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The structure of mitochondrial membranes in frozen sections
JOURNAL OF ULTRASTRUCTURE RESEARCH
56, 233-246 (1976)
The Structure of Mitochondrial Membranes in Frozen Sections I FRITIOF
S. SJOSTRAND AND W I L H E L M BERNHARD
Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024, and Institut de Recherches Scientifiques sur le Cancer, Villejuif, France Received April 9, I976 Cryomicrotomy u n d e r conditions t h a t would involve reduced risks for protein d e n a t u r a t i o n was applied in a n analysis of the structure of the i n n e r mitochondrial m e m b r a n e s of r a t h e a r t muscle tissue. In order to prevent extensive conformational changes, the tissue was crosslinked for a short time in glutaraldehyde. To minimize t h e effects of intracellular ice crystal formation d u r i n g freezing, the tissue was partially dehydrated by infusion of sucrose according to Tokuyasu (J. Cell Biol. 57, 551 (1973)) or by immersion in 30% glycerol. Caution was t a k e n to reduce the d e n a t u r a t i o n of proteins t h r o u g h surface tension during transfer of the sections to the grids. The sections were analyzed under conditions close to m i n i m u m beam damage. The i n n e r m e m b r a n e s measured about 260/~ in thickness, were closely apposed, were lacking any intracristal space, and showed a particulate substructure with the particles m e a s u r i n g from 30 to 100/~ in diameter. Prolonged fixation in glutaraldehyde at high concentration (2.5%), elimination of partial dehydration before freezing, collecting the sections on DMSO, and drying the sections before negative staining resulted in the i n n e r mitochondrial m e m b r a n e s appearing as consisting of two 50-70/~ thick m e m b r a n e elements separated by a n intracristal space and no particulate substructure. A similar appearance Of the m e m b r a n e s was observed when the tissue was dehydrated in ethanol after fixation for 1 h r in 2.5% glutaraldehyde and rehydrated before cryomicrotomy. A similar appearance was also characteristic for osmium-fixed and Epon-embedded material. It is concluded t h a t this appearance of the i n n e r mitochondrial m e m b r a n e s is due to extensive d e n a t u r a t i o n of the m e m b r a n e proteins. The first-mentioned appearance with 260/k thick i n n e r m e m b r a n e s is considered to represent a situation close to the native state of these membranes.
The improvement of the design of the electron microscope during the last 10 years and the development of the scanning transmission electron microscope have opened entirely new possibilities for the analysis of cellular structures at a molecular level. In order to make use of the improved instrumentation, it is necessary to develop preparatory procedures that make it possible to analyze complex biological structures under conditions where the structure at a molecular level is retained in a state as close as possible to that of the intact native structure. Only methods that allow preparing specimens that are thin enough to be analyzed with a minimum of This work was supported by USPHS G r a n t EY00097 and NSF G r a n t BMS 74-20390. Additional support in the form of a n NIH Special Fellowship No. i FO3 GM 55281-01 is gratefully acknowledged. Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
interference of superposition effects and with maximally favorable contrast conditions are suitable. Ultrathin sectioning is the only method that allows preparing specimens thin enough to be used for a systematic analysis of the structure of complex cellular components. It is obvious that the conventional techniques used for fixation and embedding tissues for ultrathin sectioning lead to extensive denaturation of proteins. Denaturation of tissue proteins was, in fact, aimed at in tissue preparation for light microscopy because it was advantageous to stabilize cellular structures by making the proteins insoluble by denaturation, which at the same time made the proteins accessible to intense staining. In electron microscopy, this way to stabilize structures is used when examining 233
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subcellular structural components in thin sections. It is a useful technique when examining such components at a level of resolution where the interest is confined to determining the presence of the components and their localization, extension, and amount. Such analysis can be pursued without requiring that the cellular components be preserved with their native molecular structure intact. However, when analyzing the molecular structure of these components and applying cytochemical and immunological techniques to localize various enzymes in cells, it is of basic importance that preparatory methods which do not involve denaturation of the proteins be applied. The freeze-sectioning technique has been developed as a method that theoretically appeared suitable from this point of view. This method allows preparing sections of tissues that have not been fixed or embedded. This means that the tissues have not been exposed to the denaturing effects of fixatives and of organic solvents in connection with dehydration. In a study of the structure of mitochondrial membranes, SjSstrand and Barajas (8) used a weak denaturing solvent, ethylene glycol, for dehydration according to Pease (6) before embedding in a polar embedding medium, Vestopal W. It was, however, found to be necessary to stabilize the protein conformation through intermolecular cross-linking before dehydration in ethylene glycol because even such a weak denaturing organic solvent introduced sufficient conformational changes to radically modify the appearance of the mitochondrial membranes. Furthermore, it was required that the dehydration be incomplete, allowing the proteins to retain part of the hydration water. Similar observations regarding membrane structure were made on material that had been partially dehydrated in 30% ethylene glycol after brief cross-linking with 1% glutaraldehyde and then rapidly frozen in propane chilled to -170°C,
freeze-dried at -80°C, and embedded in hydroxypropyl methacrylate at -27°C using uv light to polymerize the methacrylate (8, 11). In this case, the low temperature, which was maintained continuously after freezing until the embedding medium had solidified, reduced the risk of extensive changes in protein conformation and prevented dissolution and extraction of the lipids. Meanwhile, the freeze-sectioning technique has been worked out to allow cutting sufficiently thin sections to be used for electron microscopy at resolutions high enough to make observation of fine details in the structure of membranes possible. It then appeared justifiable to apply this technique to the problem of mitochondrial membrane structure and to compare these results with those obtained with the two methods described above. The observations made are similar to those made by SjSstrand and Barajas (8) and add some further weight to the concept of mitochondrial membrane structure proposed by these authors. MATERIALS AND METHODS Young adult r a t s were sacrificed after brief e t h e r narcosis and the hearts were removed quickly. In one series of experiments, the h e a r t tissue was cut into small pieces m e a s u r i n g about 0.2 m m in thickness. They were either frozen directly or crosslinked before freezing. In order to stabilize the structure, the tissue was cross-linked by brief exposure to a 1% solution of glutaraldehyde. The time for the cross-linking was only 5 m i n to avoid the d e n a t u r i n g effect ofglutaraldehyde (4). To avoid extensive crystallization of the water in the cells d u r i n g freezing, the tissue was partially dehydrated in 30% glycerol or immersed in sucrose before freezing according to Tokuyasu (13). The tissue was soaked first in 0.054 M and t h e n in 0.1 M sucrose for 10 m i n in each concentration of sucrose. The tissue was frozen rapidly b y i m m e r s i o n in propane chilled by means of liquid nitrogen. The cryo-ultramicrotomy was carried out according to the general principles described by B e r n h a r d and Viron (1) u s i n g a Sorval Cryokit adapted to a Sorval MT2 ultramicrotome. The tissue was cut at -80°C. The sections were either collected dry by m e a n s of
FREEZE-SECTIONED MITOCHONDRIAL MEMBRANES a hair and deposited on the filmed grid or were picked up on a drop of saturated sucrose solution (0.44 M) according to Tokuyasu (13). In another set of experiments, larger pieces of heart muscle tissue were analyzed either fresh or after glutaraldehyde or formaldehyde fixation. A 2.5% glutaraldehyde solution in buffer was used and the fixation time was 1 hr. For formaldehyde fixation, the fresh tissue was placed on a piece of large mesh gauze and fixed in air saturation with formaldehyde vapor in a glass-stoppered bottle containing an excess of solid paraformaldehyde in 1 ml of water. The fixation was done at 4°C and the fixation time was 5 min. In these experiments, the tissue was either frozen directly after fixation or partially dehydrated in 30% glycerol before freezing. The tissue was frozen in liquid nitrogen instead of in propane chilled by means of liquid nitrogen. The sections were cut at -70°C and were either collected dry in air and transferred directly to the filmed specimen grids or collected on a drop of saturated sucrose solution or on a trough filled with 50% DMSO. The sections of unfixed frozen tissue were picked up with a hair and mounted on copper grids coated with a Formvar-carbon film, gently flattened with a cold copper rod according to Christensen (2). The sections were dried by sublimation in N2 vapor at about - 80°C. The sections were stained negatively by means of 2% silicotungstate. Staining time was 5 sec. Heart muscle tissue embedded in Epon was also analyzed. It was fixed either in 2% O s Q in Cacodylate buffer, pH 7.2, for 1 hr, or in 2.5% glutaraldehyde buffered in Cacodylate, pH 7, for 30 min followed by 1 h r postfixation in O s Q . This material was dehydrated in a graded series of ethanol (70, 80, 95, and 100%). The sections were examined in a Siemens 101 electron microscope at 100 kV with a 50-t~m objective aperture. The electron optical magnification was 100,000 times. In order to minimize the effects of beam damage on the structure, the focusing was performed exposing an area in the sections adjacent to the area t h a t was photographed. This was achieved by moving the specimen stage and taking the photograph as quickly as possible after the repositioning of the stage. T h i s w a y , the photographed area had not been exposed to the beam before being photographed. RESULTS
The inner mitochondrial membranes showed greatly different appearances, depending upon what preparatory procedure had been used. The inner membranes thus
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varied with respect to the thickness of the individual membrane elements and with respect to the presence or absence of an intracristal space. We can thus distinguish between two different patterns of the profile view of the mitochondrial inner membranes. The first pattern, the type 1 pattern, was characterized by the two membranes or membrane elements of the inner membranes being closely apposed (Figs. 1 and 2). Very little or no stain had accumulated in the middle of the membranes where the two membrane elements faced each other. When this boundary was indicated by the stain, the staining was faint with the opacity of the staining considerably lower than that of the matrix space. This shows that the two membrane elements were not separated by an intracristal space." Instead, the staining only involved shallow depressions at the cut edge of the inner membranes. That the staining of the inner membranes was located in the middle of these membranes shows that the inner membranes were viewed in profile and that they were not tilted. This pattern was further characterized by rather thick inner membranes. Their thickness was measured to be 260/~. This figure represents the average thickness determined on the basis of several experiments where the experimental conditions had been varied. Since the two membrane elements were closely apposed, the thickness of the individual membrane element would be 130/~. In close to focus pictures (Figs. i and 2), the inner membranes show a particulate substructure with the smallest particles delineated by the negative stain measuring about 30 • in diameter. Larger light areas in the membranes measured up to about 100/~ in diameter. It was characteristic that the particulate substructure was lacking any simple geometrical order. The particles appeared randomly arranged. The particulate struc-
FIG. 1. C••se t• f•cus picture •frat heart musc•e mit•ch•ndri•n in tissue t h a t had been cr•ss-linked with 1% glutaraldehyde for 5 min and infiltrated with sucrose before rapid freezing. Negative staining. The inner mitochondria] m e m b r a n e s appear as compact structures lacking an intracristal space: They show a particulate substructure. This is the type 1 pattern, x 600 000. 236
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@ FIG. 2. Rat heart muscle mitochondrion in tissue that had been cross-linked for 5 min in 1% glutaraldehyde and infiltrated with 30% glycerol before rapid freezing. Negative staining. The appearance of the inner membrane is that of type 1 pattern. × 300 000. 237
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ture made the surface of the membranes appear rough. The boundary between the inner membranes and the matrix was therefore irregular. The type 2 pattern differed from the type 1 pattern by the membrane elements measuring only about 60 /~ in thickness (Figs. 3-6) and by the two membrane elements being separated by a more or less wide accumulation of stain, indicating the presence of an intracristal space. In the type 2 pattern, the two membrane elements of the inner membranes were sharply outlined by the negative stain on both the matrix side and toward the intracristal space. The boundaries of the membrane elements appeared almost smooth in contrast to the irregular boundaries at the matrix space observed in the type 1 pattern. The thickness of the inner membrane element observed in the type 2 pattern was rather uniform and measured 50 to 70/~ as compared to 130/~ estimated for the type 1 pattern. The membrane elements furthermore appeared at a high contrast in the type 2 pattern, and there were no clear indications of any particulate substructure in these membrane elements in contrast to the type 1 pattern. The average width of the intracristal space varied between 20 and 70/~ in different experiments. It was characteristic that in both type 1 and type 2 patterns, the matrix space was intensely stained, showing that little material prevented an extensive accumulation of stain in this space. Even when the intracristal space in the type 2 pattern was wide, the opacity of the stain in this space never exceeded that of the matrix space. Since the intracristal space only exceptionally contains any particulate matter, the similarity with respect to the opacity of the staining of matrix and intracristal space
shows that there must be a rather limited amount of particulate matter in the matrix space. In the type 1 pattern, there were indications of the presence of particles of a size corresponding to protein molecules, that is, in the range of 30-50/~, in the matrix space. For comparison, thin frozen sections of the optic nerve of the cat were analyzed. The layered structure of the myelin sheath was well preserved even after short crosslinking with 1% glutaraldehyde. The mean periodicity of the myelin sheath was estimated to 120 A, and the average thickness of a single layer in the myelin sheath was about 60/~. The myelin layers appeared uniformly opaque and did not show any particulate substructure. When correlating the appearance of the inner mitochondrial membranes to the preparatory treatment, it became obvious that the type 1 appearance, with thick membrane elements "closely apposed without any open intracristal space separating the membrane elements and a particulate substructure, was observed in those experiments where the tissue had been exposed to short-term cross-linking and partial dehydration either by means of sucrose (Fig. 1) or glycerol (Fig. 2) before freezing. Long-term cross-linking (Fig. 3), elimination of any protection against the formation of ice crystals intracellularly (Figs. 3 and 4) and exposure of the frozen sections to DMSO (Fig. 5) resulted in the type 2 appearance of the inner mitochondrial membranes, with thin membrane elements separated by a m o r e or less wide intracristal space. In the sections of material that had been frozen fresh, without any pretreatment, the inner membranes mostly appeared
FIG. 3. I n n e r m e m b r a n e s in r a t h e a r t muscle mitochondrion in tissue t h a t had been cross-linked for I h r in 2.5% g l u t a r a l d e h y d e which denatures the proteins. Type 2 p a t t e r n with t h i n m e m b r a n e elements separated by a n intracristal space. × 300000. FIG. 4. Type 2 p a t t e r n of inner mitochondrial m e m b r a n e s in r a t h e a r t muscle mitochondrion in tissue fixed in formaldehyde vapor for 5 rain and frozen without any cryoprotective agent. Negative staining. × 300 000.
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FREEZE-SECTIONED MITOCHONDRIAL MEMBRANES
rather irregular and ill defined. They did not show any intracristal space, and the total thickness of the membranes was measured to be 260 A. The type 2 pattern was also characteristic of the mitochondria in tissue that had been dehydrated in ethanol and then rehydrated before freeze-sectioning (Fig. 6). In the material that had been treated the conventional way with OsO4 fixation and extensive glutaraldehyde fixation followed by OsO4 fixation, ethanol dehydration, and Epon embedding, the mitochondria appearance corresponded to the type 2 pattern, although in this case the membrane elements were positively stained. DISCUSSION
In frozen-sectioned material, inner mitochondrial membranes were observed that differed drastically in appearance from inner membranes in conventionally prepared tissue. In the latter case, the individual membrane elements appear as 50/k thick, triple-layered components (10) and the membrane elements are usually separated by a wide, empty intracristal space. The layers of the membrane elements run continuously through the whole membrane elements without interruption, or the membrane elements appear to consist of a layer of globular components (7). In the frozen-sectioned material, the mitochondrial inner membranes showed two different profile patterns. The type 1 pattern was the one that most drastically deviated from that usually observed in sections of conventionally fixed and embedded m a t e r i a l The two membrane elements of the inner membranes were closely apposed, with no indications of any intracristal space separating them. The average total thickness of the inner mem-
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branes was 260 A, which would correspond to a thickness of the individual membrane elements of 130/~, versus 60 A in the type 2 pattern. In the type i pattern, the membrane elements did not show any continuous uninterrupted layer or layers. Instead, they exhibited a particulate substructure which made the surfaces of the membranes appear irregular. No indications of the presence of a continuous uninterrupted lipid bilayer were therefore found. The type 2 profile pattern was the pattern showing the greatest similarity to the pattern observed after conventional fixation and embedding of the tissue. In addition to the open intracristal space, the membrane elements measured only about 60/~ in thickness and appeared rather uniformly thick, sharply outlined, and with a uniform low opacity not revealing any particulate substructure. The width of the intracristal space varied considerably (20-70
A). When discussing these two profile patterns, it is reasonable first to consider the possibility that they are due to differences in the tilting of the membranes. The difference in thickness of the membrane elements in the type 1 and type 2 patterns and the absence of an intracristal space obviously could be explained by a tilting of the membranes in the cases where the type I pattern is observed. Usually an indication of a boundary between the two membrane elements of the inner membrane could be observed even when only certain spots in the middle of the membrane were stained. The faint and incomplete staining of this boundary shows that the negative staining involved only shallow depressions at the cut surfaces of the inner membranes. If
FIG. 5. I n n e r mitochondrial m e m b r a n e s showing type 2 p a t t e r n in r a t h e a r t muscle tissue fixed in formaldehyde vapor for 5 min. The sections were collected on DMSO. Negative staining. × 300 000. FIG. 6. I n n e r mitochondrial m e m b r a n e s in r a t h e a r t muscle tissue t h a t had been fixed in formaldehyde vapor for 5 min, dehydrated in ethanol, a n d rehydrated before cryomicrotomy. The p a t t e r n is t h a t of type 2. Negative staining. × 300 000.
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these membranes would be tilted, the staining of this boundary would be displaced laterally in the direction of the tilt and would not be projected to the middle of the inner membrane. The type 1 pattern was observed where the inner membranes appeared at a maximum contrast in these sections. This means that the negative stain in these regions was observed at a maximum thickness of the dried down stain. A maximum thickness of the stain corresponds to a minimum interference by the organic material. A maximum contrast also means that the inner membranes appeared with maximum brightness. This condition requires that the inner membranes be oriented parallel to the electron beam because any tilting of the membranes would result in a lowering of the contrast. A tilting of a membrane would mean that it would, for instance, lean over an area where the stain has accumulated. A tilting would therefore mean that in the image plane, the membrane would appear superimposed on the area containing the stain. At small degrees of tilt, this would make the membrane appear thinner and diffusely outlined. With the very dense arrangement of the inner membranes characteristic of heart muscle mitochondria, a slight tilt would result in an overlapping of the images of the individual membranes in the image plane and the disappearance of any matrix space in the image. This explains the appearance of Fig. 2, where a clear pattern showing inner membranes separated by matrix spaces is observed in the center of the picture, while the pattern is diffuse in the upper right and the lower left corners. The consistency in the dimensions of the inner membranes and of the membrane elements in pictures showing different types of profile patterns makes it justifiable to conclude that tilting of the membranes can have had only a limited effect on these measurements. This consistency
can be explained by the reduction of contrast that even slight tilting must introduce. We therefore find it justifiable to conclude that the two patterns are real profile patterns of inner mitochondrial membranes that differ structurally. It then becomes interesting to relate these patterns to the varied experimental conditions. The type 1 pattern was observed in experiments where the material had been stabilized by means of brief cross-linking with glutaraldehyde at a low concentration. This means that the exposure to glutaraldehyde had not been extensive enough to cause denaturation of the proteins. In contrast, long time cross-linking with high concentrations of glutaraldehyde resulted in mitochondria with inner membranes appearing according to the type 2 pattern. Under these conditions, glutaraldehyde denatures the proteins. The type 1 pattern was observed in material that had been partially dehydrated either with sucrose or glycerol to prevent the formation of intracellular ice crystals. This is one important factor to keep under control, since crystallization of the water leads to extensive pH changes due to differences in the solubilities of inorganic salts. Such pH changes can affect the conformation of the proteins. The type 2 pattern was observed in material that had been frozen without adding any protective agent and furthermore had been frozen slowly by immersion of relatively large pieces of tissue in liquid nitrogen. The type 2 pattern was observed in sections that had been exposed to conditions that would favor protein denaturation, such as collecting the specimens on DMSO, dehydrating the sections in ethanol followed by rehydration, osmium fixation, dehydration, and embedding in a plastic. We therefore can link the type 2 pattern
FREEZE-SECTIONED MITOCHONDRIAL MEMBRANES to conditions that lead to protein denaturation. The type 1 pattern, on the other hand, is associated with experimental conditions t h a t reduce the risks for protein denaturation. It then appears justifiable to conclude that the thin membrane elements characteristic of the type 2 pattern represent membrane elements in which the globular membrane proteins have been denatured. Since denaturation makes the globular proteins lose their defined globular shapes, it is obvious t h a t there should be little indication of a particulate substructure in these modified membranes. This explains the absence of such a substructure in the type 2 membranes. A correlation of the thickness of membranes as observed in thin sections to denaturing influences was shown by Kretzer (3) when applying the preparatory procedure of SjSstrand and Barajas (8). The partition membranes of the chloroplast of Chlarnydomonas reinhardi showed a maximum thickness of 270 A in material that had been prepared with the intention of extensively reducing the denaturation of membrane proteins. Cross-linking in 6% instead of 1% glutaraldehyde for 1 min reduced the partition membrane thickness to 150 A. Exposing the cross-linked (1 min, 1% glutaraldehyde) Chlarnydomonas to acetone for 1 min led to a reduction of the thickness of the partition membranes to 100/~. Prolonging the dehydration time in ethylene glycol from 1 to 60 min led to the same effect, as well as ethylene glycol dehydration for 1 min without previous stabilization through cross-linking. The particulate substructure observed in the type 1 pattern was of the dimensions of globular proteins molecules and of complexes of protein molecules. It therefore appears reasonable to consider that these particles are globular proteins which are the major components of these membranes. The fact t h a t the membrane particles
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appeared to be arranged in a random fashion and were not forming geometrically regular patterns is explained by the fact that these membranes, in contrast to several specialized membranes, consist of a large number of enzymes which differ with respect to molecular weights and shapes. As was pointed out by SjSstrand and Barajas (8), it is highly unlikely that such a mixture of components could be arranged according to a geometrically regular pattern. A more or less random arrangement of the membrane particles was also observed in freeze-fractured mitochondria (5). It therefore appears justifiable to conclude that the particles are globular protein molecules and complexes of protein molecules arranged either randomly or according to patterns that are too complex to be identified with any recognizable, more or less simple geometrical pattern. The absence of a continuous, uninterrupted layered structure in the mitochondrial membranes makes it justifiable to conclude that there is no continuous, uninterrupted lipid bilayer forming a backbone structure in these membranes. This agrees with the low lipid content of the inner mitochondrial membranes. The absence of an intracristal space in the mitochondria showing the type 1 pattern and the appearance of such a space in connection with denaturation of the membrane proteins make it reasonable to question the existence of such a space in the native state of the mitochondria. It seems justifiable to propose that, instead, the two membrane elements are closely apposed in the native state. It is characteristic that freeze-sectioning of fresh, non-cross-linked material is particularly difficult and results in less well organized membrane patterns, although areas showing the type 1 pattern were observed. This shows that freeze-sectioning does expose the structure to considerable strain. Stabilization of the structure by
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means of inter- and intramolecular crosslinking therefore appears to make the mitochondrial membrane resist destruction by the strain associated with the sectioning. The appearance of the inner membranes as represented by the type 1 profile pattern is similar to that observed by Sj6strand and Barajas (8) when using preparatory procedures aimed at limiting the conformational changes of membrane proteins in connection with embedding for thin sectioning. In that study, the thickness of the membrane elements of the inner membranes of kidney mitochondria was estimated to 130-150/~. No layered structure could be observed, but an irregular particulate substructure was found over the entire cross section of the inner membranes. The present observations on negatively stained frozen sections show that the inner mitochondrial membranes exclude the negative stain from penetrating into membrane elements which are about 130 /~ thick. This agrees with the concept of these membranes as consisting of proteins and lipids in a condensed state with a three-dimensional arrangement of the molecules (8).
A Critical Examination of the FreezeSectioning Technique From a theoretical point of view, the freeze-sectioning technique is likely to introduce less drastic conformational changes of the protein molecules than the conventional fixation and dehydration and embedding techniques. In the latter case, each step can contribute to the denaturation of the proteins: during fixation by exposing the proteins to heavy metal ions, during dehydration by the action of denaturing organic solvents, and during embedding by transferring the tissue to a nonpolar plastic monomer and then exposing the proteins to heat. This is, in fact, a very consistent type of treatment to achieve denaturation.
The freeze-sectioning, on the other hand, eliminates the need for dehydration by means of organic solvents and embedding in a plastic. This does not, however, mean that this procedure does not involve several steps at which the protein conformation can be affected. It seems justifiable to point to the most obvious risks when applying this method. The emphasis during the development of this technique in the past has, for obvious reasons, been on finding favorable conditions on the whole to obtain good sections even with risks that proteins would be denatured. In this study, particular attention was paid to applying the freeze-sectioning technique under conditions that would reduce the risks for conformational changes of the protein molecules. The first critical step in preparing frozen sections is the freezing of the tissue. However rapidly the !tissue is chilled down by using small pieces and the most ideal medium for quick removal of heat from the tissue, there will occur a phase separation with the formation of ice crystals. This leads to an increasing concentration of solutes in the still Iiquid phase. As a consequence, considerable pH changes can occur which can affect protein conformation. Surface tensions at the ice crystal interfaces are likely to favor denaturation of proteins (11). The pretreatment of the tissue with sucrose or glycerol leads to a reduction of the concentration of free water in the cells, and if the sucrose or glycerol concentration is sufficiently high, the crystallization of water is prevented or greatly inhibited. This does not, however, apply to low concentrations (10-20%) of glycerol. The minimum concentration is about 30% (12). The next critical step involves the sectioning. All the energy transferred from the knife to the specimen block and responsible for producing a crack through the tissue is concentrated at this crack. This energy is transformed into heat. This
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means t h a t the amount of heat transferred to be cross-linked before it could be exper unit volume of tissue when the knife posed to the strain involved in dehydration edge hits the block is high, and the tissue in ethylene glycol and Vestopal embedcan melt at the surface of the crack. The ding. The so-called inert dehydration molten surface layer will be exposed to method of Pease (6) was therefore not usesurface forces. Surface forces are also ac- ful for analysis of cell structures at a motive at the two surfaces of the frozen sec- lecular level, which does not exclude its tion in the frozen state. usefulness for studies at other levels. One of the most obvious dangers involves the collecting of the sections and Trying to Reduce the Risks for Conformational Changes during their thawing. In the case where ice crysFreeze-Sectioning tals have been formed, the thawing itself means a redistribution in the cells of the The striking experience in this study as water trapped in ice crystals, and the well as in that of SjSstrand and Barajas (8) structural appearance of the cell is recon- was the very brief cross-linking t h a t was stituted with little sign of any persisting sufficient to stabilize the mitochondrial deformation imposed by the ice crystals. membranes. This briefness, as well as the This reconstitution, however, has been low concentration of the glutaraldehyde shown to occur with respect to the struc- used, reduces the risk that glutaraldehyde ture of the cells viewed at an intermediate will change the protein conformation, as resolution, revealing the overall arrange- high concentrations of glutaraldehyde and ment of membranes in the cytoplasm and long exposure times do (4). in mitochondria. What happens at a tooThe treatment of the tissue with high lecular level, on the other hand, is not concentrations of sucrose before freezing known. according to Tokuyasu (13) aims at reducWhen drying the sections in air, surface ing the risks for extensive formation of ice tension will expose the cellular structures crystals by increasing the concentration of to enormous forces, and surface denatura- solutes in the cells. This treatment has a tion of proteins is likely to be rather exten- twofold effect. On the one hand, it makes sive. sucrose molecules enter the cells to inIt appears that the risks for conforma- crease the concentration of solutes, and on tional changes during freeze-sectioning the other hand it removes water from the that have been outlined above can explain cells by establishing a temporary osmotic the difficulties in demonstrating enzyme gradient across the cell membrane. Both activity and immunological activity in fro- these effects tend to favor vitrification of zen sections. the tissue without preceding extensive forThe risks involved in freeze-sectioning mation of ice crystals. also are illustrated clearly by the fact that With respect to sectioning, the temperait is difficult to retain the fine structure of ture was kept at -80°C which, however, is cells and, in the present case of mitochon- unlikely to eliminate the possibilities of a dria, if fresh material is freeze-sectioned. thawing of the tissue at the surface of the Cross-linking with glutaraldehyde or fixa- crack formed during sectioning. The lowtion with paraformaldehyde have been est possible temperature t h a t is compatifound to be important in reducing the risk ble with obtaining sections should be of damage to the structure of cells during aimed at. freeze-sectioning. The method introduced by Tokuyasu This parallels the experience of Sj6s- (13) to pick up the sections involves expostrand and Barajas (8) t h a t the tissue had ing the tissue to a high concentration of
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sucrose. This is a priori not to be assumed is well retained after freeze-sectioning to be a treatment without danger. From a makes it unlikely that this explanation is functional point of view, it has been shown justified. that the respiratory chain is affected by In spite of the risks for structural alterahigh concentrations of sucrose, which is tions introduced by freeze-sectioning and shown by a reduction of respiration and of by the two methods used by SjSstrand and the respiratory control index. The results Barajas (8, 9), it seems justifiable to put when the sections were picked up dry di- more weight on the observations made rectly on the filmed grid were, however, with these three methods aimed at reducsimilar to those when a drop of sucrose was ing conformational changes in protein used. The Tokuyasu technique therefore is molecules than on observations made on not likely to introduce structural changes material in which proteins have been sysat the level of resolution that presently can tematically denatured. This statement apbe achieved on frozen sections. pears rather obvious in the case where the It was characteristic that the drying of analysis involves the molecular structure the section with negative stain was neces- of cellular components. sary to retain the structure of the mitoThe very competent and skilled assistance of chondria. This is an experience similar to Miss Annie Viron is gratefully acknowledged. that for other applications of the negativeREFERENCES staining technique. Presumably, the presence of the stain approaching saturation 1. BERNHARD,W., AND VIRON, A., J. Cell Biol. 49, and precipitating on tissue surfaces re731 (1971). 2. CHRISTENSEN,A. K., J. Cell Biol. 51,772 (1971). duces the destructive influence of surface 3. KRETZER,F,, J. Ultrastruct. Res. 44, 146 (1973). tension. 4. LENARD,J., AND SINGER, S. J., J. CeUBiol. 37, From this discussion, it appears obvious 117 (1968). that it is not possible to assume that the 5. PACKER, L., Bioenergetics 3, 115 (1972). freeze-sectioned material reveals the 6. PEASE, D. C., J. Ultrastruct. Res. 14, 356 (1966). 7. SJSSTRAND, F. S., J. Ultrastruct. Res. 9, 340 structure without any modifications of the (1963). native state of cellular components at a 8. SJ6STRAND, F. S., AND BARAJAS, L., J. Ultramolecular level. struct. Res. 25, 121 (1968). The absence of a continuous layer in the 9. SJ~STRAND, F. S., AND BARAJAS, L., J. Ultrapictures showing the type 1 and type 2 struct. Res. 32, 293 (1970). profile patterns could be the consequence 10. SJ6STRAND, F. S., AND ELFVIN, L., J. Ultrastruct. Res. 7, 504 (1962). of a disorganization of a lipid bilayer by 11. SJOSTRAND, F. S., AND KRETZER, F., J. Ultrathe freeze-sectioning technique if such a struct. Res. 53, 1 (1975). bilayer were present in the native state of 12. SJ0STROM, M., AND THORNZLL, L. E., Science the membrane. The fact, however, that Tools 21, 26 (1975). the layered structure of the myelin sheath 13. TOKUYASU,K. T., Ji Cell Biol 57, 551 (1973).
56, 233-246 (1976)
The Structure of Mitochondrial Membranes in Frozen Sections I FRITIOF
S. SJOSTRAND AND W I L H E L M BERNHARD
Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024, and Institut de Recherches Scientifiques sur le Cancer, Villejuif, France Received April 9, I976 Cryomicrotomy u n d e r conditions t h a t would involve reduced risks for protein d e n a t u r a t i o n was applied in a n analysis of the structure of the i n n e r mitochondrial m e m b r a n e s of r a t h e a r t muscle tissue. In order to prevent extensive conformational changes, the tissue was crosslinked for a short time in glutaraldehyde. To minimize t h e effects of intracellular ice crystal formation d u r i n g freezing, the tissue was partially dehydrated by infusion of sucrose according to Tokuyasu (J. Cell Biol. 57, 551 (1973)) or by immersion in 30% glycerol. Caution was t a k e n to reduce the d e n a t u r a t i o n of proteins t h r o u g h surface tension during transfer of the sections to the grids. The sections were analyzed under conditions close to m i n i m u m beam damage. The i n n e r m e m b r a n e s measured about 260/~ in thickness, were closely apposed, were lacking any intracristal space, and showed a particulate substructure with the particles m e a s u r i n g from 30 to 100/~ in diameter. Prolonged fixation in glutaraldehyde at high concentration (2.5%), elimination of partial dehydration before freezing, collecting the sections on DMSO, and drying the sections before negative staining resulted in the i n n e r mitochondrial m e m b r a n e s appearing as consisting of two 50-70/~ thick m e m b r a n e elements separated by a n intracristal space and no particulate substructure. A similar appearance Of the m e m b r a n e s was observed when the tissue was dehydrated in ethanol after fixation for 1 h r in 2.5% glutaraldehyde and rehydrated before cryomicrotomy. A similar appearance was also characteristic for osmium-fixed and Epon-embedded material. It is concluded t h a t this appearance of the i n n e r mitochondrial m e m b r a n e s is due to extensive d e n a t u r a t i o n of the m e m b r a n e proteins. The first-mentioned appearance with 260/k thick i n n e r m e m b r a n e s is considered to represent a situation close to the native state of these membranes.
The improvement of the design of the electron microscope during the last 10 years and the development of the scanning transmission electron microscope have opened entirely new possibilities for the analysis of cellular structures at a molecular level. In order to make use of the improved instrumentation, it is necessary to develop preparatory procedures that make it possible to analyze complex biological structures under conditions where the structure at a molecular level is retained in a state as close as possible to that of the intact native structure. Only methods that allow preparing specimens that are thin enough to be analyzed with a minimum of This work was supported by USPHS G r a n t EY00097 and NSF G r a n t BMS 74-20390. Additional support in the form of a n NIH Special Fellowship No. i FO3 GM 55281-01 is gratefully acknowledged. Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
interference of superposition effects and with maximally favorable contrast conditions are suitable. Ultrathin sectioning is the only method that allows preparing specimens thin enough to be used for a systematic analysis of the structure of complex cellular components. It is obvious that the conventional techniques used for fixation and embedding tissues for ultrathin sectioning lead to extensive denaturation of proteins. Denaturation of tissue proteins was, in fact, aimed at in tissue preparation for light microscopy because it was advantageous to stabilize cellular structures by making the proteins insoluble by denaturation, which at the same time made the proteins accessible to intense staining. In electron microscopy, this way to stabilize structures is used when examining 233
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subcellular structural components in thin sections. It is a useful technique when examining such components at a level of resolution where the interest is confined to determining the presence of the components and their localization, extension, and amount. Such analysis can be pursued without requiring that the cellular components be preserved with their native molecular structure intact. However, when analyzing the molecular structure of these components and applying cytochemical and immunological techniques to localize various enzymes in cells, it is of basic importance that preparatory methods which do not involve denaturation of the proteins be applied. The freeze-sectioning technique has been developed as a method that theoretically appeared suitable from this point of view. This method allows preparing sections of tissues that have not been fixed or embedded. This means that the tissues have not been exposed to the denaturing effects of fixatives and of organic solvents in connection with dehydration. In a study of the structure of mitochondrial membranes, SjSstrand and Barajas (8) used a weak denaturing solvent, ethylene glycol, for dehydration according to Pease (6) before embedding in a polar embedding medium, Vestopal W. It was, however, found to be necessary to stabilize the protein conformation through intermolecular cross-linking before dehydration in ethylene glycol because even such a weak denaturing organic solvent introduced sufficient conformational changes to radically modify the appearance of the mitochondrial membranes. Furthermore, it was required that the dehydration be incomplete, allowing the proteins to retain part of the hydration water. Similar observations regarding membrane structure were made on material that had been partially dehydrated in 30% ethylene glycol after brief cross-linking with 1% glutaraldehyde and then rapidly frozen in propane chilled to -170°C,
freeze-dried at -80°C, and embedded in hydroxypropyl methacrylate at -27°C using uv light to polymerize the methacrylate (8, 11). In this case, the low temperature, which was maintained continuously after freezing until the embedding medium had solidified, reduced the risk of extensive changes in protein conformation and prevented dissolution and extraction of the lipids. Meanwhile, the freeze-sectioning technique has been worked out to allow cutting sufficiently thin sections to be used for electron microscopy at resolutions high enough to make observation of fine details in the structure of membranes possible. It then appeared justifiable to apply this technique to the problem of mitochondrial membrane structure and to compare these results with those obtained with the two methods described above. The observations made are similar to those made by SjSstrand and Barajas (8) and add some further weight to the concept of mitochondrial membrane structure proposed by these authors. MATERIALS AND METHODS Young adult r a t s were sacrificed after brief e t h e r narcosis and the hearts were removed quickly. In one series of experiments, the h e a r t tissue was cut into small pieces m e a s u r i n g about 0.2 m m in thickness. They were either frozen directly or crosslinked before freezing. In order to stabilize the structure, the tissue was cross-linked by brief exposure to a 1% solution of glutaraldehyde. The time for the cross-linking was only 5 m i n to avoid the d e n a t u r i n g effect ofglutaraldehyde (4). To avoid extensive crystallization of the water in the cells d u r i n g freezing, the tissue was partially dehydrated in 30% glycerol or immersed in sucrose before freezing according to Tokuyasu (13). The tissue was soaked first in 0.054 M and t h e n in 0.1 M sucrose for 10 m i n in each concentration of sucrose. The tissue was frozen rapidly b y i m m e r s i o n in propane chilled by means of liquid nitrogen. The cryo-ultramicrotomy was carried out according to the general principles described by B e r n h a r d and Viron (1) u s i n g a Sorval Cryokit adapted to a Sorval MT2 ultramicrotome. The tissue was cut at -80°C. The sections were either collected dry by m e a n s of
FREEZE-SECTIONED MITOCHONDRIAL MEMBRANES a hair and deposited on the filmed grid or were picked up on a drop of saturated sucrose solution (0.44 M) according to Tokuyasu (13). In another set of experiments, larger pieces of heart muscle tissue were analyzed either fresh or after glutaraldehyde or formaldehyde fixation. A 2.5% glutaraldehyde solution in buffer was used and the fixation time was 1 hr. For formaldehyde fixation, the fresh tissue was placed on a piece of large mesh gauze and fixed in air saturation with formaldehyde vapor in a glass-stoppered bottle containing an excess of solid paraformaldehyde in 1 ml of water. The fixation was done at 4°C and the fixation time was 5 min. In these experiments, the tissue was either frozen directly after fixation or partially dehydrated in 30% glycerol before freezing. The tissue was frozen in liquid nitrogen instead of in propane chilled by means of liquid nitrogen. The sections were cut at -70°C and were either collected dry in air and transferred directly to the filmed specimen grids or collected on a drop of saturated sucrose solution or on a trough filled with 50% DMSO. The sections of unfixed frozen tissue were picked up with a hair and mounted on copper grids coated with a Formvar-carbon film, gently flattened with a cold copper rod according to Christensen (2). The sections were dried by sublimation in N2 vapor at about - 80°C. The sections were stained negatively by means of 2% silicotungstate. Staining time was 5 sec. Heart muscle tissue embedded in Epon was also analyzed. It was fixed either in 2% O s Q in Cacodylate buffer, pH 7.2, for 1 hr, or in 2.5% glutaraldehyde buffered in Cacodylate, pH 7, for 30 min followed by 1 h r postfixation in O s Q . This material was dehydrated in a graded series of ethanol (70, 80, 95, and 100%). The sections were examined in a Siemens 101 electron microscope at 100 kV with a 50-t~m objective aperture. The electron optical magnification was 100,000 times. In order to minimize the effects of beam damage on the structure, the focusing was performed exposing an area in the sections adjacent to the area t h a t was photographed. This was achieved by moving the specimen stage and taking the photograph as quickly as possible after the repositioning of the stage. T h i s w a y , the photographed area had not been exposed to the beam before being photographed. RESULTS
The inner mitochondrial membranes showed greatly different appearances, depending upon what preparatory procedure had been used. The inner membranes thus
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varied with respect to the thickness of the individual membrane elements and with respect to the presence or absence of an intracristal space. We can thus distinguish between two different patterns of the profile view of the mitochondrial inner membranes. The first pattern, the type 1 pattern, was characterized by the two membranes or membrane elements of the inner membranes being closely apposed (Figs. 1 and 2). Very little or no stain had accumulated in the middle of the membranes where the two membrane elements faced each other. When this boundary was indicated by the stain, the staining was faint with the opacity of the staining considerably lower than that of the matrix space. This shows that the two membrane elements were not separated by an intracristal space." Instead, the staining only involved shallow depressions at the cut edge of the inner membranes. That the staining of the inner membranes was located in the middle of these membranes shows that the inner membranes were viewed in profile and that they were not tilted. This pattern was further characterized by rather thick inner membranes. Their thickness was measured to be 260/~. This figure represents the average thickness determined on the basis of several experiments where the experimental conditions had been varied. Since the two membrane elements were closely apposed, the thickness of the individual membrane element would be 130/~. In close to focus pictures (Figs. i and 2), the inner membranes show a particulate substructure with the smallest particles delineated by the negative stain measuring about 30 • in diameter. Larger light areas in the membranes measured up to about 100/~ in diameter. It was characteristic that the particulate substructure was lacking any simple geometrical order. The particles appeared randomly arranged. The particulate struc-
FIG. 1. C••se t• f•cus picture •frat heart musc•e mit•ch•ndri•n in tissue t h a t had been cr•ss-linked with 1% glutaraldehyde for 5 min and infiltrated with sucrose before rapid freezing. Negative staining. The inner mitochondria] m e m b r a n e s appear as compact structures lacking an intracristal space: They show a particulate substructure. This is the type 1 pattern, x 600 000. 236
~ t ~s
~
•!il &i i,j!i ~ ii!!!~i¸~ j'~
~ ~i~~~i!
~ii~i
@ FIG. 2. Rat heart muscle mitochondrion in tissue that had been cross-linked for 5 min in 1% glutaraldehyde and infiltrated with 30% glycerol before rapid freezing. Negative staining. The appearance of the inner membrane is that of type 1 pattern. × 300 000. 237
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ture made the surface of the membranes appear rough. The boundary between the inner membranes and the matrix was therefore irregular. The type 2 pattern differed from the type 1 pattern by the membrane elements measuring only about 60 /~ in thickness (Figs. 3-6) and by the two membrane elements being separated by a more or less wide accumulation of stain, indicating the presence of an intracristal space. In the type 2 pattern, the two membrane elements of the inner membranes were sharply outlined by the negative stain on both the matrix side and toward the intracristal space. The boundaries of the membrane elements appeared almost smooth in contrast to the irregular boundaries at the matrix space observed in the type 1 pattern. The thickness of the inner membrane element observed in the type 2 pattern was rather uniform and measured 50 to 70/~ as compared to 130/~ estimated for the type 1 pattern. The membrane elements furthermore appeared at a high contrast in the type 2 pattern, and there were no clear indications of any particulate substructure in these membrane elements in contrast to the type 1 pattern. The average width of the intracristal space varied between 20 and 70/~ in different experiments. It was characteristic that in both type 1 and type 2 patterns, the matrix space was intensely stained, showing that little material prevented an extensive accumulation of stain in this space. Even when the intracristal space in the type 2 pattern was wide, the opacity of the stain in this space never exceeded that of the matrix space. Since the intracristal space only exceptionally contains any particulate matter, the similarity with respect to the opacity of the staining of matrix and intracristal space
shows that there must be a rather limited amount of particulate matter in the matrix space. In the type 1 pattern, there were indications of the presence of particles of a size corresponding to protein molecules, that is, in the range of 30-50/~, in the matrix space. For comparison, thin frozen sections of the optic nerve of the cat were analyzed. The layered structure of the myelin sheath was well preserved even after short crosslinking with 1% glutaraldehyde. The mean periodicity of the myelin sheath was estimated to 120 A, and the average thickness of a single layer in the myelin sheath was about 60/~. The myelin layers appeared uniformly opaque and did not show any particulate substructure. When correlating the appearance of the inner mitochondrial membranes to the preparatory treatment, it became obvious that the type 1 appearance, with thick membrane elements "closely apposed without any open intracristal space separating the membrane elements and a particulate substructure, was observed in those experiments where the tissue had been exposed to short-term cross-linking and partial dehydration either by means of sucrose (Fig. 1) or glycerol (Fig. 2) before freezing. Long-term cross-linking (Fig. 3), elimination of any protection against the formation of ice crystals intracellularly (Figs. 3 and 4) and exposure of the frozen sections to DMSO (Fig. 5) resulted in the type 2 appearance of the inner mitochondrial membranes, with thin membrane elements separated by a m o r e or less wide intracristal space. In the sections of material that had been frozen fresh, without any pretreatment, the inner membranes mostly appeared
FIG. 3. I n n e r m e m b r a n e s in r a t h e a r t muscle mitochondrion in tissue t h a t had been cross-linked for I h r in 2.5% g l u t a r a l d e h y d e which denatures the proteins. Type 2 p a t t e r n with t h i n m e m b r a n e elements separated by a n intracristal space. × 300000. FIG. 4. Type 2 p a t t e r n of inner mitochondrial m e m b r a n e s in r a t h e a r t muscle mitochondrion in tissue fixed in formaldehyde vapor for 5 rain and frozen without any cryoprotective agent. Negative staining. × 300 000.
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FREEZE-SECTIONED MITOCHONDRIAL MEMBRANES
rather irregular and ill defined. They did not show any intracristal space, and the total thickness of the membranes was measured to be 260 A. The type 2 pattern was also characteristic of the mitochondria in tissue that had been dehydrated in ethanol and then rehydrated before freeze-sectioning (Fig. 6). In the material that had been treated the conventional way with OsO4 fixation and extensive glutaraldehyde fixation followed by OsO4 fixation, ethanol dehydration, and Epon embedding, the mitochondria appearance corresponded to the type 2 pattern, although in this case the membrane elements were positively stained. DISCUSSION
In frozen-sectioned material, inner mitochondrial membranes were observed that differed drastically in appearance from inner membranes in conventionally prepared tissue. In the latter case, the individual membrane elements appear as 50/k thick, triple-layered components (10) and the membrane elements are usually separated by a wide, empty intracristal space. The layers of the membrane elements run continuously through the whole membrane elements without interruption, or the membrane elements appear to consist of a layer of globular components (7). In the frozen-sectioned material, the mitochondrial inner membranes showed two different profile patterns. The type 1 pattern was the one that most drastically deviated from that usually observed in sections of conventionally fixed and embedded m a t e r i a l The two membrane elements of the inner membranes were closely apposed, with no indications of any intracristal space separating them. The average total thickness of the inner mem-
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branes was 260 A, which would correspond to a thickness of the individual membrane elements of 130/~, versus 60 A in the type 2 pattern. In the type i pattern, the membrane elements did not show any continuous uninterrupted layer or layers. Instead, they exhibited a particulate substructure which made the surfaces of the membranes appear irregular. No indications of the presence of a continuous uninterrupted lipid bilayer were therefore found. The type 2 profile pattern was the pattern showing the greatest similarity to the pattern observed after conventional fixation and embedding of the tissue. In addition to the open intracristal space, the membrane elements measured only about 60/~ in thickness and appeared rather uniformly thick, sharply outlined, and with a uniform low opacity not revealing any particulate substructure. The width of the intracristal space varied considerably (20-70
A). When discussing these two profile patterns, it is reasonable first to consider the possibility that they are due to differences in the tilting of the membranes. The difference in thickness of the membrane elements in the type 1 and type 2 patterns and the absence of an intracristal space obviously could be explained by a tilting of the membranes in the cases where the type I pattern is observed. Usually an indication of a boundary between the two membrane elements of the inner membrane could be observed even when only certain spots in the middle of the membrane were stained. The faint and incomplete staining of this boundary shows that the negative staining involved only shallow depressions at the cut surfaces of the inner membranes. If
FIG. 5. I n n e r mitochondrial m e m b r a n e s showing type 2 p a t t e r n in r a t h e a r t muscle tissue fixed in formaldehyde vapor for 5 min. The sections were collected on DMSO. Negative staining. × 300 000. FIG. 6. I n n e r mitochondrial m e m b r a n e s in r a t h e a r t muscle tissue t h a t had been fixed in formaldehyde vapor for 5 min, dehydrated in ethanol, a n d rehydrated before cryomicrotomy. The p a t t e r n is t h a t of type 2. Negative staining. × 300 000.
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these membranes would be tilted, the staining of this boundary would be displaced laterally in the direction of the tilt and would not be projected to the middle of the inner membrane. The type 1 pattern was observed where the inner membranes appeared at a maximum contrast in these sections. This means that the negative stain in these regions was observed at a maximum thickness of the dried down stain. A maximum thickness of the stain corresponds to a minimum interference by the organic material. A maximum contrast also means that the inner membranes appeared with maximum brightness. This condition requires that the inner membranes be oriented parallel to the electron beam because any tilting of the membranes would result in a lowering of the contrast. A tilting of a membrane would mean that it would, for instance, lean over an area where the stain has accumulated. A tilting would therefore mean that in the image plane, the membrane would appear superimposed on the area containing the stain. At small degrees of tilt, this would make the membrane appear thinner and diffusely outlined. With the very dense arrangement of the inner membranes characteristic of heart muscle mitochondria, a slight tilt would result in an overlapping of the images of the individual membranes in the image plane and the disappearance of any matrix space in the image. This explains the appearance of Fig. 2, where a clear pattern showing inner membranes separated by matrix spaces is observed in the center of the picture, while the pattern is diffuse in the upper right and the lower left corners. The consistency in the dimensions of the inner membranes and of the membrane elements in pictures showing different types of profile patterns makes it justifiable to conclude that tilting of the membranes can have had only a limited effect on these measurements. This consistency
can be explained by the reduction of contrast that even slight tilting must introduce. We therefore find it justifiable to conclude that the two patterns are real profile patterns of inner mitochondrial membranes that differ structurally. It then becomes interesting to relate these patterns to the varied experimental conditions. The type 1 pattern was observed in experiments where the material had been stabilized by means of brief cross-linking with glutaraldehyde at a low concentration. This means that the exposure to glutaraldehyde had not been extensive enough to cause denaturation of the proteins. In contrast, long time cross-linking with high concentrations of glutaraldehyde resulted in mitochondria with inner membranes appearing according to the type 2 pattern. Under these conditions, glutaraldehyde denatures the proteins. The type 1 pattern was observed in material that had been partially dehydrated either with sucrose or glycerol to prevent the formation of intracellular ice crystals. This is one important factor to keep under control, since crystallization of the water leads to extensive pH changes due to differences in the solubilities of inorganic salts. Such pH changes can affect the conformation of the proteins. The type 2 pattern was observed in material that had been frozen without adding any protective agent and furthermore had been frozen slowly by immersion of relatively large pieces of tissue in liquid nitrogen. The type 2 pattern was observed in sections that had been exposed to conditions that would favor protein denaturation, such as collecting the specimens on DMSO, dehydrating the sections in ethanol followed by rehydration, osmium fixation, dehydration, and embedding in a plastic. We therefore can link the type 2 pattern
FREEZE-SECTIONED MITOCHONDRIAL MEMBRANES to conditions that lead to protein denaturation. The type 1 pattern, on the other hand, is associated with experimental conditions t h a t reduce the risks for protein denaturation. It then appears justifiable to conclude that the thin membrane elements characteristic of the type 2 pattern represent membrane elements in which the globular membrane proteins have been denatured. Since denaturation makes the globular proteins lose their defined globular shapes, it is obvious t h a t there should be little indication of a particulate substructure in these modified membranes. This explains the absence of such a substructure in the type 2 membranes. A correlation of the thickness of membranes as observed in thin sections to denaturing influences was shown by Kretzer (3) when applying the preparatory procedure of SjSstrand and Barajas (8). The partition membranes of the chloroplast of Chlarnydomonas reinhardi showed a maximum thickness of 270 A in material that had been prepared with the intention of extensively reducing the denaturation of membrane proteins. Cross-linking in 6% instead of 1% glutaraldehyde for 1 min reduced the partition membrane thickness to 150 A. Exposing the cross-linked (1 min, 1% glutaraldehyde) Chlarnydomonas to acetone for 1 min led to a reduction of the thickness of the partition membranes to 100/~. Prolonging the dehydration time in ethylene glycol from 1 to 60 min led to the same effect, as well as ethylene glycol dehydration for 1 min without previous stabilization through cross-linking. The particulate substructure observed in the type 1 pattern was of the dimensions of globular proteins molecules and of complexes of protein molecules. It therefore appears reasonable to consider that these particles are globular proteins which are the major components of these membranes. The fact t h a t the membrane particles
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appeared to be arranged in a random fashion and were not forming geometrically regular patterns is explained by the fact that these membranes, in contrast to several specialized membranes, consist of a large number of enzymes which differ with respect to molecular weights and shapes. As was pointed out by SjSstrand and Barajas (8), it is highly unlikely that such a mixture of components could be arranged according to a geometrically regular pattern. A more or less random arrangement of the membrane particles was also observed in freeze-fractured mitochondria (5). It therefore appears justifiable to conclude that the particles are globular protein molecules and complexes of protein molecules arranged either randomly or according to patterns that are too complex to be identified with any recognizable, more or less simple geometrical pattern. The absence of a continuous, uninterrupted layered structure in the mitochondrial membranes makes it justifiable to conclude that there is no continuous, uninterrupted lipid bilayer forming a backbone structure in these membranes. This agrees with the low lipid content of the inner mitochondrial membranes. The absence of an intracristal space in the mitochondria showing the type 1 pattern and the appearance of such a space in connection with denaturation of the membrane proteins make it reasonable to question the existence of such a space in the native state of the mitochondria. It seems justifiable to propose that, instead, the two membrane elements are closely apposed in the native state. It is characteristic that freeze-sectioning of fresh, non-cross-linked material is particularly difficult and results in less well organized membrane patterns, although areas showing the type 1 pattern were observed. This shows that freeze-sectioning does expose the structure to considerable strain. Stabilization of the structure by
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means of inter- and intramolecular crosslinking therefore appears to make the mitochondrial membrane resist destruction by the strain associated with the sectioning. The appearance of the inner membranes as represented by the type 1 profile pattern is similar to that observed by Sj6strand and Barajas (8) when using preparatory procedures aimed at limiting the conformational changes of membrane proteins in connection with embedding for thin sectioning. In that study, the thickness of the membrane elements of the inner membranes of kidney mitochondria was estimated to 130-150/~. No layered structure could be observed, but an irregular particulate substructure was found over the entire cross section of the inner membranes. The present observations on negatively stained frozen sections show that the inner mitochondrial membranes exclude the negative stain from penetrating into membrane elements which are about 130 /~ thick. This agrees with the concept of these membranes as consisting of proteins and lipids in a condensed state with a three-dimensional arrangement of the molecules (8).
A Critical Examination of the FreezeSectioning Technique From a theoretical point of view, the freeze-sectioning technique is likely to introduce less drastic conformational changes of the protein molecules than the conventional fixation and dehydration and embedding techniques. In the latter case, each step can contribute to the denaturation of the proteins: during fixation by exposing the proteins to heavy metal ions, during dehydration by the action of denaturing organic solvents, and during embedding by transferring the tissue to a nonpolar plastic monomer and then exposing the proteins to heat. This is, in fact, a very consistent type of treatment to achieve denaturation.
The freeze-sectioning, on the other hand, eliminates the need for dehydration by means of organic solvents and embedding in a plastic. This does not, however, mean that this procedure does not involve several steps at which the protein conformation can be affected. It seems justifiable to point to the most obvious risks when applying this method. The emphasis during the development of this technique in the past has, for obvious reasons, been on finding favorable conditions on the whole to obtain good sections even with risks that proteins would be denatured. In this study, particular attention was paid to applying the freeze-sectioning technique under conditions that would reduce the risks for conformational changes of the protein molecules. The first critical step in preparing frozen sections is the freezing of the tissue. However rapidly the !tissue is chilled down by using small pieces and the most ideal medium for quick removal of heat from the tissue, there will occur a phase separation with the formation of ice crystals. This leads to an increasing concentration of solutes in the still Iiquid phase. As a consequence, considerable pH changes can occur which can affect protein conformation. Surface tensions at the ice crystal interfaces are likely to favor denaturation of proteins (11). The pretreatment of the tissue with sucrose or glycerol leads to a reduction of the concentration of free water in the cells, and if the sucrose or glycerol concentration is sufficiently high, the crystallization of water is prevented or greatly inhibited. This does not, however, apply to low concentrations (10-20%) of glycerol. The minimum concentration is about 30% (12). The next critical step involves the sectioning. All the energy transferred from the knife to the specimen block and responsible for producing a crack through the tissue is concentrated at this crack. This energy is transformed into heat. This
FREEZE-SECTIONED MITOCHONDRIALMEMBRANES
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means t h a t the amount of heat transferred to be cross-linked before it could be exper unit volume of tissue when the knife posed to the strain involved in dehydration edge hits the block is high, and the tissue in ethylene glycol and Vestopal embedcan melt at the surface of the crack. The ding. The so-called inert dehydration molten surface layer will be exposed to method of Pease (6) was therefore not usesurface forces. Surface forces are also ac- ful for analysis of cell structures at a motive at the two surfaces of the frozen sec- lecular level, which does not exclude its tion in the frozen state. usefulness for studies at other levels. One of the most obvious dangers involves the collecting of the sections and Trying to Reduce the Risks for Conformational Changes during their thawing. In the case where ice crysFreeze-Sectioning tals have been formed, the thawing itself means a redistribution in the cells of the The striking experience in this study as water trapped in ice crystals, and the well as in that of SjSstrand and Barajas (8) structural appearance of the cell is recon- was the very brief cross-linking t h a t was stituted with little sign of any persisting sufficient to stabilize the mitochondrial deformation imposed by the ice crystals. membranes. This briefness, as well as the This reconstitution, however, has been low concentration of the glutaraldehyde shown to occur with respect to the struc- used, reduces the risk that glutaraldehyde ture of the cells viewed at an intermediate will change the protein conformation, as resolution, revealing the overall arrange- high concentrations of glutaraldehyde and ment of membranes in the cytoplasm and long exposure times do (4). in mitochondria. What happens at a tooThe treatment of the tissue with high lecular level, on the other hand, is not concentrations of sucrose before freezing known. according to Tokuyasu (13) aims at reducWhen drying the sections in air, surface ing the risks for extensive formation of ice tension will expose the cellular structures crystals by increasing the concentration of to enormous forces, and surface denatura- solutes in the cells. This treatment has a tion of proteins is likely to be rather exten- twofold effect. On the one hand, it makes sive. sucrose molecules enter the cells to inIt appears that the risks for conforma- crease the concentration of solutes, and on tional changes during freeze-sectioning the other hand it removes water from the that have been outlined above can explain cells by establishing a temporary osmotic the difficulties in demonstrating enzyme gradient across the cell membrane. Both activity and immunological activity in fro- these effects tend to favor vitrification of zen sections. the tissue without preceding extensive forThe risks involved in freeze-sectioning mation of ice crystals. also are illustrated clearly by the fact that With respect to sectioning, the temperait is difficult to retain the fine structure of ture was kept at -80°C which, however, is cells and, in the present case of mitochon- unlikely to eliminate the possibilities of a dria, if fresh material is freeze-sectioned. thawing of the tissue at the surface of the Cross-linking with glutaraldehyde or fixa- crack formed during sectioning. The lowtion with paraformaldehyde have been est possible temperature t h a t is compatifound to be important in reducing the risk ble with obtaining sections should be of damage to the structure of cells during aimed at. freeze-sectioning. The method introduced by Tokuyasu This parallels the experience of Sj6s- (13) to pick up the sections involves expostrand and Barajas (8) t h a t the tissue had ing the tissue to a high concentration of
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sucrose. This is a priori not to be assumed is well retained after freeze-sectioning to be a treatment without danger. From a makes it unlikely that this explanation is functional point of view, it has been shown justified. that the respiratory chain is affected by In spite of the risks for structural alterahigh concentrations of sucrose, which is tions introduced by freeze-sectioning and shown by a reduction of respiration and of by the two methods used by SjSstrand and the respiratory control index. The results Barajas (8, 9), it seems justifiable to put when the sections were picked up dry di- more weight on the observations made rectly on the filmed grid were, however, with these three methods aimed at reducsimilar to those when a drop of sucrose was ing conformational changes in protein used. The Tokuyasu technique therefore is molecules than on observations made on not likely to introduce structural changes material in which proteins have been sysat the level of resolution that presently can tematically denatured. This statement apbe achieved on frozen sections. pears rather obvious in the case where the It was characteristic that the drying of analysis involves the molecular structure the section with negative stain was neces- of cellular components. sary to retain the structure of the mitoThe very competent and skilled assistance of chondria. This is an experience similar to Miss Annie Viron is gratefully acknowledged. that for other applications of the negativeREFERENCES staining technique. Presumably, the presence of the stain approaching saturation 1. BERNHARD,W., AND VIRON, A., J. Cell Biol. 49, and precipitating on tissue surfaces re731 (1971). 2. CHRISTENSEN,A. K., J. Cell Biol. 51,772 (1971). duces the destructive influence of surface 3. KRETZER,F,, J. Ultrastruct. Res. 44, 146 (1973). tension. 4. LENARD,J., AND SINGER, S. J., J. CeUBiol. 37, From this discussion, it appears obvious 117 (1968). that it is not possible to assume that the 5. PACKER, L., Bioenergetics 3, 115 (1972). freeze-sectioned material reveals the 6. PEASE, D. C., J. Ultrastruct. Res. 14, 356 (1966). 7. SJSSTRAND, F. S., J. Ultrastruct. Res. 9, 340 structure without any modifications of the (1963). native state of cellular components at a 8. SJ6STRAND, F. S., AND BARAJAS, L., J. Ultramolecular level. struct. Res. 25, 121 (1968). The absence of a continuous layer in the 9. SJ~STRAND, F. S., AND BARAJAS, L., J. Ultrapictures showing the type 1 and type 2 struct. Res. 32, 293 (1970). profile patterns could be the consequence 10. SJ6STRAND, F. S., AND ELFVIN, L., J. Ultrastruct. Res. 7, 504 (1962). of a disorganization of a lipid bilayer by 11. SJOSTRAND, F. S., AND KRETZER, F., J. Ultrathe freeze-sectioning technique if such a struct. Res. 53, 1 (1975). bilayer were present in the native state of 12. SJ0STROM, M., AND THORNZLL, L. E., Science the membrane. The fact, however, that Tools 21, 26 (1975). the layered structure of the myelin sheath 13. TOKUYASU,K. T., Ji Cell Biol 57, 551 (1973).