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The granular structure of mitochondrial membranes and of cytomembranes as demonstrated in frozen-dried tissue
S. ULTRASTRUCTURERESEARCH10, 263--292 (1964)
263
The G r o n u l a r S t r u c t u r e of M i t o c h o n d r i a l / V l e m b r a n e s and of
CytomembranesAs
D e m o n s t r a t e d in F r o z e n - D r i e d Tissue 1
FRITIOF S. SJOSTRANDAND LARS-G. ELFVlN
Department of Zoology, University of California, Los Angeles Received November 19, 1963 A regular crystal-like arrangement of what appears as globular particles was observed in face-on views of mitochondrial membrane elements, c~-cytomembranes, and presumably Golgi membranes. The globular particles were arranged in rows and appeared only lightly stained. Between these rows, strands of intensely stained material were present. In cross sections the mitochondrial membrane elements and the cytomembranes appeared as light lines reflecting a close packing of the lightly stained globular particles. The distance between the centers of the rows of particles seen in face-on views was 45-50 ~; the thickness of the light lines representing cross sections of the membranes was 40 ~. These patterns are interpreted to support the observations made on chemically fixed material (15, 16), that is, to reflect a globular substructure consisting of the membrane lipids in a micellar arrangement stabilized by proteins. The ribosomal material forms an almost complete carpet covering the membrane elements of the ~-cytomembranes. A subdivision into particles can be correlated to a high degree of dehydration of the cytoplasm due to ice crystal formation during freezing. A globular structure was demonstrated by SjSstrand (I5, 16) in mitochondrial membranes and in smooth-surfaced cytomembranes in the tubular cells of the mouse kidney after potassium permanganate fixation. Indications of a similar subdivision of these membranes into globular structural units were also found in osmium-fixed material. Since the reliability of those observations made on chemically fixed material was considered doubtful (16), it appeared important to try to reproduce them in frozen-dried material after very rapid freezing of small pieces of tissue. Mouse pancreas tissue was selected for this purpose since the cytoplasm of the exocrine pancreas cells is filled with membranous components and is structurally rather simple. The identification of cytoplasmic structural components in sections of frozen-dried material is therefore facilitated. This is important since the contrast in electron micrographs of frozen-dried material is low and the structural patterns z This research was supported by Grant No. NSF G-23392 of the National Science Foundation.
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differ from those of chemically fixed material. The differences are due to the fact that in frozen-dried tissue the more or less extensive extraction of material is eliminated, particularly when molecular distillation in v a c u u m is used as the only method for r e m o v i n g the water. A globular structure was observed in m i t o c h o n d r i a l m e m b r a n e elements, in ~-cytom e m b r a n e s , a n d in Golgi m e m b r a n e s in this material with the globular structural units showing similar dimensions to those observed in potassium p e r m a n g a n a t e fixed material. The globular c o m p o n e n t s , however, showed a n ordered arrangement frequently a p p e a r i n g with a crystal-like regularity in the frozen-dried material.
MATERIALS A N D METHODS White mice weighing about 20 g were decapitated; pieces of the pancreas were removed, cut into minute pieces with a razor blade knife, and immersed in liquid nitrogen or in isopentane chilled by means of liquid nitrogen. The tissue was frozen within 60 seconds after decapitation of the experimental animal. When liquid nitrogen was used as medium for freezing, its temperature had been lowered by vacuum evaporation to the freezing point of nitrogen. A solid phase"of nitrogen developed and filled most of the Dewar flask, with only a thin layer of liquid nitrogen on top of the frozen nitrogen. The temperature of the liquid phase was sufficiently low to prevent a rise in temperature to the boiling point of nitrogen when the small pieces of tissue were immersed. This means that no layer of nitrogen gas was formed around the tissue which would act as a thermal insulation slowing down the rate of freezing. The frozen tissue was dried in a freeze-drying apparatus designed by Sj6strand and consisting of a glass vacuum chamber, the drying chamber, connected to an oil diffusion pump by means of a wide-bore brass tube. A Welch mechanical pump was used to back the diffusion pump. A wide-bore valve on top of the diffusion pump allowed closing of the connection between vacuum chamber and diffusion pump. A side tube with a valve made it possible to connect the vacuum chamber with a glass container in which the embedding medium could be kept under vacuum. The embedding medium could be transferred to the vacuum chamber with the specimens by means of vacuum distillation. A cold finger reached from the top of the vacuum chamber down to a few centimeters from its bottom where the specimens were placed. It was kept filled with liquid nitrogen during the drying procedure. The lower part of the vacuum chamber was surrounded by a metal Dewar flask standing on the bottom of a dry-ice chest filled with dry ice. The metal Dewar flask was filled with liquid nitrogen when the specimens, submerged in liquid nitrogen, were transferred to the drying chamber and the evacuation of the freeze-drying apparatus started. The drying under vacuum was performed at a slowly rising temperature from the temperaFro. 1. Survey picture of cytoplasm of frozen-dried exocrine pancreas cell. At the bottom i s a mitochondrion (Mi). ~-Cytomembranes (e) with ribosomal material above the mitochondrion are mostly oriented perpendicular to the plane of the section and appear therefore as light lines representing cross sections of the membranes, x 130,000.
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ture of liquid nitrogen to that of dry ice, and extended over several days. The temperature was then allowed to rise slowly from dry-ice temperature to room temperature. Before embedding in Vestopal, the tissue was exposed for several hours to osmium tetroxide vapor either in a desiccator or under vacuum in a Thunberg tube used for the embedding. The embedding in Vestopal was performed under vacuum in a modified Thunberg tube allowing a thorough evacuation of air dissolved in the Vestopal before immersion of the tissue into the embedding medium. The tissue was then submerged in Vestopal under vacuum by tilting the Thunberg tube. The dried tissue was kept in the top curved part of the Thunberg tube, separated from the Vestopal, while the air was evacuated from the Vestopal. The Vestopal was allowed to set at 60°C, and sections from the hardened blocks were cut on an LKB Ultrotome. The sections were examined in a Siemens Elmiskop I at 40,000 times electron optical magnification at 80 kV with a 50-~ objective aperture and careful compensation of objective lens astigmatism to less than one step on the fine focus control. The contrast of the tissue was enhanced by means of staining in a saturated uranyl acetate solution at 60°C, according to Brody (2). RESULTS The f r o z e n - d r i e d p a n c r e a s tissue (Fig. 1) shows a structural p a t t e r n which differs very m u c h f r o m the c o n v e n t i o n a l p a t t e r n s o b s e r v e d in chemically fixed material, The g r o u n d substance of the c y t o p l a s m in f r o z e n - d r i e d m a t e r i a l contains components which are absent, owing to extraction, in chemically fixed m a t e r i a l subjected to a l c o h o l o r acetone d e h y d r a t i o n . F u r t h e r m o r e , a u n i f o r m d i s t r i b u t i o n of material m a k e s the g r o u n d substance a p p e a r relatively intensely stained in frozen-dried material as c o m p a r e d with the a l m o s t e m p t y a p p e a r a n c e in chemically fixed tissues. The c y t o p l a s m also becomes c o n c e n t r a t e d in c o n n e c t i o n with various degrees of dehyd r a t i o n due to the f o r m a t i o n of ice crystals p r e c e d i n g the freezing of the entire cells. This p h a s e s e p a r a t i o n c o n t r i b u t e s to a n increase in c o n c e n t r a t i o n of stainable material in the still liquid phase of the c y t o p l a s m until c o m p l e t e freezing has been achieved. A characteristic feature of sections t h r o u g h f r o z e n - d r i e d m a t e r i a l is t h a t cross sections of various types of m e m b r a n e s a p p e a r as light lines against a d a r k backg r o u n d (Fig. 1). This p a t t e r n was originally o b s e r v e d b y Sj6strand a n d Baker (17) a n d described as a negative p a t t e r n . The a-cytomembranes a p p e a r e d in cross sections as light lines with an average thickness of 40 A (Table I). They were a r r a n g e d in pairs, each m e m b r a n e pair deliFIG. 2. Cytoplasm of frozen-dried exocrine pancreas cell with g-cytomembranes of different orientations. In the lower part of the picture the c~-cytomembranes are cross-sectioned; in the upper part of the picture they are cut more or less tangentially. The ribosomal material forms a layer associated with the cross-sectioned membranes in the former case (A) and irregularly forms arranged opaque regions in the latter case (B). A granular pattern is observed in the tangentially sectioned c~-cytomembranes. × 220,000.
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FRITIOF S. SJ~JSTRAND AND LARS-G. ELFVIN
miring a space which varied considerably in width between 50 and 200 • or more. This space was filled with opaque material that appeared homogeneous (Figs. t-4). The e-cytomembranes were easily identified by their association with ribosomal material. This material appeared as a continuous opaque layer coating one surface of the membrane element of the ~-cytomembranes. Frequently this coating showed an indication of a division into subunits by the presence of fairly regularly distributed but diffusely outlined areas which were particularly intensely stained (Figs. t, 3, and 4). This indication of a subdivision of the ribosomal material is interpreted to correspond to ribosomal particles as observed in osmium-fixed material. The variation in the opacity in the layer of ribosomal material did not make this material appear completely subdivided into discrete particles separated by spaces free from ribosomal material. On the contrary, the ribosomal material appeared as a continuous layer with slightly varying opacity. Exceptions to this rule could be observed in cases where shrinkage of the cytoplasm, due to dehydration in connection with excessive ice crystal formation inside the cell, had taken place (Fig. 5). The more opaque regions of the ribosomal material were frequently located at shallow depressions in the membrane surface (Fig. 4). In membranes that were oriented more or less parallel to the plane of the section, the ribosomal material formed an almost complete continuous carpet with the more opaque regions vaguely indicated (Fig. 2). It was uncertain whether any regular pattern of rows of opaque regions could be seen in this carpet. The width of the depressions in the membrane surface where the more opaque regions of the ribosomal material were located was about 200 A; the thickness of the layer of ribosomal material at the membrane surface was 150 A. In the cases where the ribosomal material showed fairly uniformly distributed more opaque regions in a continuous layer of slightly less opaque material, it was possible to measure in a direction parallel to the membrane surface the average distance between the centers of these opaque regions presumably representing the centers of ribosomes. This was done by punching minute holes in prints by means of a needle through the centers of the most opaque regions and measuring the distances between the holes on the backs of the prints. The average distance between the opaque centers along the surface of the membranes was about 200 A, which is identical to the width of the depressions in the ~-cytomembranes.
FIG. 3. c~-Cytomembranesof different orientations in relation to the plane of the section. Crosssectioned membranes (A) with continuous layer of ribosomal material appear as light lines. Tangentially sectioned membranes (B) show a granular structure and irregularly arranged ribosomal material, x 380,000.
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FRITIOF S. SJ()STRAND AND LARS-G. ELFVIN
In the case mentioned above where discrete ribosomes could be seen, they showed a circular cross section and measured 150-200 A in diameter. The average diameter of these ribosomes measured parallel to the plane of the membranes was therefore, in this case, slightly smaller than in the majority of the pictures. A substructure in the membrane element of the o~-cytomembranes showed up very clearly when these membranes were oriented more or less parallel to the plane of the section (Figs. 2, 3, 6-9). A gradual transition from an orientation perpendicular to the plane of the section to one more or less oblique can frequently be seen, as in Figs. 2 and 4. With increasing tilt of the membrane, more and more of a characteristic, regular structural pattern becomes visible. It can be described as consisting of rows of lightly stained dots separated by more intensely stained irregularly dotted lines. Lightly stained cross bridges extend between adjacent dotted lines and make the light area between these lines appear subdivided into a row of light globules. The rows of dots could form different patterns with either several parallel lines running fairly straight (Fig. 6) or curved (Fig. 3). The regularity of the patterns also varied considerably, a variation that can be due either to superposition of similar patterns in two membranes on top of each other or to deformation of the pattern through the preparatory procedure. The average spacing of the lines in these patterns when exhibiting a high degree of regularity was strikingly constant and varied in different pictures within a narrow range of 45-50 A. The size of the light dots was about 40 A; the opaque dots between the light ones had a minimum diameter within the range of 12-25 A. They could appear asymmetric and rod shaped. The length of these elongate opaque dots varied between 30 and 50 A. Their long axis could be oriented at various angles in relation to the direction of the row of dots (Figs. 6-9). This asymmetry was not due to distortion through astigmatism or specimen drift. The structure of the mitochondrial membranes. The mitochondria showed a rather opaque matrix against which the mitochondrial membranes appeared as two parallel light lines separated by opaque material (Figs. 1, 10, 11). Each light line corresponded to one membrane element of the mitochondrial membrane. It was striking that the separation of the two light lines was very constant. The opacity of the matrix was uniform and there was no indication of any opaque layer at the boundary between the matrix and the mitochondrial membranes in cross sections of the membranes. This is in contrast to the picture of, for instance, osmium-fixed mitochondria (16) Where the mitochondrial membrane elements face the matrix with an opaque layer.
FIG. 4. Higher magnification of part of Fig. 1 showing partially cross-sectioned c~-cytomembranes and partially obliquely sectioned regions with a granular structure (arrows). (Mi, mitochondrion). x 240,000.
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FRITIOF S. SJOSTRAND AND LARS-G. ELFVIN
FIG. 5. e-Cytomembranes in exocrine pancreas cell where the cytoplasm has been extensively dehydrated due to intracellular ice crystal formation. Ice crystal vacuoles are marked IV. Ribosomal particles can be observed associated with the ~-cytomembranes. × 230,000.
The total thickness of the mitochondrial membranes was 115 ~ (Table II). Each light line measured 30-40 A in thickness (Table I). The average width of the opaque space separating the two light lines was 30-35 A (Table 1I). At high magnification the light lines could show an indication of a globular substructure (Fig. 11). When the outer mitochondrial membrane was properly oriented, it showed the same structural pattern as the inner mitochondrial membranes. When the mitochondrial membranes were oriented obliquely or parallel to the plane of the section, a structural pattern similar to that of the c~-cytomembranes was observed (Figs. 10 and 11). Rows of opaque particles were separated by light spaces. These light spaces appeared as rows of lightly stained or unstained globular subunits. The spacing between the rows of particles was identical to that observed for the ~-cytomembranes. The light dots measured about 30 A in diameter and the opaque dots 12--15 ~ in diameter when measured perpendicular to the length of the rows of dots. The resolution as determined by the minimum separation of object points in two consecutive pictures was estimated to about 15 A.
MITOCHONDRIAL MEMBRANESAND CYTOMEMBRANES
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TABLE I THE THICKNESS OF THE LIGHT LAYER OF CROSS-SECTIONED MITOCHONDRIAL MEMBRANE ]~LEMENTS AND OF ~-CYTOMEMBRANES a Mitochondrial Membrane Elements
¢~-Cytomembranes
43 42 38 34 34 41 29 M = 37 ~
49 35 38 38 43 35 45 M=40
a Each figure represents the mean value from measurements on 10 membranes with one measurement made at a randomly chosen point on each membrane. TABLE II TOTAL THICKNESS OF MITOCHONDRIAL MEMBRANES AND OF OPAQUE MIDDLE LAYERa" Total Thickness (A)
Opaque Middle Layer (N)
118 105 128
33 31 33
M=l17~
M-32~
Each figure represents the mean value from measurements on 10 membranes with one measurement made at a randomly chosen point on each membrane.
The Golgi membranes. W i t h i n certain regions particularly close to the z y m o g e n granules the cytoplasm appeared free f r o m r i b o s o m a l material (Fig. 12). Also i n these regions light lines a n d the regular g r a n u l a r patterns described above could be observed. Since the cytoplasm of the exocrine pancreas cells does n o t show any regions of these dimensions free from m i t o c h o n d r i a or cytomembranes, either of the a-type or of the Golgi type, it was assumed that they represented the site of the Golgi apparatus. This conclusion was supported by the characteristic localization of these regions in the cell which corresponded to that of the Golgi apparatus. FIGS. 6 and 7. Some e-cytomembranes oriented partially more or less parallel to the plane of the section showing a granular substructure. Fig. 6 is slightly underfocused and Fig. 7 close to focus. x 600,000. 18--641834 J . Ultrast~'uctu~e JResearcl~
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The structural patterns observed in these regions were therefore tentatively interpreted to reveal the structure of Golgi membranes. These patterns are similar to those described for c~-cytomembranes and mitochondrial membrane elements but frequently are particularly regular and distinct (Figs. 13-15). The spacing of the rows of particles was more frequently 50 fit than in the cases of mitochondrial membrane elements and c~-cytomembranes, but this material did not allow a statistical evaluation of this difference. DISCUSSION
The "negative" pattern observed in frozen-dried material was first described by Sj6strand and Baker (17) in frozen-dried mouse pancreas. It is a negative pattern only in appearance according to the information regarding membrane structure that has been collected since that time. The conclusion that the pattern was negative was based on an identification of the lightly stained or unstained lines representing cross sections of membranes in frozen-dried tissue as corresponding to the single (about 50 A-wide) opaque lines representing the cross sections of membranes or membrane elements in osmium-fixed material studied at that time. Since then it has been shown that both after potassium permanganate fixation (7) and osmium fixation combined with section staining (16) the membranes or membrane elements appear triplelayered and that in the case of mitochondrial and cytoplasmic membranes the triplelayered pattern corresponds in position to the single, 50-60 A-thick osmiophilic structure observed in osmium-fixed material studied earlier which had not been section stained. This is in contrast to the situation with respect to the plasma membrane where the osmiophilic structure described earlier represents only one layer (its cytoplasmic layer) of the asymmetric triple-layered plasma membrane (18). After potassium permanganate fixation and section staining, the triple-layered pattern of mitochondrial membrane elements and of cytomembranes appears to be complicated by the presence of stained cross bridges extending between the two opaque layers at the surface of the membranes (15, 16). The presence of these cross bridges has not been clearly demonstrated in osmium-fixed material but is sometimes vaguely indicated. This could mean that they are not as distinctly and easily stained after osmium fixation as the two opaque surface layers or that they are not well preserved after this fixation. A diffuse staining of this material could explain the fact that the triple-layered structure of the membrane elements of mitochondria and of FIGS. 8 and 9. Face-on view of e-cytomembrane showing the regular substructure. The size and shape of the opaque particles can be observed and the specimen resolution estimated to 15 K or better in these pictures. Fig. 8 is slightly underfocused and Fig. 9 close to focus. Within encircled areas a point resolution of 15 A can be observed in both pictures, x 1,000,000.
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FRITIOF S. SJOSTRAND AND LARS-G. ELFVIN
the cytomembranes regularly appeared as a single osmiophilic layer in earlier studies before the application of efficient section-staining methods. The thickness of the single osmiophilic layer in cytoplasmic membranes observed earlier, in contrast to the situation in the plasma membrane, does correspond to the total thickness of the triple-layered counterpart in section-stained, osmium-fixed or in unstained potassium permanganate-fixed material. It is therefore likely that the single osmiophilic layer pattern depends upon the presence of osmiophilic material in the middle of the membrane elements which corresponds to the cross bridges observed in potassium permanganate section-stained material but which is not discretely enough stained to be resolved. If we start out with these considerations we arrive at an alternative interpretation of the structural patterns observed in frozen-dried material according to which the observed structural patterns are only seemingly negative patterns. This new interpretation recognizes the light lines observed in frozen-dried material as corresponding to the middle part of the membrane elements and of the cytomembranes which contain less stainable material than the two opaque surface layers of these components (Fig. 16 A). This interpretation is supported by the structural pattern of mitochondrial membranes. This pattern can be superimposed onto the five-layered pattern of these membranes frequently observed after potassium permanganate fixation in cases where the cross bridges in the membrane elements are not revealed or after osmium fixation when the two membrane elements of the mitochondrial membranes are closely packed. The thickness of the opaque middle layer of the mitochondrial membranes observed in frozen-dried material corresponds to the thickness of the opaque middle layer of the five-layered pattern observed in the chemically fixed material. The thickness of the light layers in frozen-dried material (30-40 A) corresponds fairly closely to that of the light middle layer of chemically fixed material (20-30 A). A discrepancy between these dimensions can be explained either by some material remaining unstained after freeze-drying preservation and section staining or by a partial disorganization of the structure in connection with chemical fixation with partial collapsing of the light layer. A partial disorganization could release reactive groups of the involved molecules for interaction with the stain. In the mitochondrial membranes we could in this way account for one opaque surface layer of the membrane elements as contained in the middle opaque line in the observed triple-layered pattern of cross-sectioned mitochondrial membranes. FIG. 10. Mitochondrion (Mi) with cross-sectioned and tangentially sectioned membranes. The outer mitochondrial membrane along the upper contour of the mitochondrion (A) is tangentially sectioned and shows a regular substructure. At B, inner mitochondrial membrane is viewed face on and shows a similar structural pattern, x 310,000.
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FRITIOF S. SJOSTRAND AND LARS-G. ELFVIN
We can also account for the light middle layer of the triple-layered membrane elements, but we observe no opaque layer delimiting the mitochondrial membranes at the surface facing the mitochondrial matrix as we do in chemically fixed material. This circumstance can be explained by the fact that the matrix is intensely stained in the frozen-dried material showing the same opacity as that, for instance, of the middle opaque layer of the mitochondrial membranes, that is, of the two opaque surface layers of the membrane elements which have fused in the middle of the mitochondrial membranes. If an opaque layer exists at the matrix side of the membrane elements, the presence of this layer therefore could be masked by the identical opacity of the dense matrix. In chemically fixed material the matrix shows a low opacity, presumably due to extraction of matrix material during the fixation and embedding procedure. Any stained layer at the matrix surface of the membrane elements will therefore show up easily against the lightly stained matrix. It should also be kept in mind that the affinity of the various structural components to stains is very much weaker in frozen-dried than in chemically fixed material. This makes it more difficult to differentiate structural components due to a considerably lower contrast of the pictures of frozen-dried material. Identification of membranous components. The conclusion that the light fines observed in frozen-dried material indicate cross sections of membranes appears rather obvious both in the case of mitochondrial membranes and ~-cytomembranes. In the latter case the ribosomal material represents a reliable label for the membranes. The identification of Golgi membranes is based On the absence of ribosomal material and the knowledge that no extensive cytoplasmic membranous material other than e-cytomembranes and Golgi membranes exists in the exocrine pancreas cells. With respect to the structural patterns that have been referred to as membranous components oriented more or less parallel to the plane of the sections, we usually do not have an identical counterpart in chemically fixed material where seldom a structure is seen in tangentially sectioned membranes. Potassium permanganatefixed material is an exception since in this case a globular pattern could be observed in tangentially sectioned membranes. The globular structure did not, however, exhibit the same regular geometry as the patterns observed in the present material. In order to use the observations made on frozen-dried material as an argument that the pattern in potassium permanganate-fixed material might reflect a real and not an artifactitious structure, we must be able to prove that these patterns refer to the same structural components. We cannot use, therefore, the similarity of the patterns
FIG. 1!. Same mitochondrion as in Fig. 10 in a close-to-focus picture showing indications of globular structure of cross sections of membrane elements (arrows). The size of the opaque particles in the globular substructure of the mitochondrial membrane elements can also be observed, x 600,000.
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as a means of identifying the components showing these patterns in the frozen-dried material. The association of the observed globular patterns with mitochondrial membrane elements and with cytomembranes is based on the fact that these patterns are observed only in places where cross sections of membranes are absent. The ribosomal material assists in indicating where the ~-cytomembranes are located and where they are oriented more or less parallel to the plane of the sections, by showing a rather random arrangement or by forming a large opaque carpet instead of being arranged in even rows of opaque regions in uniformly thick layers. The globular structural patterns referred to as representing face-on views of the ~-cytomembranes have been observed in regions where ribosomal material was present and did not show a layered arrangement but a more r a n d o m distribution in carpets. In the mitochondria the inner membranes are so regularly arranged all through their interior that any region free of cross-sectioned mitochondrial membranes must contain such membranes oriented more or less parallel to the plane of the section. The globular patterns were observed in such regions. The identification of Golgi membranes oriented obliquely or parallel to the plane of the section was based on the absence in those regions of ribosomal material and the vicinity to zymogen granules. No spaces of the dimensions of these regions are free from cytomembranes in the exocrine pancreas cells. The observed patterns therefore are most likely related to cytomembranes. Direct transition from a light line pattern representing cross sections of membranes to the globular patterns could also be observed linking both patterns to the membrane. Regions lacking any patterns of either cross-sectioned or tangentially sectioned membranes were frequently found in mitochondria. This can be explained by the fact that the mitochondrial membranes consist of two tightly packed membrane elements as can be seen in cross sections of the membranes. The total thickness of this two-element structure is 100-150 A. This means that there is a high probability of both membrane elements being contained in the section even when oriented parallel to the plane of the section since the minimum thickness of these sections of frozendried material certainly is not less than 200 A. The globular structural patterns of both membrane elements will therefore be superimposed and if not in perfect register the resulting image pattern will be that of more or less randomly arranged dots. In the case of the ~-cytomembranes, on the other hand, a sufficiently wide space
FIG. 12. Survey picture of cytoplasm of exocrine pancreas cell showing c~-cytomembranesin lower part and zymogen granules (Z) in the upper part of the picture. The zone in between is interpreted to correspond to the location of Golgi apparatus (G) because it lacks ribosomal material. × 140,000.
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separates the two membranes of each membrane pair to introduce a high probability that only one membrane will be present in a tangential section to contribute to the observed pattern. Geometric interpretation of face-on views of membranes. Structural patterns associated with face-on views of the mitochondrial membrane elements and the cytomembranes are interpreted to reflect a rather regular linear arrangement of globular structural units represented by the less intensely stained parts of the patterns. These rows of globular particles appear discretely outlined due to the presence of intensely stained material associated with the globules and concentrated primarily between these rows. Stained material also extends across the rows at regular intervals, delimiting the individual globules within each row of globules. T h e globules appear arranged in a square packing when showing the regular patterns. Modifications in the arrangement of the opaque material can be accounted for by different degrees of tilt of the membranes in relation to the plane of the section. The observed patterns are in principle identical irrespective of whether the pictures were taken very close to focus or were slightly underfocused. This means that the patterns are neither distorted by diffraction effects nor due to such effects. The opaque material is assumed to be concentrated preferentially at the two surfaces of the membrane elements since these elements appear as light lines in cross sections. The lightly stained globular components are therefore assumed to be more or less closely packed. The distance between the centers of the globular units is similar in a direction parallel and perpendicular to the row, and this distance corresponds closely to the thickness of the lightly stained part of the membranes when observed in cross sections (40 A). This dimension is considered to reflect the diameter of the globular components, 40-50 A. An indication of a globular structure can also be observed in cross sections of the membranes in close-toCfocus pictures, although this is not always the case. If the opaque material present between the rows of lightly stained material in the face-on views of the membrane elements extended across the entire thickness of the membranes, we would expect to see more indications of opaque cross bridges in the cross sections of the membranes or these cross sections would not appear as lightly stained as they do. It is clear, however, from the face-on view of the membrane elements that there is faintly stained material, which indicates a subdivision into particles within the lightly stained material in a direction parallel to the rows of particles. This material could
FIG. 13. Higher magnification of the Golgi region in Fig. 12 showing transversally (A) and tangentially (B) sectioned membranes, presumably Golgi membranes, with a regular globular substructure. × 280,000.
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I~RITIOF S. SJ()STRAND AND LARS-G. ELFVIN
either form complete septa between individual globular elements which then might be extremely thin, or it could be confined to the surfaces of the membrane elements filling the grooves between adjacent globules. The low opacity of this material makes it unlikely, however, that the globular structure will be observed very frequently in cross sections of membranes. The diameter of the globular components is small in comparison to the thickness of the sections. Many globular components will therefore be superimposed and contribute to a general low opacity of the middle part of the membrane elements. Only when the globules are perfectly lined up parallel to the electron beam will the globular structure appear in cross sections in spite of the superposition effect. Interpretation of positive staining. The interpretation of what the intensely stained spots represent is rather difficult and reflects a basic problem Of image interpretation when dealing with very high resolution pictures of positively stained material. The positive staining efficiently increases the contrast because the staining gives rise to localized regions of high concentration of heavy metal-containing molecules. From the high scattering power of the stained spots it appears likely that they represent clusters of stain molecules in a compact arrangement with no or very little organic material mixed with the stain molecules within these clusters. Such cluster or stain particle formation is very obvious when lead staining methods are used. The stain particles then frequently prevent the achievement of very high specimen resolution due to their coarseness. Uranyl acetate proves to. be a more favorable stain in this situation. The clusters of stain molecules representing the very opaque dots certainly reflect the location of reactive sites in the membranes and could reflect an accumulation of such sites at the places where the opaque spots are found. The spots could, however, also represent mass precipitation of the stain in connection with its interaction with such reactive sites. This seems a reasonable explanation particularly for the very large stain particles observed after lead staining. In this latter case the size of the stained spots does not necessarily reflect accurately the density, extension, and precise location of reactive sites. The size of the stain particles presumably depends on factors affecting stain precipitation, and this precipitation could be asymmetric in relation to the active sites triggering the precipitation. The size of the intensely stained spots therefore might reflect both the concentration of reactive sites in the membranes and the tendency of the stain ' molecules to aggregate into particles of high density. It seems questionable, however, whether the size of these stained spots reflects dimensions of the molecules that are stained, but much more likely that the spots correspond only to a particular part of the molecules F~. 14. Higher magnification of part of Fig. 13. × 600,000.
MITOCHONDRIAL MEMBRANES AND CYTOMEMBRANES
5o~
289
Jsoh
K M n O4
FREEZE-DRYING
FIG. 16. Schematic drawings to illustrate the relationship between the patterns of mitochondrial membranes after permanganate fixation with section staining (left) and after freeze-drying (right) (A) and the relationship between the classical osmium picture of (B) e-cytomembranes, (C) sectionstained osmium-fixed membranes, (D) frozen-dried c~-cytomembranes.
FIG. 15. Another example of membrane patterns in what was tentatively identified as a Golgi region. x 400,000. 1 9 - 641834 J . Ultrastructure t~esearch
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FRITIOF S. SJ(JSTRAND AND LARS-G. ELFVIN
which can interact intensely with the stain, the actual size of the molecules exceeding that of the stained spots. The reality of the observed structural pattern. The fact that a globular substructure could be observed both in chemically fixed and frozen-dried material, and that the dimensions of these structural components are similar, can be used as a rather strong argument in favor of this structure reflecting a real structural organization of the mitochondrial membrane elements and of cytomembranes. The regularity of the patterns observed after freeze-drying also appears more consistent with a real structure than an artifact. The lack of this regular geometry in chemically fixed material, on the other hand, can easily be accepted as an artifact due to partial disorganization of the globular structure. The clear-cut staining of cross bridges in potassium permanganate-fixed material in contrast to frozen-dried and osmium-fixed material can be accounted for by particularly favorable staining conditions in the former case. The fact that it is easier to preserve a simple triple-layered structure than a globular structure when using chemical fixation methods might indicate that the globular structure is labile and easily transformed into such a layered structure. This is a reasonable assumption since it would mean that the surface energy of the system would be reduced from the high energy state of the globular structure to a lower energy state of a layered structure. Interpretation of pattern in terms of molecular architecture. Since the structural components that have been observed are in the molecular range of dimensions, it seems pertinent to try to interpret the observed patterns in terms of molecular architecture. In previous papers (15, 16) it was proposed that the light globular components of the chemically fixed membrane elements represents the lipids of these elements and that the stained material corresponds to proteins and possibly polar ends of the lipid molecules, according to an interpretation presented by SjSstrand (11-14) and supported by the study of model systems by Stoeckenius (19, 20). This interpretation can be applied to the present material as well. From the above discussion it seems justifiable to propose that the protein molecules are arranged in rows and that the major part of the protein is located in the furrows between the rows of globular lipid mieelles. We would then be dealing with rows or chains of protein molecules stabilized in location by the lipid components. In the mitochondrial membrane elements, these chains could correspond to a linear arrangement of the respiratory enzymes. The elementary structure of the mitochondrial membrane elements would therefore not be a particle but a kind of weaving element where each respiratory chain shares lipid components with neighboring chains. The ribosomal material shows a rather different morphology in frozen-dried material as compared to osmium-fixed tissue (Figs. 16 B-D). The delimiting of patti-
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291
cular elements is impossible in ceils where there has not occurred a certain amount of dehydration and shrinkage of the cytoplasm in connection with freezing, as first pointed out by Sj/3strand and Baker (17). These observations were confirmed and extended by Hanzon et al. (4, 5). If dehydration has taken place as a result of ice crystal formation, the R N A protein appears subdivided into rounded, diffusely outlined "particles," which, however, are in mutual contact and form continuous strands or sheets of ribosomal material. The volume of these "particles" exceeds that of the ribosomes observed in chemically fixed material, decreasing with increasing shrinkage of the cytoplasm in connection with the freezing associated with ice crystal formation. Hanzon and Hermodsson (4) showed that particles were clearly visible in badly preserved frozen-dried material. This appearance of the particles can be correlated to ice-crystal formation causing dehydration of the cytoplasm. The results reported by Seno and Yoshizawa (8-10), who demonstrated ribosomal particles in frozendried material, can be explained in this way. It seems justifiable to interpret these observations to show that the compact particulate form of ribosomal material as observed in sections of osmium-fixed tissue is an artifact and that this material appears more uniformly distributed over the surface of the 0~-cytomembranes in the living cell than is apparent from the analysis of chemically fixed material, in agreement with the conclusions of Sj~Sstrand and Baker (17) and Hanzon et al. (4, 5). In several papers where freeze substitution has been applied for fixation (3, 6) a structural picture of the cytoplasm similar to that of osmium-fixed material has been presented. It is questionable whether the material in these studies really has been dried in a frozen state by substitution. More likely, it seems that the fixation has involved freeze-thawing. As has been shown by Baker (1), the thawing in an osmium tetroxide solution of frozen tissue results in a reconstitution of an appearance of the cytoplasmic components identical to what is observed after fixation of fresh tissue in osmium tetroxide. This is the case even under conditions where extensive ice crystal formation has taken place in the cells before thawing. The advantage of freeze-drying by means of molecular distillation is that we can ascertain that the tissue has been dried before exposure to the embedding medium, eliminating the risks of any thawing-reconstitution of the cells involving the disappearance of artifacts due to ice crystal formation. Such a reconstitution means that the tissue, in fact, has not been fixed by a physical method of freeze-drying but by a chemical method, the preceding freezing and thawing only changing the conditions for chemical fixation. The possibility that the ribosomal RNA is arranged as a more or less continuous carpet covering the membrane elements of the c~-cytomembranes appears important in connection with the interaction between messenger R N A and ribosomal RNA.
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A discontinuous system of ribosomal particles would m e a n that the messenger R N A would have to move over rows of such particles in connection with protein synthesis to allow successive base groups to interact with ribosomes and transfer R N A . ' A n open spread-out configuration of the ribosomal R N A in a continuous carpetlike structure could minimize the requirements for repositioning of messenger R N A in connection with protein synthesis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20.
BAKER, R. F., J. Ultrastructure. Res. 7, 173 (1962). BROD¥, I., J. Ultrastruct. Res. 2, 482 (1959). BULLIVANT,S., J. Biophys. Biochem. CytoI. 8, 639 (1960). HANZON, V. and HERraODSSON,L. H., Y. Uhrastruct. Res. 4, 332 (1960). HANZON, V., HERMODSSON, L. H. and T o s c ~ , G., Y. Ultrastruct. Res. 3, 216 (1959). REBHUN, L. I., Y. Biophys. Biochem. Cytol. 9, 785 (1961). ROBERTSON,J. D., Biochem. Soe. Symp. (Cambridge, EngI.) 16, 3 (1959). SENO, S. and YOSHIZAWA,K., Saibo Kagaku Shimpoziumu 8, 29 (1958). -Naturwissensehaften 46, 19 (1959). -J. Biophys. Biochem. Cytol. 8, 617 (1960). SJ6STRAND, F. S., J. Cellular Comp. Physiol. 42, 15 (1953). -Nature 171, 30 (1953). -ibid. 171, 31 (1953). -Verhandl. Anat. Ges. 53. Versamml., Stockholm, 1956, p. 3. Gustav Fischer Verlag, Jena, 1957. -Nature 199, 1262 (1963). -J. Ultrastruct. Res. 9, 340 (1963). SJOSTRAND,F. S. and BAKER,R. F:, J. Ultrastruct. Res. 1, 239 (1958). SJI~STRAND,F. S. and ELEVIN, L.-G., J. Uttrastruct. Res. 7, 504 (1962). STOECKENtUS,W., J. Biophys. Biochem. CytoI. 5, 491 (1959). -Proc. European Regional Conf. Electron Microscopy, Delft, 1960, Vol. 2, p. 716 (1961).
263
The G r o n u l a r S t r u c t u r e of M i t o c h o n d r i a l / V l e m b r a n e s and of
CytomembranesAs
D e m o n s t r a t e d in F r o z e n - D r i e d Tissue 1
FRITIOF S. SJOSTRANDAND LARS-G. ELFVlN
Department of Zoology, University of California, Los Angeles Received November 19, 1963 A regular crystal-like arrangement of what appears as globular particles was observed in face-on views of mitochondrial membrane elements, c~-cytomembranes, and presumably Golgi membranes. The globular particles were arranged in rows and appeared only lightly stained. Between these rows, strands of intensely stained material were present. In cross sections the mitochondrial membrane elements and the cytomembranes appeared as light lines reflecting a close packing of the lightly stained globular particles. The distance between the centers of the rows of particles seen in face-on views was 45-50 ~; the thickness of the light lines representing cross sections of the membranes was 40 ~. These patterns are interpreted to support the observations made on chemically fixed material (15, 16), that is, to reflect a globular substructure consisting of the membrane lipids in a micellar arrangement stabilized by proteins. The ribosomal material forms an almost complete carpet covering the membrane elements of the ~-cytomembranes. A subdivision into particles can be correlated to a high degree of dehydration of the cytoplasm due to ice crystal formation during freezing. A globular structure was demonstrated by SjSstrand (I5, 16) in mitochondrial membranes and in smooth-surfaced cytomembranes in the tubular cells of the mouse kidney after potassium permanganate fixation. Indications of a similar subdivision of these membranes into globular structural units were also found in osmium-fixed material. Since the reliability of those observations made on chemically fixed material was considered doubtful (16), it appeared important to try to reproduce them in frozen-dried material after very rapid freezing of small pieces of tissue. Mouse pancreas tissue was selected for this purpose since the cytoplasm of the exocrine pancreas cells is filled with membranous components and is structurally rather simple. The identification of cytoplasmic structural components in sections of frozen-dried material is therefore facilitated. This is important since the contrast in electron micrographs of frozen-dried material is low and the structural patterns z This research was supported by Grant No. NSF G-23392 of the National Science Foundation.
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FRITIOF S. SJOSTRAND AND LARS-G, ELFVIN
differ from those of chemically fixed material. The differences are due to the fact that in frozen-dried tissue the more or less extensive extraction of material is eliminated, particularly when molecular distillation in v a c u u m is used as the only method for r e m o v i n g the water. A globular structure was observed in m i t o c h o n d r i a l m e m b r a n e elements, in ~-cytom e m b r a n e s , a n d in Golgi m e m b r a n e s in this material with the globular structural units showing similar dimensions to those observed in potassium p e r m a n g a n a t e fixed material. The globular c o m p o n e n t s , however, showed a n ordered arrangement frequently a p p e a r i n g with a crystal-like regularity in the frozen-dried material.
MATERIALS A N D METHODS White mice weighing about 20 g were decapitated; pieces of the pancreas were removed, cut into minute pieces with a razor blade knife, and immersed in liquid nitrogen or in isopentane chilled by means of liquid nitrogen. The tissue was frozen within 60 seconds after decapitation of the experimental animal. When liquid nitrogen was used as medium for freezing, its temperature had been lowered by vacuum evaporation to the freezing point of nitrogen. A solid phase"of nitrogen developed and filled most of the Dewar flask, with only a thin layer of liquid nitrogen on top of the frozen nitrogen. The temperature of the liquid phase was sufficiently low to prevent a rise in temperature to the boiling point of nitrogen when the small pieces of tissue were immersed. This means that no layer of nitrogen gas was formed around the tissue which would act as a thermal insulation slowing down the rate of freezing. The frozen tissue was dried in a freeze-drying apparatus designed by Sj6strand and consisting of a glass vacuum chamber, the drying chamber, connected to an oil diffusion pump by means of a wide-bore brass tube. A Welch mechanical pump was used to back the diffusion pump. A wide-bore valve on top of the diffusion pump allowed closing of the connection between vacuum chamber and diffusion pump. A side tube with a valve made it possible to connect the vacuum chamber with a glass container in which the embedding medium could be kept under vacuum. The embedding medium could be transferred to the vacuum chamber with the specimens by means of vacuum distillation. A cold finger reached from the top of the vacuum chamber down to a few centimeters from its bottom where the specimens were placed. It was kept filled with liquid nitrogen during the drying procedure. The lower part of the vacuum chamber was surrounded by a metal Dewar flask standing on the bottom of a dry-ice chest filled with dry ice. The metal Dewar flask was filled with liquid nitrogen when the specimens, submerged in liquid nitrogen, were transferred to the drying chamber and the evacuation of the freeze-drying apparatus started. The drying under vacuum was performed at a slowly rising temperature from the temperaFro. 1. Survey picture of cytoplasm of frozen-dried exocrine pancreas cell. At the bottom i s a mitochondrion (Mi). ~-Cytomembranes (e) with ribosomal material above the mitochondrion are mostly oriented perpendicular to the plane of the section and appear therefore as light lines representing cross sections of the membranes, x 130,000.
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FRITIOF S. SJ~JSTRAND AND LARS-G. ELFVIN
ture of liquid nitrogen to that of dry ice, and extended over several days. The temperature was then allowed to rise slowly from dry-ice temperature to room temperature. Before embedding in Vestopal, the tissue was exposed for several hours to osmium tetroxide vapor either in a desiccator or under vacuum in a Thunberg tube used for the embedding. The embedding in Vestopal was performed under vacuum in a modified Thunberg tube allowing a thorough evacuation of air dissolved in the Vestopal before immersion of the tissue into the embedding medium. The tissue was then submerged in Vestopal under vacuum by tilting the Thunberg tube. The dried tissue was kept in the top curved part of the Thunberg tube, separated from the Vestopal, while the air was evacuated from the Vestopal. The Vestopal was allowed to set at 60°C, and sections from the hardened blocks were cut on an LKB Ultrotome. The sections were examined in a Siemens Elmiskop I at 40,000 times electron optical magnification at 80 kV with a 50-~ objective aperture and careful compensation of objective lens astigmatism to less than one step on the fine focus control. The contrast of the tissue was enhanced by means of staining in a saturated uranyl acetate solution at 60°C, according to Brody (2). RESULTS The f r o z e n - d r i e d p a n c r e a s tissue (Fig. 1) shows a structural p a t t e r n which differs very m u c h f r o m the c o n v e n t i o n a l p a t t e r n s o b s e r v e d in chemically fixed material, The g r o u n d substance of the c y t o p l a s m in f r o z e n - d r i e d m a t e r i a l contains components which are absent, owing to extraction, in chemically fixed m a t e r i a l subjected to a l c o h o l o r acetone d e h y d r a t i o n . F u r t h e r m o r e , a u n i f o r m d i s t r i b u t i o n of material m a k e s the g r o u n d substance a p p e a r relatively intensely stained in frozen-dried material as c o m p a r e d with the a l m o s t e m p t y a p p e a r a n c e in chemically fixed tissues. The c y t o p l a s m also becomes c o n c e n t r a t e d in c o n n e c t i o n with various degrees of dehyd r a t i o n due to the f o r m a t i o n of ice crystals p r e c e d i n g the freezing of the entire cells. This p h a s e s e p a r a t i o n c o n t r i b u t e s to a n increase in c o n c e n t r a t i o n of stainable material in the still liquid phase of the c y t o p l a s m until c o m p l e t e freezing has been achieved. A characteristic feature of sections t h r o u g h f r o z e n - d r i e d m a t e r i a l is t h a t cross sections of various types of m e m b r a n e s a p p e a r as light lines against a d a r k backg r o u n d (Fig. 1). This p a t t e r n was originally o b s e r v e d b y Sj6strand a n d Baker (17) a n d described as a negative p a t t e r n . The a-cytomembranes a p p e a r e d in cross sections as light lines with an average thickness of 40 A (Table I). They were a r r a n g e d in pairs, each m e m b r a n e pair deliFIG. 2. Cytoplasm of frozen-dried exocrine pancreas cell with g-cytomembranes of different orientations. In the lower part of the picture the c~-cytomembranes are cross-sectioned; in the upper part of the picture they are cut more or less tangentially. The ribosomal material forms a layer associated with the cross-sectioned membranes in the former case (A) and irregularly forms arranged opaque regions in the latter case (B). A granular pattern is observed in the tangentially sectioned c~-cytomembranes. × 220,000.
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FRITIOF S. SJ~JSTRAND AND LARS-G. ELFVIN
miring a space which varied considerably in width between 50 and 200 • or more. This space was filled with opaque material that appeared homogeneous (Figs. t-4). The e-cytomembranes were easily identified by their association with ribosomal material. This material appeared as a continuous opaque layer coating one surface of the membrane element of the ~-cytomembranes. Frequently this coating showed an indication of a division into subunits by the presence of fairly regularly distributed but diffusely outlined areas which were particularly intensely stained (Figs. t, 3, and 4). This indication of a subdivision of the ribosomal material is interpreted to correspond to ribosomal particles as observed in osmium-fixed material. The variation in the opacity in the layer of ribosomal material did not make this material appear completely subdivided into discrete particles separated by spaces free from ribosomal material. On the contrary, the ribosomal material appeared as a continuous layer with slightly varying opacity. Exceptions to this rule could be observed in cases where shrinkage of the cytoplasm, due to dehydration in connection with excessive ice crystal formation inside the cell, had taken place (Fig. 5). The more opaque regions of the ribosomal material were frequently located at shallow depressions in the membrane surface (Fig. 4). In membranes that were oriented more or less parallel to the plane of the section, the ribosomal material formed an almost complete continuous carpet with the more opaque regions vaguely indicated (Fig. 2). It was uncertain whether any regular pattern of rows of opaque regions could be seen in this carpet. The width of the depressions in the membrane surface where the more opaque regions of the ribosomal material were located was about 200 A; the thickness of the layer of ribosomal material at the membrane surface was 150 A. In the cases where the ribosomal material showed fairly uniformly distributed more opaque regions in a continuous layer of slightly less opaque material, it was possible to measure in a direction parallel to the membrane surface the average distance between the centers of these opaque regions presumably representing the centers of ribosomes. This was done by punching minute holes in prints by means of a needle through the centers of the most opaque regions and measuring the distances between the holes on the backs of the prints. The average distance between the opaque centers along the surface of the membranes was about 200 A, which is identical to the width of the depressions in the ~-cytomembranes.
FIG. 3. c~-Cytomembranesof different orientations in relation to the plane of the section. Crosssectioned membranes (A) with continuous layer of ribosomal material appear as light lines. Tangentially sectioned membranes (B) show a granular structure and irregularly arranged ribosomal material, x 380,000.
i i i i
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FRITIOF S. SJ()STRAND AND LARS-G. ELFVIN
In the case mentioned above where discrete ribosomes could be seen, they showed a circular cross section and measured 150-200 A in diameter. The average diameter of these ribosomes measured parallel to the plane of the membranes was therefore, in this case, slightly smaller than in the majority of the pictures. A substructure in the membrane element of the o~-cytomembranes showed up very clearly when these membranes were oriented more or less parallel to the plane of the section (Figs. 2, 3, 6-9). A gradual transition from an orientation perpendicular to the plane of the section to one more or less oblique can frequently be seen, as in Figs. 2 and 4. With increasing tilt of the membrane, more and more of a characteristic, regular structural pattern becomes visible. It can be described as consisting of rows of lightly stained dots separated by more intensely stained irregularly dotted lines. Lightly stained cross bridges extend between adjacent dotted lines and make the light area between these lines appear subdivided into a row of light globules. The rows of dots could form different patterns with either several parallel lines running fairly straight (Fig. 6) or curved (Fig. 3). The regularity of the patterns also varied considerably, a variation that can be due either to superposition of similar patterns in two membranes on top of each other or to deformation of the pattern through the preparatory procedure. The average spacing of the lines in these patterns when exhibiting a high degree of regularity was strikingly constant and varied in different pictures within a narrow range of 45-50 A. The size of the light dots was about 40 A; the opaque dots between the light ones had a minimum diameter within the range of 12-25 A. They could appear asymmetric and rod shaped. The length of these elongate opaque dots varied between 30 and 50 A. Their long axis could be oriented at various angles in relation to the direction of the row of dots (Figs. 6-9). This asymmetry was not due to distortion through astigmatism or specimen drift. The structure of the mitochondrial membranes. The mitochondria showed a rather opaque matrix against which the mitochondrial membranes appeared as two parallel light lines separated by opaque material (Figs. 1, 10, 11). Each light line corresponded to one membrane element of the mitochondrial membrane. It was striking that the separation of the two light lines was very constant. The opacity of the matrix was uniform and there was no indication of any opaque layer at the boundary between the matrix and the mitochondrial membranes in cross sections of the membranes. This is in contrast to the picture of, for instance, osmium-fixed mitochondria (16) Where the mitochondrial membrane elements face the matrix with an opaque layer.
FIG. 4. Higher magnification of part of Fig. 1 showing partially cross-sectioned c~-cytomembranes and partially obliquely sectioned regions with a granular structure (arrows). (Mi, mitochondrion). x 240,000.
J !
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FRITIOF S. SJOSTRAND AND LARS-G. ELFVIN
FIG. 5. e-Cytomembranes in exocrine pancreas cell where the cytoplasm has been extensively dehydrated due to intracellular ice crystal formation. Ice crystal vacuoles are marked IV. Ribosomal particles can be observed associated with the ~-cytomembranes. × 230,000.
The total thickness of the mitochondrial membranes was 115 ~ (Table II). Each light line measured 30-40 A in thickness (Table I). The average width of the opaque space separating the two light lines was 30-35 A (Table 1I). At high magnification the light lines could show an indication of a globular substructure (Fig. 11). When the outer mitochondrial membrane was properly oriented, it showed the same structural pattern as the inner mitochondrial membranes. When the mitochondrial membranes were oriented obliquely or parallel to the plane of the section, a structural pattern similar to that of the c~-cytomembranes was observed (Figs. 10 and 11). Rows of opaque particles were separated by light spaces. These light spaces appeared as rows of lightly stained or unstained globular subunits. The spacing between the rows of particles was identical to that observed for the ~-cytomembranes. The light dots measured about 30 A in diameter and the opaque dots 12--15 ~ in diameter when measured perpendicular to the length of the rows of dots. The resolution as determined by the minimum separation of object points in two consecutive pictures was estimated to about 15 A.
MITOCHONDRIAL MEMBRANESAND CYTOMEMBRANES
273
TABLE I THE THICKNESS OF THE LIGHT LAYER OF CROSS-SECTIONED MITOCHONDRIAL MEMBRANE ]~LEMENTS AND OF ~-CYTOMEMBRANES a Mitochondrial Membrane Elements
¢~-Cytomembranes
43 42 38 34 34 41 29 M = 37 ~
49 35 38 38 43 35 45 M=40
a Each figure represents the mean value from measurements on 10 membranes with one measurement made at a randomly chosen point on each membrane. TABLE II TOTAL THICKNESS OF MITOCHONDRIAL MEMBRANES AND OF OPAQUE MIDDLE LAYERa" Total Thickness (A)
Opaque Middle Layer (N)
118 105 128
33 31 33
M=l17~
M-32~
Each figure represents the mean value from measurements on 10 membranes with one measurement made at a randomly chosen point on each membrane.
The Golgi membranes. W i t h i n certain regions particularly close to the z y m o g e n granules the cytoplasm appeared free f r o m r i b o s o m a l material (Fig. 12). Also i n these regions light lines a n d the regular g r a n u l a r patterns described above could be observed. Since the cytoplasm of the exocrine pancreas cells does n o t show any regions of these dimensions free from m i t o c h o n d r i a or cytomembranes, either of the a-type or of the Golgi type, it was assumed that they represented the site of the Golgi apparatus. This conclusion was supported by the characteristic localization of these regions in the cell which corresponded to that of the Golgi apparatus. FIGS. 6 and 7. Some e-cytomembranes oriented partially more or less parallel to the plane of the section showing a granular substructure. Fig. 6 is slightly underfocused and Fig. 7 close to focus. x 600,000. 18--641834 J . Ultrast~'uctu~e JResearcl~
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FRITIOF S. SJOSTRAND AND LARS-F. ELFVIN
The structural patterns observed in these regions were therefore tentatively interpreted to reveal the structure of Golgi membranes. These patterns are similar to those described for c~-cytomembranes and mitochondrial membrane elements but frequently are particularly regular and distinct (Figs. 13-15). The spacing of the rows of particles was more frequently 50 fit than in the cases of mitochondrial membrane elements and c~-cytomembranes, but this material did not allow a statistical evaluation of this difference. DISCUSSION
The "negative" pattern observed in frozen-dried material was first described by Sj6strand and Baker (17) in frozen-dried mouse pancreas. It is a negative pattern only in appearance according to the information regarding membrane structure that has been collected since that time. The conclusion that the pattern was negative was based on an identification of the lightly stained or unstained lines representing cross sections of membranes in frozen-dried tissue as corresponding to the single (about 50 A-wide) opaque lines representing the cross sections of membranes or membrane elements in osmium-fixed material studied at that time. Since then it has been shown that both after potassium permanganate fixation (7) and osmium fixation combined with section staining (16) the membranes or membrane elements appear triplelayered and that in the case of mitochondrial and cytoplasmic membranes the triplelayered pattern corresponds in position to the single, 50-60 A-thick osmiophilic structure observed in osmium-fixed material studied earlier which had not been section stained. This is in contrast to the situation with respect to the plasma membrane where the osmiophilic structure described earlier represents only one layer (its cytoplasmic layer) of the asymmetric triple-layered plasma membrane (18). After potassium permanganate fixation and section staining, the triple-layered pattern of mitochondrial membrane elements and of cytomembranes appears to be complicated by the presence of stained cross bridges extending between the two opaque layers at the surface of the membranes (15, 16). The presence of these cross bridges has not been clearly demonstrated in osmium-fixed material but is sometimes vaguely indicated. This could mean that they are not as distinctly and easily stained after osmium fixation as the two opaque surface layers or that they are not well preserved after this fixation. A diffuse staining of this material could explain the fact that the triple-layered structure of the membrane elements of mitochondria and of FIGS. 8 and 9. Face-on view of e-cytomembrane showing the regular substructure. The size and shape of the opaque particles can be observed and the specimen resolution estimated to 15 K or better in these pictures. Fig. 8 is slightly underfocused and Fig. 9 close to focus. Within encircled areas a point resolution of 15 A can be observed in both pictures, x 1,000,000.
i
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FRITIOF S. SJOSTRAND AND LARS-G. ELFVIN
the cytomembranes regularly appeared as a single osmiophilic layer in earlier studies before the application of efficient section-staining methods. The thickness of the single osmiophilic layer in cytoplasmic membranes observed earlier, in contrast to the situation in the plasma membrane, does correspond to the total thickness of the triple-layered counterpart in section-stained, osmium-fixed or in unstained potassium permanganate-fixed material. It is therefore likely that the single osmiophilic layer pattern depends upon the presence of osmiophilic material in the middle of the membrane elements which corresponds to the cross bridges observed in potassium permanganate section-stained material but which is not discretely enough stained to be resolved. If we start out with these considerations we arrive at an alternative interpretation of the structural patterns observed in frozen-dried material according to which the observed structural patterns are only seemingly negative patterns. This new interpretation recognizes the light lines observed in frozen-dried material as corresponding to the middle part of the membrane elements and of the cytomembranes which contain less stainable material than the two opaque surface layers of these components (Fig. 16 A). This interpretation is supported by the structural pattern of mitochondrial membranes. This pattern can be superimposed onto the five-layered pattern of these membranes frequently observed after potassium permanganate fixation in cases where the cross bridges in the membrane elements are not revealed or after osmium fixation when the two membrane elements of the mitochondrial membranes are closely packed. The thickness of the opaque middle layer of the mitochondrial membranes observed in frozen-dried material corresponds to the thickness of the opaque middle layer of the five-layered pattern observed in the chemically fixed material. The thickness of the light layers in frozen-dried material (30-40 A) corresponds fairly closely to that of the light middle layer of chemically fixed material (20-30 A). A discrepancy between these dimensions can be explained either by some material remaining unstained after freeze-drying preservation and section staining or by a partial disorganization of the structure in connection with chemical fixation with partial collapsing of the light layer. A partial disorganization could release reactive groups of the involved molecules for interaction with the stain. In the mitochondrial membranes we could in this way account for one opaque surface layer of the membrane elements as contained in the middle opaque line in the observed triple-layered pattern of cross-sectioned mitochondrial membranes. FIG. 10. Mitochondrion (Mi) with cross-sectioned and tangentially sectioned membranes. The outer mitochondrial membrane along the upper contour of the mitochondrion (A) is tangentially sectioned and shows a regular substructure. At B, inner mitochondrial membrane is viewed face on and shows a similar structural pattern, x 310,000.
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FRITIOF S. SJOSTRAND AND LARS-G. ELFVIN
We can also account for the light middle layer of the triple-layered membrane elements, but we observe no opaque layer delimiting the mitochondrial membranes at the surface facing the mitochondrial matrix as we do in chemically fixed material. This circumstance can be explained by the fact that the matrix is intensely stained in the frozen-dried material showing the same opacity as that, for instance, of the middle opaque layer of the mitochondrial membranes, that is, of the two opaque surface layers of the membrane elements which have fused in the middle of the mitochondrial membranes. If an opaque layer exists at the matrix side of the membrane elements, the presence of this layer therefore could be masked by the identical opacity of the dense matrix. In chemically fixed material the matrix shows a low opacity, presumably due to extraction of matrix material during the fixation and embedding procedure. Any stained layer at the matrix surface of the membrane elements will therefore show up easily against the lightly stained matrix. It should also be kept in mind that the affinity of the various structural components to stains is very much weaker in frozen-dried than in chemically fixed material. This makes it more difficult to differentiate structural components due to a considerably lower contrast of the pictures of frozen-dried material. Identification of membranous components. The conclusion that the light fines observed in frozen-dried material indicate cross sections of membranes appears rather obvious both in the case of mitochondrial membranes and ~-cytomembranes. In the latter case the ribosomal material represents a reliable label for the membranes. The identification of Golgi membranes is based On the absence of ribosomal material and the knowledge that no extensive cytoplasmic membranous material other than e-cytomembranes and Golgi membranes exists in the exocrine pancreas cells. With respect to the structural patterns that have been referred to as membranous components oriented more or less parallel to the plane of the sections, we usually do not have an identical counterpart in chemically fixed material where seldom a structure is seen in tangentially sectioned membranes. Potassium permanganatefixed material is an exception since in this case a globular pattern could be observed in tangentially sectioned membranes. The globular structure did not, however, exhibit the same regular geometry as the patterns observed in the present material. In order to use the observations made on frozen-dried material as an argument that the pattern in potassium permanganate-fixed material might reflect a real and not an artifactitious structure, we must be able to prove that these patterns refer to the same structural components. We cannot use, therefore, the similarity of the patterns
FIG. 1!. Same mitochondrion as in Fig. 10 in a close-to-focus picture showing indications of globular structure of cross sections of membrane elements (arrows). The size of the opaque particles in the globular substructure of the mitochondrial membrane elements can also be observed, x 600,000.
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FRITIOF S. SJ(JSTRAND AND LARS-G. ELFVIN
as a means of identifying the components showing these patterns in the frozen-dried material. The association of the observed globular patterns with mitochondrial membrane elements and with cytomembranes is based on the fact that these patterns are observed only in places where cross sections of membranes are absent. The ribosomal material assists in indicating where the ~-cytomembranes are located and where they are oriented more or less parallel to the plane of the sections, by showing a rather random arrangement or by forming a large opaque carpet instead of being arranged in even rows of opaque regions in uniformly thick layers. The globular structural patterns referred to as representing face-on views of the ~-cytomembranes have been observed in regions where ribosomal material was present and did not show a layered arrangement but a more r a n d o m distribution in carpets. In the mitochondria the inner membranes are so regularly arranged all through their interior that any region free of cross-sectioned mitochondrial membranes must contain such membranes oriented more or less parallel to the plane of the section. The globular patterns were observed in such regions. The identification of Golgi membranes oriented obliquely or parallel to the plane of the section was based on the absence in those regions of ribosomal material and the vicinity to zymogen granules. No spaces of the dimensions of these regions are free from cytomembranes in the exocrine pancreas cells. The observed patterns therefore are most likely related to cytomembranes. Direct transition from a light line pattern representing cross sections of membranes to the globular patterns could also be observed linking both patterns to the membrane. Regions lacking any patterns of either cross-sectioned or tangentially sectioned membranes were frequently found in mitochondria. This can be explained by the fact that the mitochondrial membranes consist of two tightly packed membrane elements as can be seen in cross sections of the membranes. The total thickness of this two-element structure is 100-150 A. This means that there is a high probability of both membrane elements being contained in the section even when oriented parallel to the plane of the section since the minimum thickness of these sections of frozendried material certainly is not less than 200 A. The globular structural patterns of both membrane elements will therefore be superimposed and if not in perfect register the resulting image pattern will be that of more or less randomly arranged dots. In the case of the ~-cytomembranes, on the other hand, a sufficiently wide space
FIG. 12. Survey picture of cytoplasm of exocrine pancreas cell showing c~-cytomembranesin lower part and zymogen granules (Z) in the upper part of the picture. The zone in between is interpreted to correspond to the location of Golgi apparatus (G) because it lacks ribosomal material. × 140,000.
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separates the two membranes of each membrane pair to introduce a high probability that only one membrane will be present in a tangential section to contribute to the observed pattern. Geometric interpretation of face-on views of membranes. Structural patterns associated with face-on views of the mitochondrial membrane elements and the cytomembranes are interpreted to reflect a rather regular linear arrangement of globular structural units represented by the less intensely stained parts of the patterns. These rows of globular particles appear discretely outlined due to the presence of intensely stained material associated with the globules and concentrated primarily between these rows. Stained material also extends across the rows at regular intervals, delimiting the individual globules within each row of globules. T h e globules appear arranged in a square packing when showing the regular patterns. Modifications in the arrangement of the opaque material can be accounted for by different degrees of tilt of the membranes in relation to the plane of the section. The observed patterns are in principle identical irrespective of whether the pictures were taken very close to focus or were slightly underfocused. This means that the patterns are neither distorted by diffraction effects nor due to such effects. The opaque material is assumed to be concentrated preferentially at the two surfaces of the membrane elements since these elements appear as light lines in cross sections. The lightly stained globular components are therefore assumed to be more or less closely packed. The distance between the centers of the globular units is similar in a direction parallel and perpendicular to the row, and this distance corresponds closely to the thickness of the lightly stained part of the membranes when observed in cross sections (40 A). This dimension is considered to reflect the diameter of the globular components, 40-50 A. An indication of a globular structure can also be observed in cross sections of the membranes in close-toCfocus pictures, although this is not always the case. If the opaque material present between the rows of lightly stained material in the face-on views of the membrane elements extended across the entire thickness of the membranes, we would expect to see more indications of opaque cross bridges in the cross sections of the membranes or these cross sections would not appear as lightly stained as they do. It is clear, however, from the face-on view of the membrane elements that there is faintly stained material, which indicates a subdivision into particles within the lightly stained material in a direction parallel to the rows of particles. This material could
FIG. 13. Higher magnification of the Golgi region in Fig. 12 showing transversally (A) and tangentially (B) sectioned membranes, presumably Golgi membranes, with a regular globular substructure. × 280,000.
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either form complete septa between individual globular elements which then might be extremely thin, or it could be confined to the surfaces of the membrane elements filling the grooves between adjacent globules. The low opacity of this material makes it unlikely, however, that the globular structure will be observed very frequently in cross sections of membranes. The diameter of the globular components is small in comparison to the thickness of the sections. Many globular components will therefore be superimposed and contribute to a general low opacity of the middle part of the membrane elements. Only when the globules are perfectly lined up parallel to the electron beam will the globular structure appear in cross sections in spite of the superposition effect. Interpretation of positive staining. The interpretation of what the intensely stained spots represent is rather difficult and reflects a basic problem Of image interpretation when dealing with very high resolution pictures of positively stained material. The positive staining efficiently increases the contrast because the staining gives rise to localized regions of high concentration of heavy metal-containing molecules. From the high scattering power of the stained spots it appears likely that they represent clusters of stain molecules in a compact arrangement with no or very little organic material mixed with the stain molecules within these clusters. Such cluster or stain particle formation is very obvious when lead staining methods are used. The stain particles then frequently prevent the achievement of very high specimen resolution due to their coarseness. Uranyl acetate proves to. be a more favorable stain in this situation. The clusters of stain molecules representing the very opaque dots certainly reflect the location of reactive sites in the membranes and could reflect an accumulation of such sites at the places where the opaque spots are found. The spots could, however, also represent mass precipitation of the stain in connection with its interaction with such reactive sites. This seems a reasonable explanation particularly for the very large stain particles observed after lead staining. In this latter case the size of the stained spots does not necessarily reflect accurately the density, extension, and precise location of reactive sites. The size of the stain particles presumably depends on factors affecting stain precipitation, and this precipitation could be asymmetric in relation to the active sites triggering the precipitation. The size of the intensely stained spots therefore might reflect both the concentration of reactive sites in the membranes and the tendency of the stain ' molecules to aggregate into particles of high density. It seems questionable, however, whether the size of these stained spots reflects dimensions of the molecules that are stained, but much more likely that the spots correspond only to a particular part of the molecules F~. 14. Higher magnification of part of Fig. 13. × 600,000.
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Jsoh
K M n O4
FREEZE-DRYING
FIG. 16. Schematic drawings to illustrate the relationship between the patterns of mitochondrial membranes after permanganate fixation with section staining (left) and after freeze-drying (right) (A) and the relationship between the classical osmium picture of (B) e-cytomembranes, (C) sectionstained osmium-fixed membranes, (D) frozen-dried c~-cytomembranes.
FIG. 15. Another example of membrane patterns in what was tentatively identified as a Golgi region. x 400,000. 1 9 - 641834 J . Ultrastructure t~esearch
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FRITIOF S. SJ(JSTRAND AND LARS-G. ELFVIN
which can interact intensely with the stain, the actual size of the molecules exceeding that of the stained spots. The reality of the observed structural pattern. The fact that a globular substructure could be observed both in chemically fixed and frozen-dried material, and that the dimensions of these structural components are similar, can be used as a rather strong argument in favor of this structure reflecting a real structural organization of the mitochondrial membrane elements and of cytomembranes. The regularity of the patterns observed after freeze-drying also appears more consistent with a real structure than an artifact. The lack of this regular geometry in chemically fixed material, on the other hand, can easily be accepted as an artifact due to partial disorganization of the globular structure. The clear-cut staining of cross bridges in potassium permanganate-fixed material in contrast to frozen-dried and osmium-fixed material can be accounted for by particularly favorable staining conditions in the former case. The fact that it is easier to preserve a simple triple-layered structure than a globular structure when using chemical fixation methods might indicate that the globular structure is labile and easily transformed into such a layered structure. This is a reasonable assumption since it would mean that the surface energy of the system would be reduced from the high energy state of the globular structure to a lower energy state of a layered structure. Interpretation of pattern in terms of molecular architecture. Since the structural components that have been observed are in the molecular range of dimensions, it seems pertinent to try to interpret the observed patterns in terms of molecular architecture. In previous papers (15, 16) it was proposed that the light globular components of the chemically fixed membrane elements represents the lipids of these elements and that the stained material corresponds to proteins and possibly polar ends of the lipid molecules, according to an interpretation presented by SjSstrand (11-14) and supported by the study of model systems by Stoeckenius (19, 20). This interpretation can be applied to the present material as well. From the above discussion it seems justifiable to propose that the protein molecules are arranged in rows and that the major part of the protein is located in the furrows between the rows of globular lipid mieelles. We would then be dealing with rows or chains of protein molecules stabilized in location by the lipid components. In the mitochondrial membrane elements, these chains could correspond to a linear arrangement of the respiratory enzymes. The elementary structure of the mitochondrial membrane elements would therefore not be a particle but a kind of weaving element where each respiratory chain shares lipid components with neighboring chains. The ribosomal material shows a rather different morphology in frozen-dried material as compared to osmium-fixed tissue (Figs. 16 B-D). The delimiting of patti-
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cular elements is impossible in ceils where there has not occurred a certain amount of dehydration and shrinkage of the cytoplasm in connection with freezing, as first pointed out by Sj/3strand and Baker (17). These observations were confirmed and extended by Hanzon et al. (4, 5). If dehydration has taken place as a result of ice crystal formation, the R N A protein appears subdivided into rounded, diffusely outlined "particles," which, however, are in mutual contact and form continuous strands or sheets of ribosomal material. The volume of these "particles" exceeds that of the ribosomes observed in chemically fixed material, decreasing with increasing shrinkage of the cytoplasm in connection with the freezing associated with ice crystal formation. Hanzon and Hermodsson (4) showed that particles were clearly visible in badly preserved frozen-dried material. This appearance of the particles can be correlated to ice-crystal formation causing dehydration of the cytoplasm. The results reported by Seno and Yoshizawa (8-10), who demonstrated ribosomal particles in frozendried material, can be explained in this way. It seems justifiable to interpret these observations to show that the compact particulate form of ribosomal material as observed in sections of osmium-fixed tissue is an artifact and that this material appears more uniformly distributed over the surface of the 0~-cytomembranes in the living cell than is apparent from the analysis of chemically fixed material, in agreement with the conclusions of Sj~Sstrand and Baker (17) and Hanzon et al. (4, 5). In several papers where freeze substitution has been applied for fixation (3, 6) a structural picture of the cytoplasm similar to that of osmium-fixed material has been presented. It is questionable whether the material in these studies really has been dried in a frozen state by substitution. More likely, it seems that the fixation has involved freeze-thawing. As has been shown by Baker (1), the thawing in an osmium tetroxide solution of frozen tissue results in a reconstitution of an appearance of the cytoplasmic components identical to what is observed after fixation of fresh tissue in osmium tetroxide. This is the case even under conditions where extensive ice crystal formation has taken place in the cells before thawing. The advantage of freeze-drying by means of molecular distillation is that we can ascertain that the tissue has been dried before exposure to the embedding medium, eliminating the risks of any thawing-reconstitution of the cells involving the disappearance of artifacts due to ice crystal formation. Such a reconstitution means that the tissue, in fact, has not been fixed by a physical method of freeze-drying but by a chemical method, the preceding freezing and thawing only changing the conditions for chemical fixation. The possibility that the ribosomal RNA is arranged as a more or less continuous carpet covering the membrane elements of the c~-cytomembranes appears important in connection with the interaction between messenger R N A and ribosomal RNA.
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A discontinuous system of ribosomal particles would m e a n that the messenger R N A would have to move over rows of such particles in connection with protein synthesis to allow successive base groups to interact with ribosomes and transfer R N A . ' A n open spread-out configuration of the ribosomal R N A in a continuous carpetlike structure could minimize the requirements for repositioning of messenger R N A in connection with protein synthesis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20.
BAKER, R. F., J. Ultrastructure. Res. 7, 173 (1962). BROD¥, I., J. Ultrastruct. Res. 2, 482 (1959). BULLIVANT,S., J. Biophys. Biochem. CytoI. 8, 639 (1960). HANZON, V. and HERraODSSON,L. H., Y. Uhrastruct. Res. 4, 332 (1960). HANZON, V., HERMODSSON, L. H. and T o s c ~ , G., Y. Ultrastruct. Res. 3, 216 (1959). REBHUN, L. I., Y. Biophys. Biochem. Cytol. 9, 785 (1961). ROBERTSON,J. D., Biochem. Soe. Symp. (Cambridge, EngI.) 16, 3 (1959). SENO, S. and YOSHIZAWA,K., Saibo Kagaku Shimpoziumu 8, 29 (1958). -Naturwissensehaften 46, 19 (1959). -J. Biophys. Biochem. Cytol. 8, 617 (1960). SJ6STRAND, F. S., J. Cellular Comp. Physiol. 42, 15 (1953). -Nature 171, 30 (1953). -ibid. 171, 31 (1953). -Verhandl. Anat. Ges. 53. Versamml., Stockholm, 1956, p. 3. Gustav Fischer Verlag, Jena, 1957. -Nature 199, 1262 (1963). -J. Ultrastruct. Res. 9, 340 (1963). SJOSTRAND,F. S. and BAKER,R. F:, J. Ultrastruct. Res. 1, 239 (1958). SJI~STRAND,F. S. and ELEVIN, L.-G., J. Uttrastruct. Res. 7, 504 (1962). STOECKENtUS,W., J. Biophys. Biochem. CytoI. 5, 491 (1959). -Proc. European Regional Conf. Electron Microscopy, Delft, 1960, Vol. 2, p. 716 (1961).