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Structural changes in swollen ascites tumor mitochondria
© 1967 by Academic Press Inc.
j. ULTRASTRUCTURERESEARCH18, 257--276 (1967)
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Structural Changes in Swollen Ascites T u m o r Mitochondria R. F. MITCHELL
Australian Atomic Energy Commission Research Establishment, Sutherland, N.S. W. Received September 27, 1965 The structure of Ehrlich ascites tumor mitochondria suspended in a medium containing 0.5 M sucrose, and the changes which occur in them as the sucrose concentration is progressively reduced, are studied by electron microscopic examination of thin sections and negatively stained preparations. The changes involve swelling of the matrix compartment and lead to the spontaneous appearance of either lamellar, particulate, or tubular forms. In negatively stained specimens, the latter show substructural organization identical to that seen in preparations of the electron transfer particle and in the structures, found in negatively stained preparations of a variety of mitochondria, that are widely regarded as being the mitochondrial cristae. Fernfindez-Morfin (7, 8), Parsons (19-21), and others (17, 33), have presented electron micrographs of mitochondria negatively stained with potassium phosphotungstate (PPT) which are widely considered accurately to depict, in much more detail than is possible by thin sectioning, their fine structural organization. The mitochondrion is regarded (8, 12) as a fluid-filled system of membranes, which collapse and are penetrated by the stain once the fluid is removed. Three separate membrane systems have been described in these preparations; an outer membrane, characterized in some cases (20) by a very fine surface pattern; an inner membrane which has particles approximately 100 A in diameter associated with its inner surface (33); and mitochondrial cristae, with similar particles attached to their outer surfaces by short stems. These stemmed particles were called "elementary particles" by Fernfindez-Morfin (7) and they are believed (8) to be functional in the electron transfer activity of the mitochondrion. The structures described as cristae may be long narrow tubules of uniform diameter, or may be extremely variable in width. They m a y or may not branch. Some segments are ribbonlike, there being no penetration of stain into a central core. While the preparation of negatively stained specimens has been claimed to produce no significant change in the form of these membranes (8), Sj6strand and co-workers have pointed out (28) that the natural existence of thin ribbons or tubules within the
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various mitochondria is difficult to reconcile with the rather large m e m b r a n e surfaces demonstrated in the same mitochondrial types by sectioning. They considered that both the tubules and their associated particles were the products of degenerative changes in the mitochondrial membranes, resulting f r o m osmotic shock and, perhaps, f r o m exposure to high P P T concentrations during drying of the specimens. That the inner mitochondrial membranes m a y be altered by negative staining seems to be shown by the studies of Smith (31) on flight muscle sarcosomes of the blowfly. He showed by means of sectioning that the cristae are extensive parallel plates. In negatively stained preparations, forms which are interpreted as these plates are sometimes seen to be confluent with arrays of ribbonlike structures of identical appearance to the "cristae" seen in other mitochondrial types. These structures are presumed (31) to represent fragments of the cristae. The possibility that P P T m a y play a part in the production of these structures appears to be excluded by the fact that fixation with osmium tetroxide or formalin before the addition of the P P T does not prevent their appearance (19, 31). However, all the methods that have been used to demonstrate them involve dilution of the n o r m a l cell sap, or concentrated sucrose solutions, media which are optimal for the preservation of mitochondrial architecture, with 1-2 % P P T in water, and in some cases with distilled water as well. If this dilution produces changes in mitochondrial structure, then one might expect to detect them by gradually diluting the suspension m e d i u m and examining the mitochondria at a n u m b e r of stages. The results of such studies are the subject of this paper. MATERIALS A N D METHODS The mitochondria used were obtained from a Lettr6 hyperdiploid strain of the Ehrlich ascites tumor cell. After a single washing in Ca ++- and Mg++-free Hanks basic salt solution, pH 7.4, containing 0.02 % EDTA, the cell suspension was transferred to flat-bottom polythene tubes and centrifuged at 20,000 g for 10 minutes. The residual suspension fluid in the resulting pellet, measured with 1~1Ihuman serum albumin, occupied only 10% of the total volume. The supernatant was taken off with a Pasteur pipette, residual fluid being removed by a wad of filter paper. The bottom was cut out of the tube, and the cells were transferred to a siliconized stainless steel tube of the same internal diameter, using the filter paper as All figures are electron micrographs. F~o. 1. Negatively stained mitochondria in 0.5 M sucrose medium containing 1% PPT. The stain has penetrated the outer mitochondrial membrane (Mo), but is excluded from the inner compartment and intracristal spaces. The cristal orifices (C) can be seen. x 70,000. FIc. 2. Similar preparation to Fig. 1, except that the mitochondria were formalin fixed before staining. The rod-shaped body (P1) is in the process of distending and rounding up. P2 has swollen considerably to show the complex branching form of the inner compartment (I) and the granular nature of the matrix (ma). x 70,000.
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FIG. 3. A thin section of mitochondria in 0.5 M sucrose medium. The inner compartment (/) is filled with a finely granular matrix and forms a branched saccular and tubular system. The outer compartment (O) is the space between the inner compartment and the outer mitochondrial membrane (Mo). The intracristal spaces (C) are continuous with this outer compartment. Methacrylate embedded, lead stained, x 85,000.
a piston. The bottom of this tube was sealed with a plastic plug, a stainless steel piston having a diameter 0.1 m m less than the internal diameter of the tube was placed on top of the cells, and the assembly was swung at 1200 g in the swing-out head of a refrigerated centrifuge. As the plunger was driven down into the mass of cells, they were forced up through the gap between it and the tube, their external membranes being broken in the process. These cells were suspended in 10 volumes of a medium containing 0.5 M sucrose in 0.01 M phosphate buffer, p H 7.0, and cell disruption was completed by one passage of a PotterElvehjem homogenizer. Nuclei and cell debris were removed by centrifugation at 1000 g for 10 minutes, the supernatant then being spun at 10,000 g for 10 minutes. The resulting mitochondrial pellet was resuspended by hand with a loose-fitting homogenizer, and the preceding sequence was repeated. The initial cell wash was carried out at room temperature (15°C) but for all subsequent procedures the temperature was maintained at 0--4°C.
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F~G. 4. Thin section of part of a mitochondrion in 0.5 M sucrose. The matrix (ma) is bounded by a membrane (Mi). Part of the margin of the mitochondrion, and the outer membrane (Mo), are visible at the upper right corner. Araldite embedded, lead stained. Section thickness less than 100 A. x 250,000.
The resulting suspension of mitochondria in 0.5 M sucrose medium was diluted with either distilled water, isotonic salt solution buffered at pH 7.0, or 1 To PPT in water, p H 6.5. In this way the sucrose concentration was reduced progressively through the values 0.25 M, 0.125 M, to 0.06 M, the preparation being allowed to stand for 10 minutes at each stage before further dilution. Alternatively, the concentration was altered to 0.125 M and 0.06 M directly, without standing at the intermediate values. Negatively stained preparations of the PPT-diluted material were made by simply spraying the suspension onto grids. Negative staining of the water- and salt-diluted preparations was complicated by having to fix them before exposure to the PPT. This was done by spreading the mitochondria on the surface of a sucrose medium which was the same as the one in which they had been suspended except that it contained 1 To methanol-free formaldehyde. The spread mitochondria were picked up on grids after 10 minutes and negatively stained. Specimens examined by sectioning were double fixed with 2 % formaldehyde and 1 TO osmium tetroxide at 4 ° for 24 hours. They were dehydrated with ethyl alcohol, embedded in Araldite (IO) or 4:1 butyl:methyl methacrylate containing 0.75 mg of uranyl nitrate (35) per milliliter and stained with lead by the method of Karnovsky (1 I).
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All preparations were examined with a Siemens Elmiskop I, using an accelerating voltage of 80 kv, beam current 4-8/~A, and plate magnifications between 20,000 and 40,000. OBSERVATIONS Structure of mitochondria in 0.5 M sucrose When the mitochondria are negatively stained by spraying a suspension in 0.5 M sucrose containing 1% PPT (Fig. 1), they appear mainly as flattened round or oval forms, rather than the rod-shaped bodies common in sections of intact cells. They consist of a central body, appearing lighter than the background because it excludes the stain, surrounded by a single light line interpreted here as an outer membrane? These structures are separated by a narrow gap into which the stain has penetrated. The body of the mitochondrion is usually without much feature but there may be signs of surface creases or furrows. In places these have a regular pattern, suggesting that they may represent surface openings of the intracristal spaces, i.e., the cristal orifices. There is apparently little penetration of stain into such spaces in these preparations. If the mitochondria are fixed by addition of formaldehyde (final strength 1%) to the suspension, the stain penetrates the intracristal spaces, revealing (Fig. 2) the mitochondrial body to be a complex membrane system. In the few remaining elongated forms (e.g., P1), there is a largely transverse arrangement of these membranes, but this is lost in those which have become distended (e.g., P2). The granular appearance of this system (seen clearly in P2) in the fixed preparations could represent membrane structure, but it seems more likely to reflect the granularity of the contents, shown by penetration of the stain into the interior. Thin sections of these mitochondria (Fig. 3) show how the structure of isolated mitochondria differs from those examined in situ. They are predominantly rounded, lack the normal cristal pattern, and in fact correspond to what one would expect on sectioning a structure such as P2 in Fig. 2. Each mitochondrion is bounded by a single membrane which is presumably continuous, although where it is in direct contact with the mitochondrial body it can only occasionally be distinguished (see Fig. 4 for detail), and corresponds to the outer membrane seen in negatively stained preparations. The body of the mitochondrion is composed of two parts; an apparently closed compartment (the inner or matrix compartment) of complex branching form filled with a finely granular (particles less than 50 A) or homogeneous matrix, and the apparently empty spaces. The latter are seen in places to be continuous with the space between the matrix compartment and the outer membrane, and are interpreted as greatly distended intracristal spaces. 1 The term "membrane" is used descriptively, the structure seen may be regarded either as a "membrane element" (25) or as a "unit membrane" (•8); see Discussion.
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Fia. 5. Negatively stained mitochondria in 0.125 M sucrose. The inner compartment of P1 is not penetrated by the stain, but the others are penetrated to varying extents. A portion of this compartment appears to have a lamellar structure (L), the lighter (stain excluding) bands being 30-40 thick and the dark ones narrower. The inner compartment of P2 shows marked localized distention, while in P3, with the apparent exception of some small unswollen parts (U), the whole compartment has swollen to form a saccular body. The membrane or contents of this structure are markedly granular, and indistinct ribbonlike assemblies can be seen (arrows) at some points. The outer membrane of P3 (Mo) has broken. A number of cristal orifices are indicated (C). x 70,000.
I n v e r y t h i n sections the m a t r i x c o m p a r t m e n t (Fig. 4) is seen to be b o u n d e d b y a n a p p a r e n t l y c o n t i n u o u s m e m b r a n e 3 0 - 4 0 A across, s e p a r a t e d f r o m t h e m a t r i x b y a n e l e c t r o n - l u c i d g a p of a b o u t the s a m e w i d t h . T h i s gap is n o t c o n t i n u o u s b u t is b r i d g e d at intervals. T h e b r i d g e s a p p e a r to c o n n e c t particles in t h e m e m b r a n e w i t h o t h e r s of similar size in the m a t r i x . H o w e v e r , the i n t e r p r e t a t i o n of such d e t a i l is h a z a r d o u s
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Fro. 6. A negatively stained preparation of mitochondria in 0.06 M sucrose medium. The irregular saccular structures $1 and $2 appear to have a granular content or membrane. Aggregation of this granular material can be seen (A), and organized tubular structures (T) are present at other places. The two mitochondria P1 and P2 are only moderately distended. The matrix compartment has partly herniated from P1, and this has swollen at one place to form $3. The margins of the matrix compartments in P1 and P2, as well as most of the margins of SI, $2, and $3, can be clearly seen to be smooth. However, part of the margin of $1 (arrow) has 100 A particles attached to it. x 85,000. for two reasons. I n the absence of a through-focal series, a little u n d e r f o c u s i n g can cause a spurious particulate appearance in the image (16). Again, the e m b e d d i n g material m a y become finely globular in the electron beam, a l t h o u g h Araldite is relatively resistant to this change (22).
Morphological changes in negatively stained mitochondria on diluting the suspension medium There is considerable variation in the extent of the reaction of individual m i t o c h o n d r i a to the reduction in sucrose c o n c e n t r a t i o n of the suspension m e d i u m , a n d a variety of changes will be seen in a n y one preparation. Consequently, changes
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FIG. 7. A higher magnification of the tubules seen in Fig. 6. The dumbbell type of substructure described in the text can be seen at the point marked with the arrow. × 250,000. FIG. 8. A higher magnification of tubular and saccular forms such as those seen in Fig. 9. In this case the stain has not entered the central core, which is presumably solid. Particles approximately 100 • in diameter (arrows) are attached by stems to the surfaces of both the ribbons and the saccules. Whether the helical arrangement of particles on many of the ribbons is caused by twisting of the ribbons, is not known, x 250,000. of f o r m seen in 0.25 M sucrose are also seen in 0.125 M sucrose, a n d only the latter p r e p a r a t i o n need be illustrated. The wide range of structural changes which occur in this m e d i u m is illustrated in Fig. 5. M o s t of the m i t o c h o n d r i a are m o r e swollen t h a n w h e n seen in 0.5 M sucrose, b u t they are still s u r r o u n d e d b y a limiting m e m b r a n e s e p a r a t e d f r o m the central b o d y b y only a n a r r o w gap. I n m a n y m i t o c h o n d r i a the stain a p p e a r s to have p e n e t r a t e d the distended intracristal spaces. I n the spread a n d flattened f o r m in which these m i t o c h o n d r i a are observed, this intracristal stain serves to outline the relatively dye-excluding m a t r i x c o m p a r t m e n t which a p p e a r s to have the f o r m of b r a n c h i n g tubules of variable size. A t several points the stainfilled gaps are seen to be c o n t i n u o u s with the outer c o m p a r t m e n t (the n a r r o w space betweeen the outer m e m b r a n e a n d the b o d y of the m i t o c h o n d r i o n ) , as w o u l d be expected if they were derived f r o m the original intracristal spaces. There are differences in the detailed a p p e a r a n c e s of m i t o c h o n d r i a subjected to these media. The dye is excluded f r o m some, which resemble the bodies seen in 0.5 M sucrose, while it has p e n e t r a t e d others to show w h a t is i n t e r p r e t e d here as the finely g r a n u l a r matrix. I n some m i t o c h o n d r i a there a p p e a r s to be local distension of the m a t r i x c o m p a r t ment, while in others (e.g., P3 in Fig. 5) virtually the whole c o m p a r t m e n t has dist e n d e d to f o r m w h a t a p p e a r s to be a large sac. These sacs have s m o o t h edges b u t the
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FIG. 9. Similar preparation to Fig. 6, showing ribbon or tubular structures (T) attached to small saccular bodies (s). The fragments of granular membrane (S) presumably represent the remains of the saccular form of the matrix compartment illustrated in Figs. 5 and 6. x 46,000.
m e m b r a n e or c o n t e n t a p p e a r s g r a n u l a r , the m e a n particle size being of the o r d e r of 100 A. This is to be c o m p a r e d with the estimate of less t h a n 50 A for particles in t h e m a t r i x of m i t o c h o n d r i a s u s p e n d e d in 0.5 M sucrose a n d e x a m i n e d b y thin sectioning. A t places, p o o r l y defined r i b b o n l i k e structures can be seen within this g r a n u l a r material. T h e outer m e m b r a n e is r u p t u r e d as the m i t o c h o n d r i a l b o d y swells. I n 0.06 M sucrose large bodies of irregular outline are often seen (Fig. 6), p r e s u m a b l y representing a further stage of distension of the m a t r i x c o m p a r t m e n t . These bodies a p p e a r g r a n u l a r in such p r e p a r a t i o n s , a n d this is considered to reflect t h e
FIo. 10. Similar to Fig. 6. The membrane fragments (S) are considered to have the same origin as those seen in Fig. 9. The branching membrane system (T) has stemmed particles (arrows) attached to its outer surface, x 70,000. Fr~. 11. Similar preparation to Fig. 6. This structure contains masses of membranes with associated 100/~ particles, presumably of the branching tubular form (T) seen. A large angular membranous structure is connected to the tubules, x 70,000.
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nature of the matrix material enclosed within the saccular forms. In many areas it is fairly uniform, but in others the appearance suggests aggregation. Within these bodies, suggesting some relationship with the granular areas by continuity, organized tubular structures may be seen. These appear (Fig. 7) as parallel lines having a particulate structure, and particles of about 100 A diameter are connected to these subunits by a narrow stem. This has been clearly shown (8, 31) to be the organization of the structures commonly referred to as cristae in negatively stained mitochondrial preparations. The conclusion that they are tubular is supported by comparing their appearance with tubular assemblies seen in negatively stained phospholipid complexes (13, Plates 20-30). The failure to visualize particles on the upper or lower surface is presumably a contrast effect due to the tube being filled with stain. If the stain is excluded from the core, as in Fig. 8, particles can be seen on the surface. The outer margins of the large sacs are usually smooth, but in places they may have 100 A particles attached. Other structures seen in 0.06 M sucrose are shown in Figs. 9-11. They illustrate the diversity of forms which can result from one mitochondrial type when subjected to such treatment, but, with differences in detail, all contain ribbonlike or tubular structures which have particles attached to their outer surface. In Fig. 9 there appears to be fragmentation of a mitochondrion, producing a crisscross pattern of ribbonlike forms. These may be tubular structures similar to those shown in Fig. 7, or may have (Fig. 8) a solid core. Attached to the tubules and rods are small saccular bodies, some of which also have particles associated with their outer surfaces. The tubular forms seen in Fig. 10 are branched and variable in diameter. Stemmed particles line their margins and presumably cover their entire outer surface. This tubular system has the same granular appearance as the saccular particles of Figs. 5 and 6, although the latter usually have a smooth outer surface. The granular horseshoe-shaped structure, together with fragments of varying size and shape, seems to represent remnants of the enveloping sac. The presence of these saccular fragments, as well as the presence of particles on the surface of the tubules, would appear to preclude the possibility that they represent the unaltered matrix compartment. In all cases in which the inner compartment has been clearly seen and identified, the margins appear smooth (see, for example, PI and P2 in Fig. 6). The masses of membranes present in the body shown in Fig. 11, with which 100 A particles can be seen associated, are presumably of the same branching tubular and saccular form as seen in the upper part of the structure. The same basic arrangement of tubules and associated particles is still present in preparations which have been diluted from 0.5 M sucrose medium in a single step rather than progressively. This is true also of those preparations fixed with formalin prior to negative staining.
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FIG. 12. Thin section of part of a mitochondrion in 0.5 M sucrose, showing lamellar structure within the matrix (ma). The electron dense layers are 30-40 A across, and are separated by a slightly wider electron-lucid gap. The globular nature of the stained layer is strongly suggested at a number of points (arrows), in spite of the fact that the section thickness interferes with the clarity of such detail. Araldite embedded, lead stained, less than 100 A thick, x 260,000.
Morphological changes in thin sections of mitochondria on dilution of the suspension medium The mitochondrial matrix in 0.5 M sucrose is seen in thin sections to be finely granular and usually of uniform density (Fig. 4). In places, however the matrix may show a lamellar structure (Fig. 12). As the sucrose concentration is decreased the matrix compartment swells, with reduction in the overall density of the matrix and the appearance of local density changes (Figs. 13 and 14). Lamellar forms are more frequent in these moderately swollen mitochondria. Density variations become more marked as swelling proceeds (Fig. 14), leading in some cases (Fig. 15) to the appearance of clusters of 150-250 • particles within the distended matrix compartment. Frequently, there is complete reorganization of this compartment with the appearance of ribbon or tubular forms (Fig. 16). The tubules vary considerably in cross section and may branch. 18 - 671823 J. Ultrastructure Research
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Preparations of the mitochondria in 0.06 M sucrose show predominantly vesicular or saccular forms, as in Fig. 17. These may have thick walls, and frequently they contain tubular structures. The mitochondrion in Fig. 18 appears to be in the process of conversion to a saccular body and suggests that the reorganization involves both matrix and membranes. Sections through the tubules shown in these sacs do not show the 100 A particles which are present with negative staining, either in the substructure of the tube or projecting from it by a stem. However, they do frequently show (Fig. 17) a bilaminar wall structure, each lamina being 30-40 A thick. DISCUSSION The mitochondrial forms seen in sections of intact Ehrlich ascites tumor cells fixed with buffered osmium tetroxide are predominantly rod-shaped and conform to the classical descriptions given by Sj6strand (25) or Palade (18). However, after they have been isolated in 0.5 M sucrose medium by the method described, most have a considerably different morphology, which appears to result from changes similar to those commonly observed in preparations of isolated mitochondria (12). As shown in Figs. 1-3, they have become rounded, and the characteristically bilaminar outer membrane and cristae are no longer apparent. The essential change appears to be a very considerable opening of the intracristal spaces, with complete loss of the original orderly arrangement. Where these spaces occur, the limiting membrane appears as a single unit membrane (18) or membrane element (25). No distinction between these being possible from the observations described here, the term membrane is used in a purely descriptive manner and does not reflect the status of this structure in unaltered mitochondria. Elsewhere, it is closely applied to the matrix compartment, and is only clearly visualized in places (see Fig. 4). In negatively stained preparations, although it is usually clearly separate from the central mitochondrial body (see Fig. 1), it follows closely the shape of the body, even when this is irregular. It is difficult to FrG. 13. Thin sections of three mitochondria in 0.25 M sucrose, each showing a different degree of swelling. P1 resembles those in 0.5 M sucrose, having a dense matrix and open intracristal spaces. P2 has a more distended matrix compartment, with a reduction in the overall density of the matrix and local variations in density. P3 is considerably swollen, and the matrix now shows an ill defined internal pattern. Methacrylate embedded, lead stained, x 125,000. FIG. 14. Thin section of a mitochondrion in 0.25 M sucrose showing an intermediate stage in the development of a pattern in the swelling matrix (ma). Methacrylate embedded, lead stained. x 145,000. Fro. 15. Thin section of a greatly swollen mitochondrion in 0.06 M sucrose. The large saccular structure, of which only a part is shown, contains clusters of particles 150-250 A in diameter, x 145,000. FIG. 16. A mitochondrion in 0.125 M sucrose medium, showing a moderately distended matrix at the upper end (ma); the remainder of the matrix compartment has become rearranged in the form of uniform ribbons or tubules (T1), and vesicles or tubules of varying section (T2). x 110,000.
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see how marked distension of the intracristal spaces can arise without related distension of the outer compartment with which they communicate. At the same time there are indications, particularly with negatively stained preparations (see Fig. 2), that the matrix compartment has distended to some extent in most of the mitochondria in 0.5 M sucrose. This is difficult to assess in sections, especially since differences in fixation affect the apparent density of the matrix. In cells fixed with buffered osmium tetroxide, the mitochondria appear relatively empty except for a variable number of granules tending to adhere to the cristae. However, if double fixation with formalin and osmium tetroxide is used [see (4) for references to other cell types], the matrix appears much denser. The addition of sucrose to the fixative enhances this effect, the mitochondrial matrix in cells fixed in this way resembling that in frozen-dried (26, 29) or aldehyde-fixed (4, 22) tissue. Thus, sucrose appears to have a stabilizing effect on the matrix components during fixation and embedding, this being responsible for the dense appearance of the matrix in mitochondria isolated in sucrose. These considerations suggest that the opening up of the intracristal spaces m a y in fact be secondary to swelling of the matrix compartment, which distends in the manner illustrated in the line diagrams used by Whittaker (36) to describe the changes produced in negatively stained mitochondria by hypotonic media. When the mitochondria are in situ, the membranes of the matrix compartment are arranged in an orderly manner and both the outer compartment and the intracristal spaces have only a potential existence, as Sjrstrand has suggested (26, 27, 30). This concept is supported by observations on mitochondria of varying origin processed by methods not involving osmium tetroxide fixation (2, 15, 24, 29). The observations made as the medium was progressively diluted, clearly show that structures such as those seen in Figs. 6-11, which are at present widely accepted as normal mitochondrial components, are not seen in Ehrlich aseites cell mitochondria if the sucrose concentration of the medium in which they are suspended is kept at or near 0.5 M, and they do not appear under the conditions used here, until it is reduced to the vicinity of 0.125-0.06 M. That sucrose concentration is the important factor in determining the appearance of the mitochondria is shown by the fact that it is immaterial if dilution of the medium is carried out with distilled water, buffered salt solution, or a 1% PPT solution. Sucrose is generally regarded as being osmotically active (6), although there is evidence (34) that it m a y prevent swelling by some other mechanism. FrG. 17. A thin section of a saccular form in 0.06 M sucrose. It has relatively thick walls and contains branching tubular structures of varying cross section. The wall of these tubules can be seen to be bilaminar (arrow). Many small particles (P) are not clearly associated with the tubular structures. x 110,000. FIG. 18. Section of a mitochondrion in 0.125 M sucrose, which appears to be in the process of swelling into one of the saccular forms, with complete rearrangement of the matrix compartment, x 110,000.
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0.1 p. I
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The mitochondria are likely to be exposed to conditions of osmotic stress in all negative staining methods commonly used. The extent and duration of this exposure will vary, but it is always considerable in every method but one, the microdroplet cross spray technique of Fernfindez-Morgm (8), although even here there is no certainty that the duration of exposure will be short enough to prevent change in the mitochondrial structure. The observations also suggest that most of the structures seen in negatively stained preparations of mitochondria have been through a sequence in which the matrix swells and the matrix compartment is rearranged, with the frequent appearance of branching ribbons or tubules. However, the possibility that a small number may have bypassed these changes must be considered, and this will be discussed in a separate communication. Whittaker (36) has described ballooning of the inner compartment in swollen mitochondria examined with negative staining, and the appearance within this saccular structure of what he described as "disintegrating tubular membranous clumps." He suggested that the matrix normally contains a complex membrane system which becomes visible only after the loss of the soluble constituents of the matrix. The observations described here do not support the view that the structures seen in dilute sucrose media are present at all times. On the contrary, they suggest their spontaneous appearance within the matrix compartment under conditions of osmotic stress which cause it to become considerably distended. The alternative interpretation, that the appearance of these structures derives from a patchy breakdown of the surrounding membrane, does not appear compatible with the observation that such bodies have a continuous, clearly defined margin which suggests an unbroken surrounding membrane, On the other hand, the formation of a simple saccular body by distension of the original complex matrix compartment must involve considerable rearrangement of the membrane. The fact that tubular structures spontaneously appear under these circumstances might well suggest that those parts of the membrane not incorporated in the wall of the sac give rise to them. However, the thin sections suggest (Figs. 13-18) that not only the membranes, but the whole matrix compartment may play a part in the structural changes which occur. The ability of the matrix to form ordered structure is shown (Fig. 12) by the demonstration in moderately swollen mitochondria of lamellar patterns. Similar lamellae have been described by a number of workers in both negatively stained (5, 7, 13) and osmic fixed (9, 23, 32) phospholipid preparations. Lucy and Glauert (13) demonstrated the presence of small subunits in the lipid-lamellae examined by them, and postulated that these structures form by the spontaneous association of globular micelles of the lipid. Although not sufficient is known about the nature or properties of the mitochondrial matrix to permit a detailed comparison, there is a remarkable similarity
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between some of the structural forms seen in mitochondrial preparations and certain of the "macromolecular assemblies" which form spontaneously under appropriate conditions in watery dispersions both of lipids (5, 13) and of proteins (1, 3). Furthermore, a capacity for spontaneous development of structures resembling "intact cristae" or electron transfer particles (which have identical appearances in negatively stained preparations) has been demonstrated (8) in mitochondrial subunits. The four complexes which constitute the electron transfer chain were separated from beef heart mitochondria and then recombined in vitro to form 120-140 ~, particles which showed a marked tendency to spontaneously form linear assemblies similar to those seen in Figs. 7 and 8. The present observations suggest that when the matrix is dispersed with imbibed water, certain of its components (whether lipid, lipoprotein, or protein cannot be stated) interact to spontaneously produce, perhaps after prior formation of micelles, the structural assemblies which have been described. Luzzati and Husson (14) have shown that in simple lipid-water systems, which can exist in a number of different structural phases, one of the factors determining the phase present at any time is lipid concentration. The laminar form is always seen at the highest lipid concentrations, and the present observations suggest the possibility that a similar situation exists in the mitochondrial matrix. Why the tubular forms, when seen in thin sections, do not have the 100 A particles associated with their outer surface is not clear. In places their walls appear to be bilaminar, with layers 30-40 A thick, so shrinkage might be suggested as a possible explanation. However, the lamellar structures seen in the matrix in Fig. 12 (thin section) and Fig. 5 (negative stain) both have bands between 30 and 40 • wide. If, as is postulated, the tubular forms have the same origin as the lamellae, one would not expect shrinkage differences of sufficient magnitude to account for the discrepancy. It may be, as suggested by Stoeckenius (33), that osmium tetroxide destroys these particles. Alternatively, they may be destroyed during dehydration and embedding. Although they were never seen when osmic fixation was used in this work, several workers (8, 19, 31) have seen the particles in negatively stained preparations after prior osmic fixation, so the first possibility seems unlikely. Similarly, these observations make it unlikely that the passage of a drying front across the negatively stained specimens caused spurious and orderly association of free particles (seen both in sections and negative staining) with the surface of the tubules in the negatively stained preparations. REFERENCES 1. ABRAM, D. and KOFFLER, H., Y. Cell Biol. 19, 3A (1963). 2. AFZELIUS,B. J., Symp. Intern. Soc. Cell Biol. 1, 1 (1962). 3. ANSEVlN,A. T. and LAUFFLER,M. A., Biophys. J. 3, 239 (1963).
276 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25. 26. 27. 28.
29. 30. 31. 32. 33. 34. 35. 36.
R.F. MITCHELL BAKER, J. R., J. Roy. Microscop. Soc. 84, 115 (1965). BANGHAM,A. D. and HORNE, R. W., J. Mol. Biol. 8, 660 (1964). BRIERLEY,G. and GREEN, D. E., Proc. Natl. Acad. Sci. U.S. 53, 73 (1965). FERN.~NDEZ-MORXN, H., Circulation 26, 1039 (1962). FERN.~NDEZ-MOR.~N,H., ODA, T., BLAIR, P. V. and GREEN, D. E., J. Ceil Biol. 22, 63 (1964). GEREN, B. B. and SCHMITT, F. O., J. Appl. Phys. 24, 1421 (t953). GLAUERT,A. M., ROGERS, G. E. and GLAUERT, R. H., Nature 178, 803 (1956). KARNOVSKY,M. J., J. Cell Biol. 11, 729 (1961). LEHNINGER,A. L., The Mitochondrion. Benjamin, New York, 1964. LucY, J. A. and GLAUERT, A. M., J. MoI. Biol. 8, 727 (1964). LUZZATI, V. and HussoN, F., J. Cell Biol. 12, 207 (1962). MALHOTRA,S. K. and VAN HARREVELD,A., J. Ultrastruct. Res. 12, 473 (1965). MITCHELL, R. F., Biochim. Biophys. Acta 9, 430 (1952). NADAKAVUKAREN,M. J., J. Cell Biol. 23, 193 (1964). PALADE, G. E., In GAEBLER, O. H. (Ed.), Enzymes: Units of Biological Structure and Function, p. 185, Academic Press, New York, 1956. PARSONS,D. F., J. Cell Biol. 16, 620 (1963). -Science 140, 985 (1963). -Lab. lnvest. 14, 431/1169 (1965). PEASE, D. C., Histological Techniques for Electron Microscopy. Academic Press, New York, 1964. REVEL, J. P., ITO, S. and FAWCETT, D. W., J. Biophys. Biochem. Cytol. 4, 495 (1958). ROBERTSON, J. D., in LOCKE, M. (Ed.), Cellular Membranes in Development, p. 1. Academic Press, New York, 1964. SJOSTRAND,F. S., Intern. Rev. CytoL 5, 455, 1956. -Radiation Res., Suppl. 2, 349 (1960). -in HARRIS, R. J. C. (ed.), The Interpretation of Ultrastructure, p. 47. Academic Press, New York, 1962. SJ()STRAND, F. S., ANDERSSON-CEDERGREN,E. and KARLSSON, U., Nature 202, 1075 (1964). SJOSTRAND,F. S. and BAKER, R. F., J. Ultrastruct. Res. 1, 239 (1958). SJOSTRAND,F. S. and RHODIN, J., Exptl. Cell Res. 4, 426 (1953). SMITH,D. S., J. CellBiol. 19, 115 (1963). STOECKENIUS,W., J. Cell Biol. 12, 221 (1962). -ibid. 17, 443 (1963). TEDESCHI,H., J. Cell Biol. 25, 229 (1965). WARD, R. T., J. Histochem. Cytochem. 6, 398 (1958). WHITTAKER,V. P., in GRANT, J. K. (Ed.), Methods of Separation of Subcellular Structural Components, p. 91. Cambridge Univ. Press, London and New York, 1963.
j. ULTRASTRUCTURERESEARCH18, 257--276 (1967)
257
Structural Changes in Swollen Ascites T u m o r Mitochondria R. F. MITCHELL
Australian Atomic Energy Commission Research Establishment, Sutherland, N.S. W. Received September 27, 1965 The structure of Ehrlich ascites tumor mitochondria suspended in a medium containing 0.5 M sucrose, and the changes which occur in them as the sucrose concentration is progressively reduced, are studied by electron microscopic examination of thin sections and negatively stained preparations. The changes involve swelling of the matrix compartment and lead to the spontaneous appearance of either lamellar, particulate, or tubular forms. In negatively stained specimens, the latter show substructural organization identical to that seen in preparations of the electron transfer particle and in the structures, found in negatively stained preparations of a variety of mitochondria, that are widely regarded as being the mitochondrial cristae. Fernfindez-Morfin (7, 8), Parsons (19-21), and others (17, 33), have presented electron micrographs of mitochondria negatively stained with potassium phosphotungstate (PPT) which are widely considered accurately to depict, in much more detail than is possible by thin sectioning, their fine structural organization. The mitochondrion is regarded (8, 12) as a fluid-filled system of membranes, which collapse and are penetrated by the stain once the fluid is removed. Three separate membrane systems have been described in these preparations; an outer membrane, characterized in some cases (20) by a very fine surface pattern; an inner membrane which has particles approximately 100 A in diameter associated with its inner surface (33); and mitochondrial cristae, with similar particles attached to their outer surfaces by short stems. These stemmed particles were called "elementary particles" by Fernfindez-Morfin (7) and they are believed (8) to be functional in the electron transfer activity of the mitochondrion. The structures described as cristae may be long narrow tubules of uniform diameter, or may be extremely variable in width. They m a y or may not branch. Some segments are ribbonlike, there being no penetration of stain into a central core. While the preparation of negatively stained specimens has been claimed to produce no significant change in the form of these membranes (8), Sj6strand and co-workers have pointed out (28) that the natural existence of thin ribbons or tubules within the
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various mitochondria is difficult to reconcile with the rather large m e m b r a n e surfaces demonstrated in the same mitochondrial types by sectioning. They considered that both the tubules and their associated particles were the products of degenerative changes in the mitochondrial membranes, resulting f r o m osmotic shock and, perhaps, f r o m exposure to high P P T concentrations during drying of the specimens. That the inner mitochondrial membranes m a y be altered by negative staining seems to be shown by the studies of Smith (31) on flight muscle sarcosomes of the blowfly. He showed by means of sectioning that the cristae are extensive parallel plates. In negatively stained preparations, forms which are interpreted as these plates are sometimes seen to be confluent with arrays of ribbonlike structures of identical appearance to the "cristae" seen in other mitochondrial types. These structures are presumed (31) to represent fragments of the cristae. The possibility that P P T m a y play a part in the production of these structures appears to be excluded by the fact that fixation with osmium tetroxide or formalin before the addition of the P P T does not prevent their appearance (19, 31). However, all the methods that have been used to demonstrate them involve dilution of the n o r m a l cell sap, or concentrated sucrose solutions, media which are optimal for the preservation of mitochondrial architecture, with 1-2 % P P T in water, and in some cases with distilled water as well. If this dilution produces changes in mitochondrial structure, then one might expect to detect them by gradually diluting the suspension m e d i u m and examining the mitochondria at a n u m b e r of stages. The results of such studies are the subject of this paper. MATERIALS A N D METHODS The mitochondria used were obtained from a Lettr6 hyperdiploid strain of the Ehrlich ascites tumor cell. After a single washing in Ca ++- and Mg++-free Hanks basic salt solution, pH 7.4, containing 0.02 % EDTA, the cell suspension was transferred to flat-bottom polythene tubes and centrifuged at 20,000 g for 10 minutes. The residual suspension fluid in the resulting pellet, measured with 1~1Ihuman serum albumin, occupied only 10% of the total volume. The supernatant was taken off with a Pasteur pipette, residual fluid being removed by a wad of filter paper. The bottom was cut out of the tube, and the cells were transferred to a siliconized stainless steel tube of the same internal diameter, using the filter paper as All figures are electron micrographs. F~o. 1. Negatively stained mitochondria in 0.5 M sucrose medium containing 1% PPT. The stain has penetrated the outer mitochondrial membrane (Mo), but is excluded from the inner compartment and intracristal spaces. The cristal orifices (C) can be seen. x 70,000. FIc. 2. Similar preparation to Fig. 1, except that the mitochondria were formalin fixed before staining. The rod-shaped body (P1) is in the process of distending and rounding up. P2 has swollen considerably to show the complex branching form of the inner compartment (I) and the granular nature of the matrix (ma). x 70,000.
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FIG. 3. A thin section of mitochondria in 0.5 M sucrose medium. The inner compartment (/) is filled with a finely granular matrix and forms a branched saccular and tubular system. The outer compartment (O) is the space between the inner compartment and the outer mitochondrial membrane (Mo). The intracristal spaces (C) are continuous with this outer compartment. Methacrylate embedded, lead stained, x 85,000.
a piston. The bottom of this tube was sealed with a plastic plug, a stainless steel piston having a diameter 0.1 m m less than the internal diameter of the tube was placed on top of the cells, and the assembly was swung at 1200 g in the swing-out head of a refrigerated centrifuge. As the plunger was driven down into the mass of cells, they were forced up through the gap between it and the tube, their external membranes being broken in the process. These cells were suspended in 10 volumes of a medium containing 0.5 M sucrose in 0.01 M phosphate buffer, p H 7.0, and cell disruption was completed by one passage of a PotterElvehjem homogenizer. Nuclei and cell debris were removed by centrifugation at 1000 g for 10 minutes, the supernatant then being spun at 10,000 g for 10 minutes. The resulting mitochondrial pellet was resuspended by hand with a loose-fitting homogenizer, and the preceding sequence was repeated. The initial cell wash was carried out at room temperature (15°C) but for all subsequent procedures the temperature was maintained at 0--4°C.
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F~G. 4. Thin section of part of a mitochondrion in 0.5 M sucrose. The matrix (ma) is bounded by a membrane (Mi). Part of the margin of the mitochondrion, and the outer membrane (Mo), are visible at the upper right corner. Araldite embedded, lead stained. Section thickness less than 100 A. x 250,000.
The resulting suspension of mitochondria in 0.5 M sucrose medium was diluted with either distilled water, isotonic salt solution buffered at pH 7.0, or 1 To PPT in water, p H 6.5. In this way the sucrose concentration was reduced progressively through the values 0.25 M, 0.125 M, to 0.06 M, the preparation being allowed to stand for 10 minutes at each stage before further dilution. Alternatively, the concentration was altered to 0.125 M and 0.06 M directly, without standing at the intermediate values. Negatively stained preparations of the PPT-diluted material were made by simply spraying the suspension onto grids. Negative staining of the water- and salt-diluted preparations was complicated by having to fix them before exposure to the PPT. This was done by spreading the mitochondria on the surface of a sucrose medium which was the same as the one in which they had been suspended except that it contained 1 To methanol-free formaldehyde. The spread mitochondria were picked up on grids after 10 minutes and negatively stained. Specimens examined by sectioning were double fixed with 2 % formaldehyde and 1 TO osmium tetroxide at 4 ° for 24 hours. They were dehydrated with ethyl alcohol, embedded in Araldite (IO) or 4:1 butyl:methyl methacrylate containing 0.75 mg of uranyl nitrate (35) per milliliter and stained with lead by the method of Karnovsky (1 I).
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All preparations were examined with a Siemens Elmiskop I, using an accelerating voltage of 80 kv, beam current 4-8/~A, and plate magnifications between 20,000 and 40,000. OBSERVATIONS Structure of mitochondria in 0.5 M sucrose When the mitochondria are negatively stained by spraying a suspension in 0.5 M sucrose containing 1% PPT (Fig. 1), they appear mainly as flattened round or oval forms, rather than the rod-shaped bodies common in sections of intact cells. They consist of a central body, appearing lighter than the background because it excludes the stain, surrounded by a single light line interpreted here as an outer membrane? These structures are separated by a narrow gap into which the stain has penetrated. The body of the mitochondrion is usually without much feature but there may be signs of surface creases or furrows. In places these have a regular pattern, suggesting that they may represent surface openings of the intracristal spaces, i.e., the cristal orifices. There is apparently little penetration of stain into such spaces in these preparations. If the mitochondria are fixed by addition of formaldehyde (final strength 1%) to the suspension, the stain penetrates the intracristal spaces, revealing (Fig. 2) the mitochondrial body to be a complex membrane system. In the few remaining elongated forms (e.g., P1), there is a largely transverse arrangement of these membranes, but this is lost in those which have become distended (e.g., P2). The granular appearance of this system (seen clearly in P2) in the fixed preparations could represent membrane structure, but it seems more likely to reflect the granularity of the contents, shown by penetration of the stain into the interior. Thin sections of these mitochondria (Fig. 3) show how the structure of isolated mitochondria differs from those examined in situ. They are predominantly rounded, lack the normal cristal pattern, and in fact correspond to what one would expect on sectioning a structure such as P2 in Fig. 2. Each mitochondrion is bounded by a single membrane which is presumably continuous, although where it is in direct contact with the mitochondrial body it can only occasionally be distinguished (see Fig. 4 for detail), and corresponds to the outer membrane seen in negatively stained preparations. The body of the mitochondrion is composed of two parts; an apparently closed compartment (the inner or matrix compartment) of complex branching form filled with a finely granular (particles less than 50 A) or homogeneous matrix, and the apparently empty spaces. The latter are seen in places to be continuous with the space between the matrix compartment and the outer membrane, and are interpreted as greatly distended intracristal spaces. 1 The term "membrane" is used descriptively, the structure seen may be regarded either as a "membrane element" (25) or as a "unit membrane" (•8); see Discussion.
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Fia. 5. Negatively stained mitochondria in 0.125 M sucrose. The inner compartment of P1 is not penetrated by the stain, but the others are penetrated to varying extents. A portion of this compartment appears to have a lamellar structure (L), the lighter (stain excluding) bands being 30-40 thick and the dark ones narrower. The inner compartment of P2 shows marked localized distention, while in P3, with the apparent exception of some small unswollen parts (U), the whole compartment has swollen to form a saccular body. The membrane or contents of this structure are markedly granular, and indistinct ribbonlike assemblies can be seen (arrows) at some points. The outer membrane of P3 (Mo) has broken. A number of cristal orifices are indicated (C). x 70,000.
I n v e r y t h i n sections the m a t r i x c o m p a r t m e n t (Fig. 4) is seen to be b o u n d e d b y a n a p p a r e n t l y c o n t i n u o u s m e m b r a n e 3 0 - 4 0 A across, s e p a r a t e d f r o m t h e m a t r i x b y a n e l e c t r o n - l u c i d g a p of a b o u t the s a m e w i d t h . T h i s gap is n o t c o n t i n u o u s b u t is b r i d g e d at intervals. T h e b r i d g e s a p p e a r to c o n n e c t particles in t h e m e m b r a n e w i t h o t h e r s of similar size in the m a t r i x . H o w e v e r , the i n t e r p r e t a t i o n of such d e t a i l is h a z a r d o u s
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Fro. 6. A negatively stained preparation of mitochondria in 0.06 M sucrose medium. The irregular saccular structures $1 and $2 appear to have a granular content or membrane. Aggregation of this granular material can be seen (A), and organized tubular structures (T) are present at other places. The two mitochondria P1 and P2 are only moderately distended. The matrix compartment has partly herniated from P1, and this has swollen at one place to form $3. The margins of the matrix compartments in P1 and P2, as well as most of the margins of SI, $2, and $3, can be clearly seen to be smooth. However, part of the margin of $1 (arrow) has 100 A particles attached to it. x 85,000. for two reasons. I n the absence of a through-focal series, a little u n d e r f o c u s i n g can cause a spurious particulate appearance in the image (16). Again, the e m b e d d i n g material m a y become finely globular in the electron beam, a l t h o u g h Araldite is relatively resistant to this change (22).
Morphological changes in negatively stained mitochondria on diluting the suspension medium There is considerable variation in the extent of the reaction of individual m i t o c h o n d r i a to the reduction in sucrose c o n c e n t r a t i o n of the suspension m e d i u m , a n d a variety of changes will be seen in a n y one preparation. Consequently, changes
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FIG. 7. A higher magnification of the tubules seen in Fig. 6. The dumbbell type of substructure described in the text can be seen at the point marked with the arrow. × 250,000. FIG. 8. A higher magnification of tubular and saccular forms such as those seen in Fig. 9. In this case the stain has not entered the central core, which is presumably solid. Particles approximately 100 • in diameter (arrows) are attached by stems to the surfaces of both the ribbons and the saccules. Whether the helical arrangement of particles on many of the ribbons is caused by twisting of the ribbons, is not known, x 250,000. of f o r m seen in 0.25 M sucrose are also seen in 0.125 M sucrose, a n d only the latter p r e p a r a t i o n need be illustrated. The wide range of structural changes which occur in this m e d i u m is illustrated in Fig. 5. M o s t of the m i t o c h o n d r i a are m o r e swollen t h a n w h e n seen in 0.5 M sucrose, b u t they are still s u r r o u n d e d b y a limiting m e m b r a n e s e p a r a t e d f r o m the central b o d y b y only a n a r r o w gap. I n m a n y m i t o c h o n d r i a the stain a p p e a r s to have p e n e t r a t e d the distended intracristal spaces. I n the spread a n d flattened f o r m in which these m i t o c h o n d r i a are observed, this intracristal stain serves to outline the relatively dye-excluding m a t r i x c o m p a r t m e n t which a p p e a r s to have the f o r m of b r a n c h i n g tubules of variable size. A t several points the stainfilled gaps are seen to be c o n t i n u o u s with the outer c o m p a r t m e n t (the n a r r o w space betweeen the outer m e m b r a n e a n d the b o d y of the m i t o c h o n d r i o n ) , as w o u l d be expected if they were derived f r o m the original intracristal spaces. There are differences in the detailed a p p e a r a n c e s of m i t o c h o n d r i a subjected to these media. The dye is excluded f r o m some, which resemble the bodies seen in 0.5 M sucrose, while it has p e n e t r a t e d others to show w h a t is i n t e r p r e t e d here as the finely g r a n u l a r matrix. I n some m i t o c h o n d r i a there a p p e a r s to be local distension of the m a t r i x c o m p a r t ment, while in others (e.g., P3 in Fig. 5) virtually the whole c o m p a r t m e n t has dist e n d e d to f o r m w h a t a p p e a r s to be a large sac. These sacs have s m o o t h edges b u t the
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FIG. 9. Similar preparation to Fig. 6, showing ribbon or tubular structures (T) attached to small saccular bodies (s). The fragments of granular membrane (S) presumably represent the remains of the saccular form of the matrix compartment illustrated in Figs. 5 and 6. x 46,000.
m e m b r a n e or c o n t e n t a p p e a r s g r a n u l a r , the m e a n particle size being of the o r d e r of 100 A. This is to be c o m p a r e d with the estimate of less t h a n 50 A for particles in t h e m a t r i x of m i t o c h o n d r i a s u s p e n d e d in 0.5 M sucrose a n d e x a m i n e d b y thin sectioning. A t places, p o o r l y defined r i b b o n l i k e structures can be seen within this g r a n u l a r material. T h e outer m e m b r a n e is r u p t u r e d as the m i t o c h o n d r i a l b o d y swells. I n 0.06 M sucrose large bodies of irregular outline are often seen (Fig. 6), p r e s u m a b l y representing a further stage of distension of the m a t r i x c o m p a r t m e n t . These bodies a p p e a r g r a n u l a r in such p r e p a r a t i o n s , a n d this is considered to reflect t h e
FIo. 10. Similar to Fig. 6. The membrane fragments (S) are considered to have the same origin as those seen in Fig. 9. The branching membrane system (T) has stemmed particles (arrows) attached to its outer surface, x 70,000. Fr~. 11. Similar preparation to Fig. 6. This structure contains masses of membranes with associated 100/~ particles, presumably of the branching tubular form (T) seen. A large angular membranous structure is connected to the tubules, x 70,000.
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nature of the matrix material enclosed within the saccular forms. In many areas it is fairly uniform, but in others the appearance suggests aggregation. Within these bodies, suggesting some relationship with the granular areas by continuity, organized tubular structures may be seen. These appear (Fig. 7) as parallel lines having a particulate structure, and particles of about 100 A diameter are connected to these subunits by a narrow stem. This has been clearly shown (8, 31) to be the organization of the structures commonly referred to as cristae in negatively stained mitochondrial preparations. The conclusion that they are tubular is supported by comparing their appearance with tubular assemblies seen in negatively stained phospholipid complexes (13, Plates 20-30). The failure to visualize particles on the upper or lower surface is presumably a contrast effect due to the tube being filled with stain. If the stain is excluded from the core, as in Fig. 8, particles can be seen on the surface. The outer margins of the large sacs are usually smooth, but in places they may have 100 A particles attached. Other structures seen in 0.06 M sucrose are shown in Figs. 9-11. They illustrate the diversity of forms which can result from one mitochondrial type when subjected to such treatment, but, with differences in detail, all contain ribbonlike or tubular structures which have particles attached to their outer surface. In Fig. 9 there appears to be fragmentation of a mitochondrion, producing a crisscross pattern of ribbonlike forms. These may be tubular structures similar to those shown in Fig. 7, or may have (Fig. 8) a solid core. Attached to the tubules and rods are small saccular bodies, some of which also have particles associated with their outer surfaces. The tubular forms seen in Fig. 10 are branched and variable in diameter. Stemmed particles line their margins and presumably cover their entire outer surface. This tubular system has the same granular appearance as the saccular particles of Figs. 5 and 6, although the latter usually have a smooth outer surface. The granular horseshoe-shaped structure, together with fragments of varying size and shape, seems to represent remnants of the enveloping sac. The presence of these saccular fragments, as well as the presence of particles on the surface of the tubules, would appear to preclude the possibility that they represent the unaltered matrix compartment. In all cases in which the inner compartment has been clearly seen and identified, the margins appear smooth (see, for example, PI and P2 in Fig. 6). The masses of membranes present in the body shown in Fig. 11, with which 100 A particles can be seen associated, are presumably of the same branching tubular and saccular form as seen in the upper part of the structure. The same basic arrangement of tubules and associated particles is still present in preparations which have been diluted from 0.5 M sucrose medium in a single step rather than progressively. This is true also of those preparations fixed with formalin prior to negative staining.
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FIG. 12. Thin section of part of a mitochondrion in 0.5 M sucrose, showing lamellar structure within the matrix (ma). The electron dense layers are 30-40 A across, and are separated by a slightly wider electron-lucid gap. The globular nature of the stained layer is strongly suggested at a number of points (arrows), in spite of the fact that the section thickness interferes with the clarity of such detail. Araldite embedded, lead stained, less than 100 A thick, x 260,000.
Morphological changes in thin sections of mitochondria on dilution of the suspension medium The mitochondrial matrix in 0.5 M sucrose is seen in thin sections to be finely granular and usually of uniform density (Fig. 4). In places, however the matrix may show a lamellar structure (Fig. 12). As the sucrose concentration is decreased the matrix compartment swells, with reduction in the overall density of the matrix and the appearance of local density changes (Figs. 13 and 14). Lamellar forms are more frequent in these moderately swollen mitochondria. Density variations become more marked as swelling proceeds (Fig. 14), leading in some cases (Fig. 15) to the appearance of clusters of 150-250 • particles within the distended matrix compartment. Frequently, there is complete reorganization of this compartment with the appearance of ribbon or tubular forms (Fig. 16). The tubules vary considerably in cross section and may branch. 18 - 671823 J. Ultrastructure Research
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Preparations of the mitochondria in 0.06 M sucrose show predominantly vesicular or saccular forms, as in Fig. 17. These may have thick walls, and frequently they contain tubular structures. The mitochondrion in Fig. 18 appears to be in the process of conversion to a saccular body and suggests that the reorganization involves both matrix and membranes. Sections through the tubules shown in these sacs do not show the 100 A particles which are present with negative staining, either in the substructure of the tube or projecting from it by a stem. However, they do frequently show (Fig. 17) a bilaminar wall structure, each lamina being 30-40 A thick. DISCUSSION The mitochondrial forms seen in sections of intact Ehrlich ascites tumor cells fixed with buffered osmium tetroxide are predominantly rod-shaped and conform to the classical descriptions given by Sj6strand (25) or Palade (18). However, after they have been isolated in 0.5 M sucrose medium by the method described, most have a considerably different morphology, which appears to result from changes similar to those commonly observed in preparations of isolated mitochondria (12). As shown in Figs. 1-3, they have become rounded, and the characteristically bilaminar outer membrane and cristae are no longer apparent. The essential change appears to be a very considerable opening of the intracristal spaces, with complete loss of the original orderly arrangement. Where these spaces occur, the limiting membrane appears as a single unit membrane (18) or membrane element (25). No distinction between these being possible from the observations described here, the term membrane is used in a purely descriptive manner and does not reflect the status of this structure in unaltered mitochondria. Elsewhere, it is closely applied to the matrix compartment, and is only clearly visualized in places (see Fig. 4). In negatively stained preparations, although it is usually clearly separate from the central mitochondrial body (see Fig. 1), it follows closely the shape of the body, even when this is irregular. It is difficult to FrG. 13. Thin sections of three mitochondria in 0.25 M sucrose, each showing a different degree of swelling. P1 resembles those in 0.5 M sucrose, having a dense matrix and open intracristal spaces. P2 has a more distended matrix compartment, with a reduction in the overall density of the matrix and local variations in density. P3 is considerably swollen, and the matrix now shows an ill defined internal pattern. Methacrylate embedded, lead stained, x 125,000. FIG. 14. Thin section of a mitochondrion in 0.25 M sucrose showing an intermediate stage in the development of a pattern in the swelling matrix (ma). Methacrylate embedded, lead stained. x 145,000. Fro. 15. Thin section of a greatly swollen mitochondrion in 0.06 M sucrose. The large saccular structure, of which only a part is shown, contains clusters of particles 150-250 A in diameter, x 145,000. FIG. 16. A mitochondrion in 0.125 M sucrose medium, showing a moderately distended matrix at the upper end (ma); the remainder of the matrix compartment has become rearranged in the form of uniform ribbons or tubules (T1), and vesicles or tubules of varying section (T2). x 110,000.
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see how marked distension of the intracristal spaces can arise without related distension of the outer compartment with which they communicate. At the same time there are indications, particularly with negatively stained preparations (see Fig. 2), that the matrix compartment has distended to some extent in most of the mitochondria in 0.5 M sucrose. This is difficult to assess in sections, especially since differences in fixation affect the apparent density of the matrix. In cells fixed with buffered osmium tetroxide, the mitochondria appear relatively empty except for a variable number of granules tending to adhere to the cristae. However, if double fixation with formalin and osmium tetroxide is used [see (4) for references to other cell types], the matrix appears much denser. The addition of sucrose to the fixative enhances this effect, the mitochondrial matrix in cells fixed in this way resembling that in frozen-dried (26, 29) or aldehyde-fixed (4, 22) tissue. Thus, sucrose appears to have a stabilizing effect on the matrix components during fixation and embedding, this being responsible for the dense appearance of the matrix in mitochondria isolated in sucrose. These considerations suggest that the opening up of the intracristal spaces m a y in fact be secondary to swelling of the matrix compartment, which distends in the manner illustrated in the line diagrams used by Whittaker (36) to describe the changes produced in negatively stained mitochondria by hypotonic media. When the mitochondria are in situ, the membranes of the matrix compartment are arranged in an orderly manner and both the outer compartment and the intracristal spaces have only a potential existence, as Sjrstrand has suggested (26, 27, 30). This concept is supported by observations on mitochondria of varying origin processed by methods not involving osmium tetroxide fixation (2, 15, 24, 29). The observations made as the medium was progressively diluted, clearly show that structures such as those seen in Figs. 6-11, which are at present widely accepted as normal mitochondrial components, are not seen in Ehrlich aseites cell mitochondria if the sucrose concentration of the medium in which they are suspended is kept at or near 0.5 M, and they do not appear under the conditions used here, until it is reduced to the vicinity of 0.125-0.06 M. That sucrose concentration is the important factor in determining the appearance of the mitochondria is shown by the fact that it is immaterial if dilution of the medium is carried out with distilled water, buffered salt solution, or a 1% PPT solution. Sucrose is generally regarded as being osmotically active (6), although there is evidence (34) that it m a y prevent swelling by some other mechanism. FrG. 17. A thin section of a saccular form in 0.06 M sucrose. It has relatively thick walls and contains branching tubular structures of varying cross section. The wall of these tubules can be seen to be bilaminar (arrow). Many small particles (P) are not clearly associated with the tubular structures. x 110,000. FIG. 18. Section of a mitochondrion in 0.125 M sucrose, which appears to be in the process of swelling into one of the saccular forms, with complete rearrangement of the matrix compartment, x 110,000.
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The mitochondria are likely to be exposed to conditions of osmotic stress in all negative staining methods commonly used. The extent and duration of this exposure will vary, but it is always considerable in every method but one, the microdroplet cross spray technique of Fernfindez-Morgm (8), although even here there is no certainty that the duration of exposure will be short enough to prevent change in the mitochondrial structure. The observations also suggest that most of the structures seen in negatively stained preparations of mitochondria have been through a sequence in which the matrix swells and the matrix compartment is rearranged, with the frequent appearance of branching ribbons or tubules. However, the possibility that a small number may have bypassed these changes must be considered, and this will be discussed in a separate communication. Whittaker (36) has described ballooning of the inner compartment in swollen mitochondria examined with negative staining, and the appearance within this saccular structure of what he described as "disintegrating tubular membranous clumps." He suggested that the matrix normally contains a complex membrane system which becomes visible only after the loss of the soluble constituents of the matrix. The observations described here do not support the view that the structures seen in dilute sucrose media are present at all times. On the contrary, they suggest their spontaneous appearance within the matrix compartment under conditions of osmotic stress which cause it to become considerably distended. The alternative interpretation, that the appearance of these structures derives from a patchy breakdown of the surrounding membrane, does not appear compatible with the observation that such bodies have a continuous, clearly defined margin which suggests an unbroken surrounding membrane, On the other hand, the formation of a simple saccular body by distension of the original complex matrix compartment must involve considerable rearrangement of the membrane. The fact that tubular structures spontaneously appear under these circumstances might well suggest that those parts of the membrane not incorporated in the wall of the sac give rise to them. However, the thin sections suggest (Figs. 13-18) that not only the membranes, but the whole matrix compartment may play a part in the structural changes which occur. The ability of the matrix to form ordered structure is shown (Fig. 12) by the demonstration in moderately swollen mitochondria of lamellar patterns. Similar lamellae have been described by a number of workers in both negatively stained (5, 7, 13) and osmic fixed (9, 23, 32) phospholipid preparations. Lucy and Glauert (13) demonstrated the presence of small subunits in the lipid-lamellae examined by them, and postulated that these structures form by the spontaneous association of globular micelles of the lipid. Although not sufficient is known about the nature or properties of the mitochondrial matrix to permit a detailed comparison, there is a remarkable similarity
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between some of the structural forms seen in mitochondrial preparations and certain of the "macromolecular assemblies" which form spontaneously under appropriate conditions in watery dispersions both of lipids (5, 13) and of proteins (1, 3). Furthermore, a capacity for spontaneous development of structures resembling "intact cristae" or electron transfer particles (which have identical appearances in negatively stained preparations) has been demonstrated (8) in mitochondrial subunits. The four complexes which constitute the electron transfer chain were separated from beef heart mitochondria and then recombined in vitro to form 120-140 ~, particles which showed a marked tendency to spontaneously form linear assemblies similar to those seen in Figs. 7 and 8. The present observations suggest that when the matrix is dispersed with imbibed water, certain of its components (whether lipid, lipoprotein, or protein cannot be stated) interact to spontaneously produce, perhaps after prior formation of micelles, the structural assemblies which have been described. Luzzati and Husson (14) have shown that in simple lipid-water systems, which can exist in a number of different structural phases, one of the factors determining the phase present at any time is lipid concentration. The laminar form is always seen at the highest lipid concentrations, and the present observations suggest the possibility that a similar situation exists in the mitochondrial matrix. Why the tubular forms, when seen in thin sections, do not have the 100 A particles associated with their outer surface is not clear. In places their walls appear to be bilaminar, with layers 30-40 A thick, so shrinkage might be suggested as a possible explanation. However, the lamellar structures seen in the matrix in Fig. 12 (thin section) and Fig. 5 (negative stain) both have bands between 30 and 40 • wide. If, as is postulated, the tubular forms have the same origin as the lamellae, one would not expect shrinkage differences of sufficient magnitude to account for the discrepancy. It may be, as suggested by Stoeckenius (33), that osmium tetroxide destroys these particles. Alternatively, they may be destroyed during dehydration and embedding. Although they were never seen when osmic fixation was used in this work, several workers (8, 19, 31) have seen the particles in negatively stained preparations after prior osmic fixation, so the first possibility seems unlikely. Similarly, these observations make it unlikely that the passage of a drying front across the negatively stained specimens caused spurious and orderly association of free particles (seen both in sections and negative staining) with the surface of the tubules in the negatively stained preparations. REFERENCES 1. ABRAM, D. and KOFFLER, H., Y. Cell Biol. 19, 3A (1963). 2. AFZELIUS,B. J., Symp. Intern. Soc. Cell Biol. 1, 1 (1962). 3. ANSEVlN,A. T. and LAUFFLER,M. A., Biophys. J. 3, 239 (1963).
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