ENERGY-LINKED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED LIVER MITOCHONDRIA AND MITOPLASTS : Preservation of Configurations by Freeze-Cleaving Compared to Chemical Fixation

An investigation was carried out in which microsamples of isolated rat liver mitochondria and freshly prepared mitoplasts in defined energy states were freeze-cleaved. Parallel microsamples were fixed with osmium tetroxide and with glutaraldehyde followed by osmium tetroxide as previously used in this laboratory for the preservation of energy-linked mitochondrial configurations. The details of the orthodox configuration of energized mitochondria and the condensed configuration of de-energized mitochondria, as revealed previously by chemical fixation, are confirmed in this report for nonfixed, freeze-cleaved mitochondria. The precise agreement in preservation of configuration obtained by the physical fixation of rapid freezing and by chemical fixation establishes unequivocally that mitochondria undergo energy-linked ultrastructural transformation between the condensed and the orthodox configurations which are thus natural structural states related to the metabolic activity of the mitochondrion. Configurations observed by freeze-cleaving and by chemical fixation reveal that mitoplasts also undergo a specific and dramatic ultrastructural transformation with the induction of oxidative phosphorylation. The transformation appears to be isovolumetric and therefore is thought to be mediated through energized conformational activity in the surface electron-transport membrane of the mitoplast. Passively swollen, spherical, osmotically active mitoplasts could not be fixed rapidly enough by chemical fixatives as normally used without altering the spherical form. In this special case preservation of configurational form required rapid freezing or chemical fixatives of low osmolar concentration.     

Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. III. Loss of coupling ability of adrenal cortex mitochondria in the orthodox configuration

When the cristae of adrenal cortex mitochondria are stabilized in the orthodox configuration by the binding of 20–25 mmoles/mg protein of either Ca²⁺ or free fatty acids (oleic acid), both the capacity for carrying out coupled reactions and the capacity for undergoing energized configurational transitions are lost. The coupled reactions studied included ATP synthesis, divalent cation translocation, monovalent cation trnaslocation, and reversed electron transfer. The coupled processes and energized configurational changes are fully operative when the cristae of adrenal cortex mitochondria are in the aggregated configuration. However, two processes that have been shown to depend on conformational changes (the anaerobic-aerobic proton ejection and energized accumulation of inorganic phosphate) still proceed when mitochondria are in the orthodox configuration. When the mitochondria are initially in the orthodox configuration, addition of divalent cations (Mg²⁺ or Mn²⁺) or albumin induces a transition of the cristae to the aggregated configuration and leads to restoration of all the coupled processes. the orthodox to aggregated transition is reversible and the modulation of this reversibility appears to be one of the key points of control in the mitochondrion and possibly of cellular functions.On leave of absence from the Department of Pathology, Nagoya University School of Medicine, Nagoya, Japan.     

Conformational basis of energy transduction in membrane systems VIII. Configurational changes of mitochondria

A method has been devised for the study of configurational changes in mitochondriain situ during the transition from nonenergized to energized conditions. The method depends upon the following component features: (a) subdivision of the tissue into finely diced sections; (b) the use of a modified Krebs-Ringer phosphate solution as the suspending medium; (c) aerobic conditions as the tactic for imposing the energized state; (d) anaerobic conditions or the presence of uncoupler under aerobic conditions as the tactic for imposing the nonenergized state; and (e) rapid fixation of the diced sections by addition of a mixture of formaldehyde and glutaraldehyde at a controlled temperature. Regardless of the tissue of source (heart, liver, skeletal muscle, retina, kidney) or the species (beef, rat, canary), all mitochondria show unambiguous configurational changes during the transition from nonenergized to energized conditions. The present study has revealed various optional features of the configurational states. Thus, there are two nonenergized configurations of the crista—orthodox and aggregated. The osmotic pressure of the suspending medium determines which nonenergized configuration will be observed. There are at least two variant forms of the energized-twisted configuration—tubular and zigzag. Again the osmotic pressure of the medium is an important factor in determining the form of the crista in the energized-twisted configuration. Mitochondria, such as those of heart muscle with relatively little matrix protein, show the clearest and most regular configurational changes, whereas mitochondria, such as those of liver with an abundance of matrix protein, show a more complex and less regular pattern of configurational change. From this comparative study of mitochondriain situ, it can be concluded that no exceptions have been found to the generalization that changes in configurational state of the cristae accompany changes in the energy state; this exact correlation provides additional support for the hypothesis of the conformational basis of energy transduction in the mitochondrion.Postdoctoral Trainee of the University of Wisconsin.Established Investigator of the American Heart Association.This work was supported in part by U.S. Public Health Service Program Project Grant GM-12847 and by a training grant GM-88 from the National Institute of Medical Sciences.     

Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. II: Determinants of the orthodox-aggregated transition in adrenal cortex mitochondria

Bovine adrenal cortex mitochondria when isolated in a medium 0·25 M in sucrose contaminated (with calcium) have tubular cristae which are periodically expanded to spherical vesicles and contracted to flattened connecting sections. This scalloped tubular form of the cristae corresponds to the nonenergized orthodox configuration—a configuration in which the matrix space is maximally expanded. When adrenal cortex mitochondria are isolated in media in which the free calcium content is relatively low, e.g., a medium 0·25 M in sucrose and 0·1 mM in EDTA, the cristae assume the aggregated configuration—a nonenergized configuration in which the matrix space is maximally contracted. The composition of the isolation medium determined the configuration. Procedures have been described for isolating bovine adrenal cortex mitochondria (a) predominantly in the orthodox configuration, (b) predominantly in the aggregated configuration, or (c) in a mixed (11) population of configurations. The concentration of Ca²⁺ bound to the mitochondrion was found to be a determinant of the nonenergized configuration. When the level of bound Ca⁺⁺ was 20–25 moles/mg protein, the cristae of the mitochondria were entirely in the orthodox configuration. Addition of Ca²⁺ could induce the transition of cristae from the aggregated to the orthodox configuration whereas addition of Mg²⁺ could induce the transition of cristae from the orthodox to the aggregated configuration. The configurational transition could be followed by any of several methods—a change in 90° light scattering, a change in O.D._{520 m}, a change in pH, or examination by electron microscopy. The orthodox to aggregated transition is energy-independent since it proceeds even in presence of inhibitors both of electron transfer and of ATP hydrolysis. The binding of Ca²⁺ is independent of the binding of Mg²⁺; this independent binding is consistent with the opposite effects induced by Ca²⁺ and Mg²⁺, respectively. Whereas Ca²⁺ induces a proton release, a decrease in 90° light scattering and a decrease in O.D._{520 m} (when the cristae are initially in the aggregated configuration), Mg²⁺ induces equal and opposite changes (when the cristae are initially in the orthodox configuration).     

An analysis of the contribution of the preparatory technique to the appearance of condensed and orthodox conformations of liver mitochondria

The structure and volume of isolated mitochondria embedded for electron microscopy during different respiratory states were analyzed in thin sections. Three different embedding methods were compared; osmium tetroxide fixation/acetone dehydration, glutaraldehyde fixation/acetone dehydration, and glutaraldehyde fixation-osmium tetroxide postfixation/acetone dehydration. Analysis of fresh mitochondria, isolated in a sucrose medium, revealed the presence of a homogeneous population with respect to structure when any of the three methods were applied. After fixation with osmium alone, or in combination with glutaraldehyde, nearly 100% of the mitochondria were in a "condensed" conformation. Mitochondria fixed with glutaraldehyde alone resulted in a population of mitochondria that had large spaces separating the two membranes of the cristae which corresponds to the condensed conformation as observed after osmium fixation. Transfer of the mitochondria to the incubation medium led to the appearance of two classes of mitochondria with respect to size. One class had a volume close to that observed when suspended in sucrose, and another class was present that was 30-45% larger. In osmium fixed or in double-fixed preparations, these small and large classes corresponded to "condensed" and "orthodox" forms of mitochondria respectively. When glutaraldehyde was used alone as the fixative, the two size classes were also present. However, the mitochondria were homogeneous with respect to structure. In these mitochondria, the width of the space that separated the cristae membranes had become reduced when compared to mitochondria suspended in sucrose. The two size classes were also present in samples of mitochondria prepared during both states 3 and 4. State 4 conditions did not lead to any significant increase of the number of condensed mitochondria. In state 3 preparations, 65-70% of the population were condensed. The condensed and orthodox forms could be related to normal and swollen forms of mitochondria. Conditions that led to a swelling also led to an increase in the number of orthodox mitochondria in osmium-fixed material. The different appearance of the mitochondria is explained by the different conditions for fixation of the mitochondria that exist when nonswollen and swollen mitochondria are fixed. This difference is particularly crucial in the case of osmium tetroxide due to the unique way this fixative, among generally used fixatives, denatures proteins.     

Ultrastructure of the concentric membrane system in Arthrinium aureum

An ultrastructural study was performed on Arthrinium aureum. The fungi were treated with glutaraldehyde and osmium tetroxide fixation. The hypha and conidia has a concentric membrane system which consisted of multiple membranes of a myelinoid appearance, and continued to the conidia and hypha plasma membrane. The fungi were also treated with periodic acid-alkaline bismuth (PABi) staining after glutaraldehyde and osmium tetroxide fixation. PABi positive materials were found on the marginal glycogen granules, the concentric membrane system and the conidia plasma membrane.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

PREPARATION OF ISOLATED RAT LIVER MITOCHONDRIA FOR ELECTRON MICROSCOPY

A comparative study of the fixation of isolated rat liver mitochondria was undertaken. If the criterion is adopted that after processing, the mitochondria should resemble as closely as possible rat liver mitochondria in situ, the procedure found to produce such preservation was that of fixation in suspension in veronal-buffered 2% potassium permanganate. Fixation in osmium tetroxide produced variable results, while mitochondria fixed in glutaraldehyde were contracted. We suggest that in cases where fixation procedures modify the morphological appearance of mitochondria, the significance of such changes must be treated with caution.     

Osmium dependent argentaffin staining of lysosomes

Summary Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscope, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.Parts of these results have been presented as a poster during the 20th Congress of Electron Microscopy, joint session of the Austrian Society of Electron Microscopy and the German Society of Electron Microscopy, August 23–28, 1981, Innsbruck, Austria     

Structure of the nucleoid in cells of Streptococcus faecalis

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.     

Ultrastructural localization of calcium in prothoracic gland cells of Galleria mellonella L. (Insecta,Lepidoptera) in relation to secretory activity

Summary Localization of intracellular calcium was demonstrated by precipitation with potassium hexahydroxoantimonate in the fixation medium containing osmium tetroxide or osmium tetroxide and glutaraldehyde. The presence of calcium in the precipitates was confirmed by X-ray microanalysis. Cells from active prothoracic glands contain more calcium deposits than inactive glands. The calcium precipitates are mainly localized in the nucleus, in the smooth endoplasmic reticulum, in the hyaloplasm and to a lesser degree in the mitochondria. These findings are consistent with the proposed role of calcium in the stimulation of steroidogenesis.     

Fluidity of phospholipid bilayers and membranes after exposure to osmium tetroxide and gluteraldehyde

The response of fluid bilayer regions to osmium tetroxide and glutaraldehyde fixation was examined in phospholipid multilayers and in nerve bundles from the walking legs of the lobster Homarus americanus. The samples were spinlabeled either with 5-doxylstearic acid (the 4′4′-dimethyloxazolidine-N-ozyl derivative of 5-ketostearic acid) or the maleimide spin label, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Osmium tetroxide fixation abolishes the characteristic orientation of the spin-labeled lipid bilayer regions and virtually eliminates motion on the electron spin resonance time scale. Glutaraldehyde treatment reduces the motion of maleimide spin labels covalently attached to proteins. However, in contrast to osmium tetroxide fixation, glutaraldehyde has essentially no effect on the orientation and mobility in the fluid bilayer regions, and hence probably does not restrict directly the potential for translational motion in membrane phospholipid bilayer regions.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.     

The inhibition of mitochondrial energized processes by fluorescein mercuric acetate

Fluorescein mercuric acetate (FMA) has been shown to be a potent inhibitor of energized processes in both beef heart mitochondria and ETP_H particles. FMA reacts preferentially with a small number of specific sulfur atoms and inhibits the phosphate-dependent configurational transition. FMA enhances the anaerobic to aerobic pH changes observed in intact mitochondria and submitochondrial particles, and also enhances nonenergized swelling in 0·15 M sodium or potassium chloride. The results are interpreted in terms of a model whereby FMA, in reacting with the mitochondrion, modifies its conformation. The resulting conformational changes which occur upon energization are therefore different from those conformational changes which would occur in the absence of FMA. The net result of this process is the inhibition of some processes (e.g., oxidative phosphorylation, ATP-³²P_i exchange, etc.) and the enhancement of other processes (the proton shift and nonenergized swelling in chloride salts).This work was supported in part by U.S. Public Health Service Program Project Grant GM-12847 and by a training grant GM-88, both from the National Institute of General Medical Sciences. Meat by-products were generously furnished by Oscar Mayer and Co., Madison, Wisconsin.     

Specific inhibition of phosphate transport in mitochondria by N-ethylmaleimide

N-ethylmaleimide (NEM), a reagent that alkylates free sulfhydryl groups, was shown to be a highly effective inhibitor of the following coupled mitochondrial processes: oxidative phosphorylation, ATP-³²Pi exchange, Pi-induced light scattering and configurational changes, State III respiration, valinomycin-induced translocation of potassium with Pi as the anion, and calcium accumulation in presence of Pi. However, NEM was less effective or ineffective in inhibiting some processes that do not require inorganic Pi, namely electron transfer and ATPase activity, ADP binding, energized light scattering changes induced by arsenate and nonenergized light scattering changes induced by acetate. The rate of oxidative phosphorylation and of ATP-³²Pi exchange was normal in ETP_H particles prepared from NEM-treated mitochondria. Also NEM, even et levels 2–3 times greater than those required to inhibit oxidative phosphorylation in intact mitochondria, did not inhibit coupled processes in submitochondrial particles. We are proposing that NEM alkylates sulfhydryl groups in the mitochondrion that modulate Pi translocation, and that the suppression of Pi translocation blocks oxidative phosphorylation, the Pi-dependent energized configurational change in mitochondria and Pi-dependent transport processes.On leave of absence from the Department of Biochemistry, Cancer Institute Okayama University Medical School, Okayama, Japan.On leave of absence from the Department of Pathology, Nagoya University Medical School, Nagoya, Japan.     

An atypical crista resembling a "tight junction" in bean root mitochondria

The conformation and structure of an atypical crista found in a small percentage of the mitochondria in root tip cells of Phaseolus vulgaris L. have been studied electron microscopically in material fixed in glutaraldehyde followed by osmium tetroxide. In its transformation into an atypical crista, a normal crista elongates, broadens, and flattens, and the inner leaflets of its apposed unit membranes appear to fuse in a manner analogous to the formation of "tight junctions" between certain animal cells. The result is a large platelike, quintuple-layered structure, 240–260 A thick, whose long axis parallels that of the mitochondrion. The outer layers of the "plate," bordering on the mitochondrial matrix, are thickened and exhibit striking patterns in the micrographs. The structure of the plate is compared with that previously described for tight junctions between animal cells.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.     

Use of isoelectric focusing to determine the isoeletric point of bovine serum albumin after treatment with various common fixatives

Synopsis Isoelectric focusing was used to determine the effects of fixation on the isoelectric point of bovine serum albumin. Aqueous solutions of formaldehyde, -hydroxyadipaldehyde, osmium tetroxide and potassium dichromate produced no change or a relatively small decrease. Glutaraldehyde produced two fractions of fixed protein, the larger with a considerably lowered isoelectric point. The results are interpreted as suggesting that glutaraldehyde reacts rapidly and largely irreversibly, whereas the other fixatives react more slowly and reversibly with bovine serum albumin.