线粒体膜动态变化（英文）

<正>Dear Editor,Mitochondria acts as a cellular organelle that produces ATP and buffers Ca²⁺, and plays an important role in neuronal growth, survival and function^[1]. Loss of mitochondria will make the ATP supply insufficient, resulting in synaptic transmission dysfunction^[2]. Further, presynaptic mitochondrial dysfunctions are often associated with severe neurological diseases^[3].     

Metabolic responses to low temperature in fish muscle

For most fish, body temperature is very close to that of the habitat. The diversity of thermal habitats exploited by fish as well as their capacity to adapt to thermal change makes them excellent organisms in which to examine the evolutionary and phenotypic responses to temperature. An extensive literature links cold temperatures with enhanced oxidative capacities in fish tissues, particularly skeletal muscle. Closer examination of inter-species comparisons (i.e. the evolutionary perspective) indicates that the proportion of muscle fibres occupied by mitochondria increases at low temperatures, most clearly in moderately active demersal species. Isolated muscle mitochondria show no compensation of protein-specific rates of substrate oxidation during evolutionary adaptation to cold temperatures. During phenotypic cold acclimation, mitochondrial volume density increases in oxidative muscle of some species (striped bass Morone saxatilis, crucian carp Carassius carassius), but remains stable in others (rainbow trout Oncorhynchus mykiss). A role for the mitochondrial reticulum in distributing oxygen through the complex architecture of skeletal muscle fibres may explain mitochondrial proliferation. In rainbow trout, compensatory increases in the protein-specific rates of mitochondrial substrate oxidation maintain constant capacities except at winter extremes. Changes in mitochondrial properties (membrane phospholipids, enzymatic complement and cristae densities) can enhance the oxidative capacity of muscle in the absence of changes in mitochondrial volume density. Changes in the unsaturation of membrane phospholipids are a direct response to temperature and occur in isolated cells. This fundamental response maintains the dynamic phase behaviour of the membrane and adjusts the rates of membrane processes. However, these adjustments may have deleterious consequences. For fish living at low temperatures, the increased polyunsaturation of mitochondrial membranes should raise rates of mitochondrial respiration which would in turn enhance the formation of reactive oxygen species (ROS), increase proton leak and favour peroxidation of these membranes. Minimisation of mitochondrial oxidative capacities in organisms living at low temperatures would reduce such damage.     

Mitochondrial changes in flight muscles of normal and flightless Drosophila melanogaster with age

Fine structural changes in mitochondrial morphology pertaining to size, number and growth were examined in flight muscles of normal and experimentally dewinged male Drosphila melanogaster ranging up to 26 days of age. In the normal winged flies, the number of mitochondria decreases during the first week of adult life whereas the size of individual mitochondrial profile increases significantly. Changes in mitochondrial size and number are due to the fusion of mitochondria. Fused mitochondria are extremely large in size and irregular in shape. In 26-day old normal flies, the number of mitochondria increases while the mitochondrial size is reduced indicating mitochondrial division. In comparison to the normal flies, dewinged flies exhibit a similar degree of mitochondrial fusion and growth during the first week of life. However, the extent of mitochondrial fission in 26-day old dewinged flies is greater than in the normal flies of this age. Structural mechanisms of mitochondrial fusion and fission are described. The objective of this study was to examine the relative effects of age and flight activity on the mitochondria.     

THE INFLUENCE OF PRECURSOR POOL SIZE ON MITOCHONDRIAL COMPOSITION IN NEUROSPORA CRASSA

The chemical composition of mitochondria obtained from exponentially growing Neurospora can be varied by addition of choline or amino acids to the culture medium. The variation affects the phospholipid to protein ratio, and the density of mitochondria as determined by isopycnic centrifugation in sucrose gradients. These variations have been observed in biochemical mutant strains as well as wild type cultures. In a choline-requiring strain, two levels of choline supplementation to the medium have been defined: a low choline concentration just adequate to support maximal logarithmic growth, and a high choline concentration which permits maximal incorporation of radioactive choline into cellular lipids. Mitochondria isolated from cultures growing at the low choline concentration have one-half the phospholipid to protein ratio of those from high choline cultures, and their density is significantly higher. Artificial mixtures of the two types of mitochondria can be resolved into two populations by isopycnic centrifugation. The concentration of cytochromes (measured by mitochondrial difference spectra) and of malate and succinate dehydrogenases (measured by enzyme activity) were the same in both types of mitochondria, on a protein basis. The results suggest that during growth of the mitochondrial mass, the incorporation of phospholipid and protein components can vary independently. Direct kinetic measurements did indeed show that choline, added to a culture growing at low choline concentration, was incorporated into mitochondrial lipids at a rate faster than the incorporation of protein. The mitochondrial phospholipid to protein ratio can also be influenced by the level of leucine supplementation to a leucine-requiring mutant, so that with leucine concentrations above those required for maximal exponential growth, mitochondria of increasing density and decreasing phospholipid to protein ratio are produced. Additions of choline or amino acids to the minimal medium of wild type cultures influence mitochondrial composition in a manner directly comparable to that observed in biochemical mutant strains. The results suggest that mitochondrial composition, in general, is determined by rates of incorporation of the two major components, phospholipid and protein; that these rates can vary independently in response to precursor concentration in the culture medium; and that they normally operate at a precursor (substrate) concentration below saturation level.     

Light scattering of normal human lens I. Application of random density and orientation fluctuation theory.

Light-scattering intensities in the I parallel and I+ mode were obtained on thin sections of three human lenses. Random density and orientation fluctuation theory, without cross correlation, was employed to evaluate light-scattering parameters. Both the density correlation distances, as well as the orientation correlation distances, were related to structural elements in the lens fiber cell that have been observed by other investigators with different techniques. The magnitude of these fluctuations were evaluated, and it was demonstrated that the density fluctuations are the main contributors to light scattering in normal human lenses. Changes in the light-scattering parameters were evaluated as a function of position within the lens. The changes observed agree with the biochemical data in the literature that reflects that an aging process occurs when one proceeds from the periphery of the lens toward the center.     

A comparative study of the metabolic capacity of hearts from reptiles and mammals

The metabolic capacities of reptilian and mammalian hearts have been investigated using two methods: measurement of mitochondrial enzyme activity (cytochrome oxidase) and measurement of both mitochondrial volume density and membrane surface area. The heart tissues from the reptiles and mammals showed 2-fold "weight specific" and 3-fold total organ metabolic capacity differences. Heart mitochondria from reptiles and mammals showed 2-fold differences in the activity of their enzymes per mg of mitochondrial protein yet showed very similar mitochondrial surface areas per cm3 of mitochondria. Heart mitochondria differ from liver mitochondria which have the same enzyme activities per mg of protein and the same mitochondrial surface area per cm3 of mitochondria in both the reptiles and mammals. A wide variety of reptiles and mammals both showed relationships between total heart metabolic capacity and body weight. Mammals have larger hearts than similar sized reptiles and their hearts have a greater proportion of cellular volume occupied by mitochondria.     

The Complexing of Lysozyme with Poly C and Other homopolymers

Lysozyme forms very large complexes with poly C, in acetate buffer solutions (pH 5.4), when the ratio of lysozyme to poly C concentration is 3/2. When this is less than 3/8 there is virtually no complexing, as evidenced by the low light-scattering power of such mixtures. At such relatively high poly C concentrations, the addition of pancreatic ribonuclease causes both the intensity and dissymmetry of scattering to rise to very high values after which time the intensity falls exponentially with time and with very little change in dissymmetry. Other homopolymers also form largest complexes with lysozyme at characteristic concentration ratios.     

A histochemical study of changes in mitochondrial enzyme activities of rat liver after ischemia in vitro

Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.