J O U R N A L OF ULTRASTRUCTURE R E S E A R C H
66, 254-275 (1979)
Freeze-Fracture Analysis of Structure of Plasma Membrane of Photoreceptor Cell Outer Segments FRITIOF S. SJ()STRAND AND MICHAEL KREMAN Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024 Received December 1, 1978 The plasma membrane of the outer segments of photoreceptor cells in the guinea pig retina was analyzed by means of freeze-fracturing. Two fracture faces with greatly different structure were observed. The relationship of these two faces to the edge which was exposed when the fracture passed across the membrane made it possible to establish that they revealed the cytoplasmic and the peripheral surfaces of the plasma membrane. The thickness of the plasma membrane was measured to be about 100 A. The difference in appearance of the cytoplasmic and the peripheral surfaces of the plasma membrane was interpreted to reveal a layered structure with a predominantly lipid layer at the cytoplasmic surface, and a predominantly protein layer at the peripheral surface. A concept describing the molecular structure of the membrane is proposed. The outer segment plasma membrane differs structurally from the outer segment disk membranes. Certain aspects of disk membrane formation as invaginations of the plasma membrane are discussed. The fact t h a t only two fracture faces were observed and no complementary faces was explained by a simple analysis of some effects of the fracturing on the frozen material.
In an earlier study the structure of the photoreceptor cell outer segment disks was analyzed mainly on the basis of information obtained by freeze-fracturing (SjSstrand and Kreman, 1978). Since the disk membranes develop as infoldings of the outer segment plasma membrane (SjSstrand, 1959; Nilsson, 1964) it is of interest to compare the structures of these two types of membranes in order to find out whether such a structural analysis could shed some light on the mechanism of membrane assembly in the outer segment. The analysis of the structure of one type of plasma membrane furthermore has an interest in itself since our knowledge about the structure of plasma membranes is fragmentary. In fact, our concept regarding the molecular structure of the plasma membrane is primarily based on the hypothetical model proposed by Singer and Nicolson (1972) and on observations made on freeze-fractured material that have been interpreted to conform with this model. Unfortunately, the observations made on freeze-fractured material have been interpreted on the basis
of some hypothetical assumptions that make the interpretation of these observations uncertain. First, it has thus been assumed c~ priori that the structure of the membrane agrees with a particular hypothetical concept instead of considering the structure as being unknown. Second, it has been assumed that it is possible to predict where the fracture planes will be located relative to this unknown structure and to state that there is one particular location that applies to all membranes. This way of interpreting observations made on freeze-fractured material has not been considered acceptable in this study for the simple reason that correct conclusions regarding the location of the fracture planes are required to make it possible to interpret the observations made with this technique. In this study, therefore, the structure of the outer segment plasma membrane has been considered to be unknown and no generalizing concept regarding the structure of membranes has been allowed to influence the interpretation of the observations. The location of the fracture planes has been
254 0022-5320/79/030254-22502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
OUTER SEGMENT PLASMA MEMBRANE established on the basis of observations, and the interpretation has been carried out with p r o p e r considerations of the great limitations of the freeze-fracture technique w h e n aiming at analyzing the m e m b r a n e structure at a molecular level. Since the function of the plasma membrane is likely to be different from t h a t of m e m b r a n e s of the outer segment disks, it is of interest to establish to what extent the different functions are reflected in structural differences. T h e analysis revealed considerable structural differences between the plasma membrane and the disk membranes. It appeared, however, t h a t both types of m e m b r a n e s have a layered structure and are highly asymmetrical. MATERIALS AND METHODS Basically the same material that was analyzed in a previous paper (SjSstrand and Kreman, 1978) was studied in the present study. Although the analysis primarily involved the guinea pig retina, comparisons were made with the frog and gecko retinas.
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RESULTS T h e r e were two types of fracture faces associated with the outer segment plasma m e m b r a n e in the guinea pig retina, one convex and one concave face. All convex fracture faces showed a particulate structure with a dense a r r a n g e m e n t of the particles (Figs. 1-3). T h e particulate surface could be i n t e r r u p t e d by sharply outlined areas with a s m o o t h or pebbled s u r f a c e structure (Figs. 1 and 2). T h e s e areas h a d a r o u n d e d shape and usually measured 600 to 1100/~ in diameter, and their surface was recessed in relation to the surface of the particulate areas. T h e n u m b e r of such plaques per unit surface area varied greatly in different preparations and different receptor cells in the same preparation. T h e y could be missing completely over the entire exposed area of the plasma m e m b r a n e in some outer segments. T h e size of these areas also varied considerably with sometimes m u c h larger areas t h a n 1100 A exposing a s m o o t h surface. With respect to the
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DIAGRAM2. Size distribution of isodiametric particles in the disk membranes determined on the same electron micrographs as those on which the measurements shown in Diagram 1 were made. a p p e a r a n c e of these plaques, there was no difference observed in specimens t h a t h a d been crosslinked at _+0°C or at r o o m temperature. T h e particles in the particulate areas prot r u d e d r a t h e r high above the m e m b r a n e surface in some preparations (Fig. 3), while t h e y p r o t r u d e d considerably less in o t h e r specimens (Fig. 4). This difference was attributed to variations in distortion due to plastic flow. T h e size distribution of isodiametric particles was determined on fracture faces t h a t showed a m i n i m u m of obvious plastic deformation. T h e result is il-
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lustrated in Diagram 1. This diagram shows a maximum for diameters within the range 80-100 /k. It furthermore shows a highly asymmetrical distribution of the particle diameters with a large proportion of the diameters of the particles in the range 5070 A. For comparison, the particle size distribution was determined for disk membranes present in the same electron micrographs. Diagram 2 shows a somewhat nonrandom distribution of particle diameters within the range 50-80/~. All concave fracture faces associated with the outer segment plasma membrane exposed a rather smooth surface with a few
large particles protruding high above the surface. These particles were far apart, separated by distances of 500 to 1000 A (Figs. 1 and 5). Their diameters showed little variation, 95-110/k. Except for these particles the exposed surface appeared rather smooth as compared to the convex fracture face. The surface showed, however, some structure with particularly smooth areas surrounded by areas exposing a very fine particulate structure with the diameter of the particles measuring 40 to 60/k (Fig. 5). The smooth areas surrounded by these areas also showed a fine particulate structure with the diameters of the particles measuring 20 to 30/k, which are the dimen-
FIG. 1. Fracture involving three outer segments. The fracture exposes one smooth, (s), and one particulate, (p), face associated with the plasma membranes of these outer segments. Only a thin layer of extraceUular medium separates the two plasma membranes of two outer segments within a region marked by an arrow and the letter e. The double membrane disks of the outer segments are shown and the cytoplasm (c) next to the plasma membrane is exposed in all three outer segments. Notice on the particulate face plaques showing a smooth surface (pl). × 110 000. FIG. 2. Fracture face exposing the particulate face of an outer segment plasma membrane. Many plaques with a smooth or pebbled surface are present on this face. The parallel ridges at the edge of the particulate face show t h a t the material in the exposed surface has been subjected to considerable plastic deformation. × 130 000. Fro. 3. Particulate plasma membrane face showing considerable distortion due to plastic flow resulting in parallel ridges at the edge of the face and rather high elevations as shown by the long shadows cast on the surface of the exposed extracellular medium. An edge is indicated at the boundary of the particulate face (arrows). In lower left corner the fracture exposes a smooth membrane face. × 230 000. FIG. 4. High magnification picture of a particulate membrane face which did not show the signs of extensive plastic deformation t h a t can be observed in Figs. 2 and 3. × 460 000. FIa. 5. High magnification of the smooth membrane face showing the large particles protruding rather high above the surface and the areas with smooth surface (s) surrounded by less smooth areas. The particles observed in the smooth areas are of the dimensions of the metal grains formed during metal shadowing. The structure observed in the less smooth areas is in the 40- to 60-A range and cannot be caused by metal grains. T h e mixing of rather smooth areas with these differences in the dimensions of the observed graininess makes it possible to conclude t h a t the particulate appearance in the less smooth areas must reflect the structure of the exposed membrane surface, and t h a t it is not due to the structure of the metal coat. The smooth plaques thus are like internal standards showing the limit of resolution introduced by the structure of the metal. In the less smooth areas the real structure of the surface is partially distorted by the superimposed pattern formed by the metal grains. This makes it impossible to determine with any great precision the real size of the smallest elevations (particles} t h a t can be observed on the surface of the plasma membrane t h a t has been exposed. This is a demonstration of one limiting factor with respect to the specimen resolution t h a t can be achieved with freezefracturing. × 460 000. Fla. 6. Fracture exposing a particulate membrane face and an edge where the fracture plane has cut across the plasma membrane. This edge shows a particulate structure and measures about 100/k in width. × 160 000. Flas. 7, 8. Fracture across the outer segment plasma membrane exposing an edge with a particulate structure. Fig. 7, × 310 000; Fig. 8, x 340 000. Fla. 9. Fracture exposing an edge where it cuts across the plasma membrane. In this case the plasma membrane is viewed from the cytoplasmic side as in Fig. 8, while it was viewed from the peripheral medium in Figs. 6 and 7. Some large particles (arrows) are observed presumably reaching through the exposed part of the plasma membrane. These particles are likely to correspond to the large particles observed on the smooth membrane face. × 230 000.
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OUTER SEGMENT PLASMA MEMBRANE sions of the metal grains formed by aggregation of the metal atoms when shadowing the surface. T h e d i a m e t e r of these latter areas varied between 500 and 1300 A and t h e y could be r a t h e r densely arranged or could be missing from large areas. T h e large particles were located within the areas with particles in the size range 40-60 A. Cross and slightly oblique fractures t h r o u g h the plasma m e m b r a n e (Figs. 6-8), revealed a particulate structure of the m e m b r a n e . T h e thickness of the plasma m e m b r a n e was m e a s u r e d in cross fractures and was found to be about 100/~. Within the r o u n d e d plaques with a s m o o t h surface t h a t were exposed on the convex fracture face, the thickness of the m e m b r a n e was considerably less t h a n 100/~, and the cross fracture did not show any particulate structure. W h e n the fracture plane changed from an orientation parallel to the m e m b r a n e to cutting across the membrane, it was characteristic t h a t b o t h convex and concave fracture faces were associated with an edge with a width of a b o u t 100 A (Fig. 6-9). This edge exposed a particulate structure. T h e concave fracture face was in direct contact with the exposed cytoplasm. T h e r e was only a change in the angular orientation of the fracture plane at the transition from a fracture t h r o u g h the cytoplasm to the fracture passing along the m e m b r a n e {Fig. 10). No step could be observed which would have indicated t h a t the fracture passed t h r o u g h a layer interposed between the observed m e m b r a n e surface and the cytoplasm. At the convex m e m b r a n e face the same situation applied to the transition from the fracture passing t h r o u g h the extracellular
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m e d i u m to extending along the m e m b r a n e (Fig. 10). DISCUSSION
Location of Fracture Faces T h e locations of the two fracture faces, the particulate convex face, and the s m o o t h concave face could be established thanks to the edge structure t h a t was associated with b o t h fracture faces where the fracture planes changed from being oriented parallel to the m e m b r a n e surface to being perpendicular to this surface. Since in both cases an approximately 100-A edge with a particulate structure was observed, it can be concluded t h a t the two fracture faces were located at the two surfaces of the membrane. T h e fact t h a t no indications were found for the presence of any layer t h a t could have been coating the two exposed membrane surfaces also supports this conclusion. T h e cytoplasm exposed next to the concave fracture face reached without any interruption or any step all the way to the concave fracture face. T h e same relationship applied to the extracellular m e d i u m and the convex fracture face. No etching was done because it will always lead to the deposition of a layer of solutes t h a t can be misinterpreted to represent an additional layer belonging to the m e m b r a n e . In fact, without such a layer appearing it is difficult to be certain t h a t the surface has been etched.
The Exposed Materials With the location of the fracture faces known it is justified to refer to the two fracture faces as the cytoplasmic and the peripheral surfaces of the plasma membrane. T h e plasma m e m b r a n e thus exposes
FIG. 10. Two plasma membranes exposed running in parallel diagonally across the picture, one exposing a smooth membrane face and the other a particulate face. The smooth membrane face is viewed from the inside of the outer segment. The cytoplasm reaches all the way to the surface of this face without any step separating the fracture plane cutting through the cytoplasm and the fracture plane located along the membrane. The particulate face is viewed from outside the outer segment. The fracture face exposing the extracellular medium reaches to the particulate face without any step or rim separating them. × 230 000.
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closely arranged particles at its peripheral surface, while a relatively smooth surface with a few large particles characterizes its cytoplasmic surface. The discussion of these observations will be based on the concept that the topography of fracture faces is first hand determined by the properties of the material being exposed by fracturing. If this is not accepted as a reasonable basis for the discussion, freeze-fracturing would not allow a useful interpretation of the observations made with this technique. As a consequence of this concept, a fracture through the middle of a membrane cannot give rise to two fracture faces that are structurally different since both faces expose the same material. Double replicas showing fracture faces that are not complementary must therefore expose two different materials, which means that the fracture must be located at the boundary between materials of different properties (SjSstrand and Kreman, 1978). It then follows that a fracture through the middle of a membrane could expose two structurally different faces provided the fracture was located between two layers of materials of different composition within the membrane. Superimposed on the structural pattern at the fracture face that is determined by the properties of the exposed material is a pattern produced by plastic deformation of this material. This pattern is also likely to depend on the properties of the exposed material and can therefore contribute to reveal, and even enhance, differences in the properties of the materials at the fracture face. Plastic deformation is easily identified in the cases where it has caused certain regular patterns in the surface structure. These patterns can be related to plastic deformation because they have the characteristics of patterns according to which stress can be expected to develop in the material during fracturing. With one fracture face showing densely arranged particles, and another a rather
smooth surface, we conclude that these two faces expose materials with different properties. Since the two fracture faces were found to be located at the two surfaces of the membrane, it follows that the membrane exposes materials with rather different properties at its two surfaces. When considering the particle size distribution in the particulate face it is clear that this distribution is nonrandom, while the arrangement of the particles is random. This can either mean that the fracture has exposed particles and aggregates of particles present in the exposed material, or that plastic deformation has produced a pattern consisting of randomly arranged particles with a nonrandom size distribution. The latter alternative would require a factor that introduces a deviation from randomness with respect to the dimensions of elevations formed during plastic flow. A simple explanation for such a deviation is that the material contains particles in or close to the observed range of dimensions. We therefore conclude that the particle distribution reflects the presence at this surface of particles roughly in the range of dimensions shown by the observed size distribution of isodiametric particles. Since the range of dimensions of these particles corresponds to the dimensions of globular protein molecules, and since we know that a large proportion of the mass of the membrane consists of proteins, the simplest explanation for the particulate appearance of this face is that it exposes a material containing a high concentration of globular protein molecules. This conclusion appears even more justified when considering the fact it is highly likely that the fracture would have a higher tendency to pass along the surface of the protein molecules than to cut through the interior of the molecules. With this tendency we should expect that if the protein molecules become exposed they should show up as bumps in the fracture face. The relatively smooth cytoplasmic face revealed a material which appeared fairly
OUTER SEGMENT PLASMA MEMBRANE homogeneous with only a few large particles extending high above the surface. The relative smoothness of this surface can be explained by the presence at this surface of lipids. The plaques showing a smooth surface that were observed on the peripheral surface of the membrane are likely to correspond to areas consisting of a pure lipid bilayer. A comparison of the size, shape, and pattern of distribution of these plaques and the smooth areas at the cytoplasmic surface of the membrane shows a similar range of dimensions. The distribution of the smooth areas at the cytoplasmic surface showed the same type of irregularity as that of the plaques. It is therefore assumed that the smooth areas on the cytoplasmic surface correspond to the plaques observed on the peripheral surface of the plasma membrane. A Molecular Model
The aim of any analysis of the membrane structure, at a level of resolution at which freeze-fractured material can be analyzed, is to reveal the molecular structure of the membrane. The reliability of the concept regarding the molecular structure of a membrane that can be developed then depends upon how truthful freeze-fracturing reveals the native structure of the membrane, upon how detailed the information is that can be obtained with this technique, and how precisely the fracture planes can be located. Provided that the fracture planes have been located correctly, it then is clear that the information is limited by the limitations imposed by specimen resolution and by distortion due to plastic deformation. These factors determine the reliability of measurements of particle diameters and can make the obtained absolute values for the diameters become rather dubious. These measurements primarily fulfill the purpose of establishing that structural differences exist that characterize different types of membranes and membrane faces.
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As a consequence, even if we identify the particles as reflecting the presence of globular protein molecules, and of complexes of protein molecules, the dimensions of the particles cannot be translated directly into corresponding dimensions of the molecules from which the molecular weight could be deduced. Aggregates of protein molecules are likely to be formed artificially due to plastic deformation during facturing. The long ridges often observed at the edge of the particulate convex fracture face are likely to be such aggregates (Figs. 2 and 3). Distortion due to artificial aggregation is likely to have affected the size of particles less, the smaller the particles in the fracture face were. It therefore appears justified to consider the particles in the smaller size range as perhaps the most representative for the size range of particles present in the membrane and consequently to roughly correspond to the size range of protein molecules present in the membrane. When developing a concept regarding the molecular structure of the outer segment plasma membrane, we start out from the information which appears to be most reliable. The first piece of information belonging to this category is the thickness of the plasma membrane. This represents a very important piece of information. To this we can add the information that the two surfaces of the plasma membrane exposed materials with very different structural properties. Combined with the information regarding the thickness of the membrane this information makes it justified to consider the possibility that the membrane consists of two layers with different molecular composition. We now assume that the outer segment plasma membrane has a lipid concentration similar to that of other plasma membranes, the lipid and protein content of which has been determined. It then is reasonable to consider that the layered structure reflects a preferred location of the lipids to one layer and a domination of the proteins in the second layer. This would
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FIG. 11. Molecular model of plasma membrane that illustrates various possible types of molecular interactions in the membrane. The observed asymmetry of the membrane is interpreted to reveal the presence of two layers, one predominantly lipid and one predominantly protein layer. The interaction of protein molecules with the lipid bflayer is shown at 1 where one protein molecule interacts hydrophobically with the lipid layer, while another protein molecule interacts with the polar head of the lipid molecules. There is mutual interaction between the two protein molecules whereby a membrane bound multimolecular complex is formed. At 2, the lipid bilayer is not coated with proteins which would correspond to the situation at the plaques observed on the peripheral plasma membrane surface. At 3, a large multimolecular complex which penetrates the membrane. Such protein complexes can explain the presence of large particles on the cytoplasmic surface of the membrane. At 4, a single protein molecule bound to the lipid bilayer, and at 5, a multimolecular complex extending in the plane of the membrane. This complex could explain the fine particulate appearance of the cytoplasmic membrane surface. The perturbation of the lipid bilayer structure introduced by lipid-protein interaction can also explain this structure. At 6, a protein molecule bound through charge interaction to the polar heads of the lipid molecules in the bilayer.
explain the observation that the cytoplasmic surface of the plasma membrane showed a rather smooth surface which is characteristic for a cleavage along a lipid layer. It also explains the particulate appearance of the peripheral surface of the plasma membrane. The lipids would thus be predominantly located to the cytoplasmic surface of the membrane. We now assume that the lipid molecules cover a large part of this surface and that the basic arrangement of the lipid molecules is a bilayer arrangement. Since nonpolar interaction is characteristic for part of the membrane proteins there must be protein molecules in the membrane that tend to expose a large number of nonpolar groups at their surface. These groups can be assumed to be able to interact both with the hydrocarbon tails of the lipid molecules and with nonpolar groups exposed at the surfaces of other
protein molecules. To establish nonpolar interaction with the lipids in the bilayer it is required that there are openings in the layer of polar heads facing the protein layer. A nonpolar area on the surface of a protein molecule can then enter into contact with the hydrocarbon layer of the bilayer without sinking into the bilayer. This arrangement is illustrated in Fig. 11. It was proposed for the protein-lipid interaction in the outer segment disk membranes by SjSstrand and Kreman (1978). Consequently, most of the surface of the protein molecule is available for interaction with other protein molecules in the membrane and with ions and molecules in the surrounding medium. This arrangement must introduce a perturbation of the bilayer structure which could explain the fine particulate structure that was observed on the cytoplasmic surface of the membrane. The protein mole-
OUTER SEGMENT PLASMA MEMBRANE cules interacting this way with the lipid layer become firmly anchored to this layer. Other protein molecules can be conceived of as interacting with the polar head groups of the lipid molecules through charge interaction and with other protein molecules through contacts between nonpolar areas on the surfaces of the molecules as well as through charge interaction. A nonpolar surface on a protein molecule does not have to interact with lipids since nonpolar proteinprotein interaction is equally feasible. When other protein molecules interact with those anchored to the lipid bilayer, complexes of protein molecules are formed with the molecules of the entire complex being anchored directly or indirectly to the lipid bilayer. Protein molecules that are not anchored directly to the lipid bilayer will be more loosely bound to the membrane and will therefore be easier to solubilize than those anchored. Protein molecules linked to the membrane by charge interaction only would be particularly easy to remove. Since all these proteins are conceived of as being located to one layer about 50/~ thick (the lipid bilayer is assumed to account for about 50 A of the thickness of the membrane), differences in solubility will only reflect conditions of binding the protein molecules to the membrane and cannot be used as criterion for the location of the protein within the membrane, whether superficial or deep. The large particles observed on the cytoplasmic surface of the plasma membrane are interpreted to represent large complexes of protein molecules that penetrate the lipid bilayer. These complexes expose part of their surface to the extracellular medium and part of their surface to the cytoplasm. They seem to be suitable for transmembrane transport. The fine particulate structure observed on the cytoplasmic surface of the plasma membrane could also be caused by tightly arranged globular molecules in the 40- to 60-/~ range of dimensions. This arrange-
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ment of protein molecules is therefore considered as a second possibility to explain this appearance of the surface. It would increase the number of transmembrane proteins to accommodate a wide range of controls of membrane permeability. The packing density of the protein molecules in the protein layer cannot be estimated with any high degree of precision from the observed fracture faces because of the uncertainty regarding the extent to which plastic deformation had interfered to modify the surface topography of the convex fracture face. It is, however, likely that the protein molecules are fairly densely arranged but that they are not present in a close-packed arrangement. This means that the lateral surfaces of part of the protein molecules would be exposed to the extracellular medium. Such a loose packing would mean that the viscosity within this layer of the membrane could be low enough to allow diffusion of the protein molecules, and of complexes of protein molecules, in the plane of the membrane. Figure 11 illustrates schematically the various possible relationships between proteins and lipids that have been discussed here. This figure also includes an alternative interpretation which in certain aspects conforms with the Singer-Nicolson membrane model (1972). It differs from that model by the very asymmetrical arrangement of the protein and lipid molecules and by specifying the location of less firmly bound proteins. The two models differ with respect to the mode of interaction between the protein molecules and the lipid bilayer. The firstmentioned model leaves a larger part of the globular protein molecules in contact with an aqueous medium than the second model. In the latter model, so large parts of the protein molecules are exposed to a nonpolar, liquid environment that it seems possible that this environment could greatly affect the conformation of the protein molecules. This effect of the nonpolar environment would be less pronounced if the pro-
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tein molecules located in the lipid bilayer were associated in the form of multimolecular complexes. It is obvious that these models depict features of the membrane that are far beyond the actual observations. We therefore must distinguish between what interpretations are based on observations and what are reasonable deductions from these observations. The asymmetric arrangement of proteins and lipids in the membrane belongs to interpretations based on observations, while the various possible modes of interaction between proteins and lipids represent reasonable deductions. This illustrates the limitations of the freeze-fracturing technique and the great need of developing improved preparatory procedures that can further extend the range of specimen resolution toward molecular dimensions.
The Development of Outer Segment Disks The structural arrangement of the outer segment plasma membrane as deduced here has certain similarities to that of the disk membranes. In both cases it was concluded that the membrane was a layered structure. However, the size distribution of isodiametric particles differed in the two types of membrane. The cytoplasmic surface of both membranes exposed large particles, but these particles were larger and were considerably more numerous in the case of the plasma membrane. Areas with a fine particulate structure alternating with smooth areas were not observed in the case of the disk membranes except after extensive cross-linking. The outer segment disks develop as invaginations extending from the outer segment plasma membrane (SjSstrand 1959; Nilsson, 1964). It is then of interest to compare the two membranes in order to find some clues to the mechanism of disk formation. Both the plasma membrane and the disk membrane face the cytoplasm with a surface which is smooth in comparison to the surface facing away from the cytoplasm.
This means that during the process of formation the material in the two membranes that gives rise to smooth fracture faces is continuous. The fact that the plasma membrane differs structurally from the disk membrane in other respects shows that the disk membrane is not a simple invagination of the plasma membrane formed in connection with a general growth of the plasma membrane. Instead, it shows that a membrane that is structurally different, and structurally simpler than the plasma membrane, is formed at the invagination. This is an example where continuity does not necessarily mean that the structures involved are identical. The crista membranes and the innermost surface membrane of mitochondria represent a second example of such a relationship (SjSstrand, 1977). There are now several features that characterize the formation of the disks. First, the disk develops from a narrow zone of the plasma membrane which is extending perpendicular to the long axis of the outer segment. The length of this zone is not known, but from the fact that the invagination is easily observed in thin sections excludes that it is a tubular invagination. Second, the disk develops in one plane without any wrinkling of the newly formed membrane. Third, the growth of the disk stops when it has reached a certain dimension and the control of the size is executed with a high degree of precision. Fourth, the disk edge has a different structure when compared with that of the disk membranes. The fact that the disk grows in one plane without any random wrinkling of the newly formed membrane is here considered to require a particular organization of the components responsible for the assembly of the new membrane. The narrow zone of plasma membrane, from which the invagination develops, must during the first stage of disk formation be the growth zone where the new membrane is assembled.
O U T E R S E G M E N T PLASMA M E M B R A N E
It would be simple to explain the directionality of the membrane growth if the membrane assembly were restricted to this narrow zone and if it became part of the edge of the invagination. It will then be translocated further and further in the interior of the outer segment when the invagination increases in size. If we furthermore assume that the growth zone splits up into two zones in the guinea pig outer segment disks, it is possible to explain the formation of the incision that gives the disk its characteristic shape. The growth of the invagination involves the assemblage of lipids and proteins, and the assemblage of the edge structure which appears to be a predominantly protein structure. The proteins are synthesized in the inner segment and are transported through the cytoplasm to the invagination. The structure of the disk membrane that was proposed by SjSstrand and Kreman (1978) means that the membrane proteins are located at the side of the membrane facing away from the cytoplasm and that a lipid layer is interposed between them and the cytoplasm. This structure makes it likely that the assemblage of the membrane involves a more or less simultaneous incorporation of lipids and proteins which is possible at the edge of the invagination. This way the photopigment molecules would not have to penetrate into and pass through a lipid layer to arrive at their destination. While the site of synthesis of membrane proteins is known from the distribution of ribosomes in the receptor cell, the site of synthesis of the membrane lipids is not known. It is appealing to speculate that the lipids are synthesized in the edge structure of the disk and that this synthesis is one feature characterizing the growth zone. We then need only one mechanism for trapping the molecules in the growth zone which is one for trapping the proteins. There is one advantage of restricting the membrane assembly to the edge of the invagination. It will automatically favor a
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growth in one plane. Through protein-protein interaction between the two disk membranes, the disk would furthermore tend to form a planar compact structure with no tendency for the development of a vesicular disk. The size of the disk could possibly be controlled by the time the phospholipid synthesis can proceed before the enzyme systems involved become spontaneously inactivated.
The Absence of Complementary Fracture Faces It was characteristic for the examined material that no fracture faces were observed that could have been complementary to the two types of fracture faces described here. Every convex fracture face showed the dense particulate structure, and every concave face showed a relatively smooth surface. This means that there must be certain definite restrictions involved with respect to how the fracture proceeds at the plasma membrane when fracturing by sectioning. We will try to furnish an explanation for these observations by analyzing in a simple way how the fracturing of the material proceeds. We start out from the conclusion
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Fro. 12. Fracturing by sectioning means that pieces of the frozen material are chipped off. The two concentric rings symbolize the plasma membrane of, for instance, an outer segment.
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Fro. 13. (A) The three stages characterizingthe chipping off of a piece of the frozenmaterial. 1, A crack is formed; 2, the two surfaces at the crack becomeseparated; and 3, a piece is lifted off the newlyformedsurface through the developmentof a vertical crack. (B-D) Consecutive stages in the chipping off of a piece of the frozen material. t h a t sectioning makes pieces of material become lifted off the newly formed surface in the form of small chips. Most of the crack through the material initiated by the moving knife edge proceeds in a plane located below the plane in which the knife edge moves. Th e pieces of material that are being chipped off therefore must be lifted from the level of the crack to the front face of the knife (Fig. 12). It seems reasonable to distinguish between three phases in this chipping off of the material. T h e first phase involves the formation of a crack through the material without much separation of the two surfaces at the crack. T he second phase involves a separation of the two surfaces at the crack which leaves a wedgeshaped space between these surfaces, with
the edge of the wedge facing in the direction the crack proceeds. T h e third stage involves the separation of a piece of material through a more or less vertical crack through the material and the translocation of this piece of material to the front face of the knife (Fig. 13A). During this process the material located above the fracture plane has been exposed to considerable deformation. T he piece of the material t hat eventually is chipped off must have been exposed to a horizontal force "compression" as the wedge-shaped space between the two surfaces of the crack is developing, before a vertical crack introduces a release in the stress to which the material is exposed. (We will not include other horizontal forces in this discussion.)
OUTER SEGMENT PLASMA MEMBRANE As a consequence, any structures that are present in the pieces that are chipped off must have been exposed to stress. The deformation caused by stress is likely to be only partially reversible {elastic deformation). Because the material that is being chipped off is lifted up from the new surface formed by the crack, it is clear that the material in this surface must be exposed to a force with a large vertical component. If the crack reaches the convex outer surface of the plasma membrane the orientation of this vertical component relative to the membrane surface will vary for different parts of the surface. Where the surface is oriented parallel to the direction in which the cleavage is initiated, this vertical force will tend to pull material out of the surface as the cleavage proceeds. The effect of such a pulling force on the exposed surface structure will obviously depend on the structure of the exposed material. Since the force can be considered to be acting uniformly over fairly large areas of the surface, it will have little effect if the material at the exposed surfaces is homogeneous, while it can be expected that it can have considerable influence on the structure of the surface in the case in which the material at the crack is nonhomogeneous, for instance, when the material has a particulate structure. The action of a force with a vertical orientation explains the observation made by SjSstrand and Kreman (1978) that particles in the exposed particulate surface of the outer segment disk membranes protruded further than what corresponded to the total thickness of the particulate layer. For those parts of the plasma membrane surface, which are oriented at an angle relative to the direction in which the cleavage is initiated, the vertical force acting at the fracturing will have one component that is oriented tangential to the surface. This means that the material in these parts is exposed to a sheering force. This sheering force will be greater the more the orientation of the surface of the plasma membrane
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C FIG. 14. The permitted (arrows) and the excluded (crossed over) fracture faces associated with the plasma membrane. approaches an orientation perpendicular to the direction in which the material is being cleaved. The sheering effect means that the material at the surface will be exposed to a force acting parallel to the surface, and oriented perpendicular to the edge of the area of exposed membrane. It offers an explanation for the appearance of a pattern located at this edge which consists of paraUel ridges. This pattern is shown in Figs. 2 and 3. Also in this case there should be little deformation introduced by this force in the case in which the material is homogeneous since the force is uniformly distributed over rather large areas of the surface. No deformation that could be linked to this force was observed at smooth membrane surfaces. The observations made so far have shown that the extent to which the material is deformed due to plastic flow varies considerably in different specimens. One factor that will have considerable influence on the extent of deformation is the temperature at which the fracturing is performed. The lower this temperature, the less deformation due to plastic flow is to be expected. It therefore appears essential to pursue the
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This is illustrated in Figs. 13 and 15. As the crack reached the membrane surface this stress must have involved parts of the membrane located just above the level to which the crack had progressed. This stress followed by deformation is likely to favor the crack cutting across the membrane over the crack following the first natural cleavage plane that it reaches. The cleavage across the membrane is facilitated by the arrangement of the molecules in the membrane, with lipid molecules predominantly oriFIG. 15. Deformationof a membranedue to plastic ented perpendicular to the plane of the and elastic deformationof the frozenmaterial as the membrane and with protein molecules with two surfacesof a crack becomeseparated. a globular shape. It is obvious that the same conditions as fracturing at the lowest possible tempera- discussed for B apply to C in Fig. 14. ture. From this we can conclude that only In Fig. 14 are shown the planes along double replicas could furnish fracture faces which the plasma membranes are fractured complementary to the two types of faces and the excluded fracture planes. The fig- observed here. However, since the fracture ure shows the two possibilities, a crack ex- planes are located between materials with panding from the outside of the outer seg- different properties, it is likely that the ment, and a crack that has cut through the complementary faces differ considerably plasma membrane on one side and ap- from what would be expected from a real proaches the plasma membrane from its complementarity, that is, that elevations on cytoplasmic side on the opposite side of the one surface correspond to depressions on outer segment. There is a simple rule with the complementary face. Considerable derespect to permissible and excluded frac- viation from complementarity can be preture paths. If the angle between the plane dicted particularly for the peripheral of the cleavage and the first surface of the plasma membrane surface because the parmembrane reached by the crack is larger ticulate structure has been enhanced by than 90 ° the fracture follows the first sur- plastic deformation, while the extracellular face reached by the crack as shown at A medium is little affected by plastic deforand D in Fig. 14. If this angle is less than mation as shown by the smooth fracture 90 ° the crack cuts across the membrane faces it exposes. and follows the more distant surface of the This analysis explains the fact that only membrane. This is illustrated at B and C in two types of fracture faces were observed, Fig. 14. In A and D, the preference for the and never any that could have been comlocation of the fracture plane can be ex- plementary faces to these two faces. The plained by these locations representing the deformation to which the membrane is exfirst natural cleavage plane reached by the posed as the crack proceeds in B and C in crack. Fig. 14, makes it highly unlikely that the In B and C another explanation must be crack would continue through the middle found. Considering case B it becomes clear of the membrane. If the fracturing still that the membrane must have been ex- would have exposed inner membrane faces, posed to considerable stress and finally to the lack of complementarity of the two breakage as the crack proceeded and ma- faces would then necessitate the conclusion terial was being chipped off the surface. that the membrane consists of two layers
OUTER SEGMENT PLASMA MEMBRANE with very different properties. T h e s e layers would then, however, be located in a way opposite to t h a t described here, with the s m o o t h material at the peripheral surface and the particulate material at the cytoplasmic surface. It would then be difficult to explain the presence of large particles extending high above the surface of the concave face and the thickness of the mem~ brane would t h e n be a b o u t 200/~. We wish to express our gratitude to Dr. J. Frank, Department of Physiology,for her generosity in allow-
275
ing us to use her freeze-fracture equipment, and to Mrs. S. Beydler for her excellent technical assistance. This research was supported by NSF Grant 74-20390 and by a Biomedical Research Support Grant, University of California. REFERENCES NILSSON,S.-E. (1964) J. Ultrastruct. Res. 11, 581. SINGER, S. J., AND NICHOLSON,G. L. {1972) Science 175, 720. SJOSTRAND, F. S. (1959) Rev. Mod. Phys. 31, 301. SJOSTRAND,F. S. (1977) J. Ultrastruct. Res. 59, 292. SJOSTRAND, F. S., ANDKREMAN,M. (1978) J. Ultrastruct. Res. 65, 195.
66, 254-275 (1979)
Freeze-Fracture Analysis of Structure of Plasma Membrane of Photoreceptor Cell Outer Segments FRITIOF S. SJ()STRAND AND MICHAEL KREMAN Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024 Received December 1, 1978 The plasma membrane of the outer segments of photoreceptor cells in the guinea pig retina was analyzed by means of freeze-fracturing. Two fracture faces with greatly different structure were observed. The relationship of these two faces to the edge which was exposed when the fracture passed across the membrane made it possible to establish that they revealed the cytoplasmic and the peripheral surfaces of the plasma membrane. The thickness of the plasma membrane was measured to be about 100 A. The difference in appearance of the cytoplasmic and the peripheral surfaces of the plasma membrane was interpreted to reveal a layered structure with a predominantly lipid layer at the cytoplasmic surface, and a predominantly protein layer at the peripheral surface. A concept describing the molecular structure of the membrane is proposed. The outer segment plasma membrane differs structurally from the outer segment disk membranes. Certain aspects of disk membrane formation as invaginations of the plasma membrane are discussed. The fact t h a t only two fracture faces were observed and no complementary faces was explained by a simple analysis of some effects of the fracturing on the frozen material.
In an earlier study the structure of the photoreceptor cell outer segment disks was analyzed mainly on the basis of information obtained by freeze-fracturing (SjSstrand and Kreman, 1978). Since the disk membranes develop as infoldings of the outer segment plasma membrane (SjSstrand, 1959; Nilsson, 1964) it is of interest to compare the structures of these two types of membranes in order to find out whether such a structural analysis could shed some light on the mechanism of membrane assembly in the outer segment. The analysis of the structure of one type of plasma membrane furthermore has an interest in itself since our knowledge about the structure of plasma membranes is fragmentary. In fact, our concept regarding the molecular structure of the plasma membrane is primarily based on the hypothetical model proposed by Singer and Nicolson (1972) and on observations made on freeze-fractured material that have been interpreted to conform with this model. Unfortunately, the observations made on freeze-fractured material have been interpreted on the basis
of some hypothetical assumptions that make the interpretation of these observations uncertain. First, it has thus been assumed c~ priori that the structure of the membrane agrees with a particular hypothetical concept instead of considering the structure as being unknown. Second, it has been assumed that it is possible to predict where the fracture planes will be located relative to this unknown structure and to state that there is one particular location that applies to all membranes. This way of interpreting observations made on freeze-fractured material has not been considered acceptable in this study for the simple reason that correct conclusions regarding the location of the fracture planes are required to make it possible to interpret the observations made with this technique. In this study, therefore, the structure of the outer segment plasma membrane has been considered to be unknown and no generalizing concept regarding the structure of membranes has been allowed to influence the interpretation of the observations. The location of the fracture planes has been
254 0022-5320/79/030254-22502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
OUTER SEGMENT PLASMA MEMBRANE established on the basis of observations, and the interpretation has been carried out with p r o p e r considerations of the great limitations of the freeze-fracture technique w h e n aiming at analyzing the m e m b r a n e structure at a molecular level. Since the function of the plasma membrane is likely to be different from t h a t of m e m b r a n e s of the outer segment disks, it is of interest to establish to what extent the different functions are reflected in structural differences. T h e analysis revealed considerable structural differences between the plasma membrane and the disk membranes. It appeared, however, t h a t both types of m e m b r a n e s have a layered structure and are highly asymmetrical. MATERIALS AND METHODS Basically the same material that was analyzed in a previous paper (SjSstrand and Kreman, 1978) was studied in the present study. Although the analysis primarily involved the guinea pig retina, comparisons were made with the frog and gecko retinas.
255
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RESULTS T h e r e were two types of fracture faces associated with the outer segment plasma m e m b r a n e in the guinea pig retina, one convex and one concave face. All convex fracture faces showed a particulate structure with a dense a r r a n g e m e n t of the particles (Figs. 1-3). T h e particulate surface could be i n t e r r u p t e d by sharply outlined areas with a s m o o t h or pebbled s u r f a c e structure (Figs. 1 and 2). T h e s e areas h a d a r o u n d e d shape and usually measured 600 to 1100/~ in diameter, and their surface was recessed in relation to the surface of the particulate areas. T h e n u m b e r of such plaques per unit surface area varied greatly in different preparations and different receptor cells in the same preparation. T h e y could be missing completely over the entire exposed area of the plasma m e m b r a n e in some outer segments. T h e size of these areas also varied considerably with sometimes m u c h larger areas t h a n 1100 A exposing a s m o o t h surface. With respect to the
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DIAGRAM2. Size distribution of isodiametric particles in the disk membranes determined on the same electron micrographs as those on which the measurements shown in Diagram 1 were made. a p p e a r a n c e of these plaques, there was no difference observed in specimens t h a t h a d been crosslinked at _+0°C or at r o o m temperature. T h e particles in the particulate areas prot r u d e d r a t h e r high above the m e m b r a n e surface in some preparations (Fig. 3), while t h e y p r o t r u d e d considerably less in o t h e r specimens (Fig. 4). This difference was attributed to variations in distortion due to plastic flow. T h e size distribution of isodiametric particles was determined on fracture faces t h a t showed a m i n i m u m of obvious plastic deformation. T h e result is il-
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s J O S T R A N D AND K R E M A N
lustrated in Diagram 1. This diagram shows a maximum for diameters within the range 80-100 /k. It furthermore shows a highly asymmetrical distribution of the particle diameters with a large proportion of the diameters of the particles in the range 5070 A. For comparison, the particle size distribution was determined for disk membranes present in the same electron micrographs. Diagram 2 shows a somewhat nonrandom distribution of particle diameters within the range 50-80/~. All concave fracture faces associated with the outer segment plasma membrane exposed a rather smooth surface with a few
large particles protruding high above the surface. These particles were far apart, separated by distances of 500 to 1000 A (Figs. 1 and 5). Their diameters showed little variation, 95-110/k. Except for these particles the exposed surface appeared rather smooth as compared to the convex fracture face. The surface showed, however, some structure with particularly smooth areas surrounded by areas exposing a very fine particulate structure with the diameter of the particles measuring 40 to 60/k (Fig. 5). The smooth areas surrounded by these areas also showed a fine particulate structure with the diameters of the particles measuring 20 to 30/k, which are the dimen-
FIG. 1. Fracture involving three outer segments. The fracture exposes one smooth, (s), and one particulate, (p), face associated with the plasma membranes of these outer segments. Only a thin layer of extraceUular medium separates the two plasma membranes of two outer segments within a region marked by an arrow and the letter e. The double membrane disks of the outer segments are shown and the cytoplasm (c) next to the plasma membrane is exposed in all three outer segments. Notice on the particulate face plaques showing a smooth surface (pl). × 110 000. FIG. 2. Fracture face exposing the particulate face of an outer segment plasma membrane. Many plaques with a smooth or pebbled surface are present on this face. The parallel ridges at the edge of the particulate face show t h a t the material in the exposed surface has been subjected to considerable plastic deformation. × 130 000. Fro. 3. Particulate plasma membrane face showing considerable distortion due to plastic flow resulting in parallel ridges at the edge of the face and rather high elevations as shown by the long shadows cast on the surface of the exposed extracellular medium. An edge is indicated at the boundary of the particulate face (arrows). In lower left corner the fracture exposes a smooth membrane face. × 230 000. FIG. 4. High magnification picture of a particulate membrane face which did not show the signs of extensive plastic deformation t h a t can be observed in Figs. 2 and 3. × 460 000. FIa. 5. High magnification of the smooth membrane face showing the large particles protruding rather high above the surface and the areas with smooth surface (s) surrounded by less smooth areas. The particles observed in the smooth areas are of the dimensions of the metal grains formed during metal shadowing. The structure observed in the less smooth areas is in the 40- to 60-A range and cannot be caused by metal grains. T h e mixing of rather smooth areas with these differences in the dimensions of the observed graininess makes it possible to conclude t h a t the particulate appearance in the less smooth areas must reflect the structure of the exposed membrane surface, and t h a t it is not due to the structure of the metal coat. The smooth plaques thus are like internal standards showing the limit of resolution introduced by the structure of the metal. In the less smooth areas the real structure of the surface is partially distorted by the superimposed pattern formed by the metal grains. This makes it impossible to determine with any great precision the real size of the smallest elevations (particles} t h a t can be observed on the surface of the plasma membrane t h a t has been exposed. This is a demonstration of one limiting factor with respect to the specimen resolution t h a t can be achieved with freezefracturing. × 460 000. Fla. 6. Fracture exposing a particulate membrane face and an edge where the fracture plane has cut across the plasma membrane. This edge shows a particulate structure and measures about 100/k in width. × 160 000. Flas. 7, 8. Fracture across the outer segment plasma membrane exposing an edge with a particulate structure. Fig. 7, × 310 000; Fig. 8, x 340 000. Fla. 9. Fracture exposing an edge where it cuts across the plasma membrane. In this case the plasma membrane is viewed from the cytoplasmic side as in Fig. 8, while it was viewed from the peripheral medium in Figs. 6 and 7. Some large particles (arrows) are observed presumably reaching through the exposed part of the plasma membrane. These particles are likely to correspond to the large particles observed on the smooth membrane face. × 230 000.
258
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OUTER SEGMENT PLASMA MEMBRANE sions of the metal grains formed by aggregation of the metal atoms when shadowing the surface. T h e d i a m e t e r of these latter areas varied between 500 and 1300 A and t h e y could be r a t h e r densely arranged or could be missing from large areas. T h e large particles were located within the areas with particles in the size range 40-60 A. Cross and slightly oblique fractures t h r o u g h the plasma m e m b r a n e (Figs. 6-8), revealed a particulate structure of the m e m b r a n e . T h e thickness of the plasma m e m b r a n e was m e a s u r e d in cross fractures and was found to be about 100/~. Within the r o u n d e d plaques with a s m o o t h surface t h a t were exposed on the convex fracture face, the thickness of the m e m b r a n e was considerably less t h a n 100/~, and the cross fracture did not show any particulate structure. W h e n the fracture plane changed from an orientation parallel to the m e m b r a n e to cutting across the membrane, it was characteristic t h a t b o t h convex and concave fracture faces were associated with an edge with a width of a b o u t 100 A (Fig. 6-9). This edge exposed a particulate structure. T h e concave fracture face was in direct contact with the exposed cytoplasm. T h e r e was only a change in the angular orientation of the fracture plane at the transition from a fracture t h r o u g h the cytoplasm to the fracture passing along the m e m b r a n e {Fig. 10). No step could be observed which would have indicated t h a t the fracture passed t h r o u g h a layer interposed between the observed m e m b r a n e surface and the cytoplasm. At the convex m e m b r a n e face the same situation applied to the transition from the fracture passing t h r o u g h the extracellular
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m e d i u m to extending along the m e m b r a n e (Fig. 10). DISCUSSION
Location of Fracture Faces T h e locations of the two fracture faces, the particulate convex face, and the s m o o t h concave face could be established thanks to the edge structure t h a t was associated with b o t h fracture faces where the fracture planes changed from being oriented parallel to the m e m b r a n e surface to being perpendicular to this surface. Since in both cases an approximately 100-A edge with a particulate structure was observed, it can be concluded t h a t the two fracture faces were located at the two surfaces of the membrane. T h e fact t h a t no indications were found for the presence of any layer t h a t could have been coating the two exposed membrane surfaces also supports this conclusion. T h e cytoplasm exposed next to the concave fracture face reached without any interruption or any step all the way to the concave fracture face. T h e same relationship applied to the extracellular m e d i u m and the convex fracture face. No etching was done because it will always lead to the deposition of a layer of solutes t h a t can be misinterpreted to represent an additional layer belonging to the m e m b r a n e . In fact, without such a layer appearing it is difficult to be certain t h a t the surface has been etched.
The Exposed Materials With the location of the fracture faces known it is justified to refer to the two fracture faces as the cytoplasmic and the peripheral surfaces of the plasma membrane. T h e plasma m e m b r a n e thus exposes
FIG. 10. Two plasma membranes exposed running in parallel diagonally across the picture, one exposing a smooth membrane face and the other a particulate face. The smooth membrane face is viewed from the inside of the outer segment. The cytoplasm reaches all the way to the surface of this face without any step separating the fracture plane cutting through the cytoplasm and the fracture plane located along the membrane. The particulate face is viewed from outside the outer segment. The fracture face exposing the extracellular medium reaches to the particulate face without any step or rim separating them. × 230 000.
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closely arranged particles at its peripheral surface, while a relatively smooth surface with a few large particles characterizes its cytoplasmic surface. The discussion of these observations will be based on the concept that the topography of fracture faces is first hand determined by the properties of the material being exposed by fracturing. If this is not accepted as a reasonable basis for the discussion, freeze-fracturing would not allow a useful interpretation of the observations made with this technique. As a consequence of this concept, a fracture through the middle of a membrane cannot give rise to two fracture faces that are structurally different since both faces expose the same material. Double replicas showing fracture faces that are not complementary must therefore expose two different materials, which means that the fracture must be located at the boundary between materials of different properties (SjSstrand and Kreman, 1978). It then follows that a fracture through the middle of a membrane could expose two structurally different faces provided the fracture was located between two layers of materials of different composition within the membrane. Superimposed on the structural pattern at the fracture face that is determined by the properties of the exposed material is a pattern produced by plastic deformation of this material. This pattern is also likely to depend on the properties of the exposed material and can therefore contribute to reveal, and even enhance, differences in the properties of the materials at the fracture face. Plastic deformation is easily identified in the cases where it has caused certain regular patterns in the surface structure. These patterns can be related to plastic deformation because they have the characteristics of patterns according to which stress can be expected to develop in the material during fracturing. With one fracture face showing densely arranged particles, and another a rather
smooth surface, we conclude that these two faces expose materials with different properties. Since the two fracture faces were found to be located at the two surfaces of the membrane, it follows that the membrane exposes materials with rather different properties at its two surfaces. When considering the particle size distribution in the particulate face it is clear that this distribution is nonrandom, while the arrangement of the particles is random. This can either mean that the fracture has exposed particles and aggregates of particles present in the exposed material, or that plastic deformation has produced a pattern consisting of randomly arranged particles with a nonrandom size distribution. The latter alternative would require a factor that introduces a deviation from randomness with respect to the dimensions of elevations formed during plastic flow. A simple explanation for such a deviation is that the material contains particles in or close to the observed range of dimensions. We therefore conclude that the particle distribution reflects the presence at this surface of particles roughly in the range of dimensions shown by the observed size distribution of isodiametric particles. Since the range of dimensions of these particles corresponds to the dimensions of globular protein molecules, and since we know that a large proportion of the mass of the membrane consists of proteins, the simplest explanation for the particulate appearance of this face is that it exposes a material containing a high concentration of globular protein molecules. This conclusion appears even more justified when considering the fact it is highly likely that the fracture would have a higher tendency to pass along the surface of the protein molecules than to cut through the interior of the molecules. With this tendency we should expect that if the protein molecules become exposed they should show up as bumps in the fracture face. The relatively smooth cytoplasmic face revealed a material which appeared fairly
OUTER SEGMENT PLASMA MEMBRANE homogeneous with only a few large particles extending high above the surface. The relative smoothness of this surface can be explained by the presence at this surface of lipids. The plaques showing a smooth surface that were observed on the peripheral surface of the membrane are likely to correspond to areas consisting of a pure lipid bilayer. A comparison of the size, shape, and pattern of distribution of these plaques and the smooth areas at the cytoplasmic surface of the membrane shows a similar range of dimensions. The distribution of the smooth areas at the cytoplasmic surface showed the same type of irregularity as that of the plaques. It is therefore assumed that the smooth areas on the cytoplasmic surface correspond to the plaques observed on the peripheral surface of the plasma membrane. A Molecular Model
The aim of any analysis of the membrane structure, at a level of resolution at which freeze-fractured material can be analyzed, is to reveal the molecular structure of the membrane. The reliability of the concept regarding the molecular structure of a membrane that can be developed then depends upon how truthful freeze-fracturing reveals the native structure of the membrane, upon how detailed the information is that can be obtained with this technique, and how precisely the fracture planes can be located. Provided that the fracture planes have been located correctly, it then is clear that the information is limited by the limitations imposed by specimen resolution and by distortion due to plastic deformation. These factors determine the reliability of measurements of particle diameters and can make the obtained absolute values for the diameters become rather dubious. These measurements primarily fulfill the purpose of establishing that structural differences exist that characterize different types of membranes and membrane faces.
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As a consequence, even if we identify the particles as reflecting the presence of globular protein molecules, and of complexes of protein molecules, the dimensions of the particles cannot be translated directly into corresponding dimensions of the molecules from which the molecular weight could be deduced. Aggregates of protein molecules are likely to be formed artificially due to plastic deformation during facturing. The long ridges often observed at the edge of the particulate convex fracture face are likely to be such aggregates (Figs. 2 and 3). Distortion due to artificial aggregation is likely to have affected the size of particles less, the smaller the particles in the fracture face were. It therefore appears justified to consider the particles in the smaller size range as perhaps the most representative for the size range of particles present in the membrane and consequently to roughly correspond to the size range of protein molecules present in the membrane. When developing a concept regarding the molecular structure of the outer segment plasma membrane, we start out from the information which appears to be most reliable. The first piece of information belonging to this category is the thickness of the plasma membrane. This represents a very important piece of information. To this we can add the information that the two surfaces of the plasma membrane exposed materials with very different structural properties. Combined with the information regarding the thickness of the membrane this information makes it justified to consider the possibility that the membrane consists of two layers with different molecular composition. We now assume that the outer segment plasma membrane has a lipid concentration similar to that of other plasma membranes, the lipid and protein content of which has been determined. It then is reasonable to consider that the layered structure reflects a preferred location of the lipids to one layer and a domination of the proteins in the second layer. This would
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FIG. 11. Molecular model of plasma membrane that illustrates various possible types of molecular interactions in the membrane. The observed asymmetry of the membrane is interpreted to reveal the presence of two layers, one predominantly lipid and one predominantly protein layer. The interaction of protein molecules with the lipid bflayer is shown at 1 where one protein molecule interacts hydrophobically with the lipid layer, while another protein molecule interacts with the polar head of the lipid molecules. There is mutual interaction between the two protein molecules whereby a membrane bound multimolecular complex is formed. At 2, the lipid bilayer is not coated with proteins which would correspond to the situation at the plaques observed on the peripheral plasma membrane surface. At 3, a large multimolecular complex which penetrates the membrane. Such protein complexes can explain the presence of large particles on the cytoplasmic surface of the membrane. At 4, a single protein molecule bound to the lipid bilayer, and at 5, a multimolecular complex extending in the plane of the membrane. This complex could explain the fine particulate appearance of the cytoplasmic membrane surface. The perturbation of the lipid bilayer structure introduced by lipid-protein interaction can also explain this structure. At 6, a protein molecule bound through charge interaction to the polar heads of the lipid molecules in the bilayer.
explain the observation that the cytoplasmic surface of the plasma membrane showed a rather smooth surface which is characteristic for a cleavage along a lipid layer. It also explains the particulate appearance of the peripheral surface of the plasma membrane. The lipids would thus be predominantly located to the cytoplasmic surface of the membrane. We now assume that the lipid molecules cover a large part of this surface and that the basic arrangement of the lipid molecules is a bilayer arrangement. Since nonpolar interaction is characteristic for part of the membrane proteins there must be protein molecules in the membrane that tend to expose a large number of nonpolar groups at their surface. These groups can be assumed to be able to interact both with the hydrocarbon tails of the lipid molecules and with nonpolar groups exposed at the surfaces of other
protein molecules. To establish nonpolar interaction with the lipids in the bilayer it is required that there are openings in the layer of polar heads facing the protein layer. A nonpolar area on the surface of a protein molecule can then enter into contact with the hydrocarbon layer of the bilayer without sinking into the bilayer. This arrangement is illustrated in Fig. 11. It was proposed for the protein-lipid interaction in the outer segment disk membranes by SjSstrand and Kreman (1978). Consequently, most of the surface of the protein molecule is available for interaction with other protein molecules in the membrane and with ions and molecules in the surrounding medium. This arrangement must introduce a perturbation of the bilayer structure which could explain the fine particulate structure that was observed on the cytoplasmic surface of the membrane. The protein mole-
OUTER SEGMENT PLASMA MEMBRANE cules interacting this way with the lipid layer become firmly anchored to this layer. Other protein molecules can be conceived of as interacting with the polar head groups of the lipid molecules through charge interaction and with other protein molecules through contacts between nonpolar areas on the surfaces of the molecules as well as through charge interaction. A nonpolar surface on a protein molecule does not have to interact with lipids since nonpolar proteinprotein interaction is equally feasible. When other protein molecules interact with those anchored to the lipid bilayer, complexes of protein molecules are formed with the molecules of the entire complex being anchored directly or indirectly to the lipid bilayer. Protein molecules that are not anchored directly to the lipid bilayer will be more loosely bound to the membrane and will therefore be easier to solubilize than those anchored. Protein molecules linked to the membrane by charge interaction only would be particularly easy to remove. Since all these proteins are conceived of as being located to one layer about 50/~ thick (the lipid bilayer is assumed to account for about 50 A of the thickness of the membrane), differences in solubility will only reflect conditions of binding the protein molecules to the membrane and cannot be used as criterion for the location of the protein within the membrane, whether superficial or deep. The large particles observed on the cytoplasmic surface of the plasma membrane are interpreted to represent large complexes of protein molecules that penetrate the lipid bilayer. These complexes expose part of their surface to the extracellular medium and part of their surface to the cytoplasm. They seem to be suitable for transmembrane transport. The fine particulate structure observed on the cytoplasmic surface of the plasma membrane could also be caused by tightly arranged globular molecules in the 40- to 60-/~ range of dimensions. This arrange-
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ment of protein molecules is therefore considered as a second possibility to explain this appearance of the surface. It would increase the number of transmembrane proteins to accommodate a wide range of controls of membrane permeability. The packing density of the protein molecules in the protein layer cannot be estimated with any high degree of precision from the observed fracture faces because of the uncertainty regarding the extent to which plastic deformation had interfered to modify the surface topography of the convex fracture face. It is, however, likely that the protein molecules are fairly densely arranged but that they are not present in a close-packed arrangement. This means that the lateral surfaces of part of the protein molecules would be exposed to the extracellular medium. Such a loose packing would mean that the viscosity within this layer of the membrane could be low enough to allow diffusion of the protein molecules, and of complexes of protein molecules, in the plane of the membrane. Figure 11 illustrates schematically the various possible relationships between proteins and lipids that have been discussed here. This figure also includes an alternative interpretation which in certain aspects conforms with the Singer-Nicolson membrane model (1972). It differs from that model by the very asymmetrical arrangement of the protein and lipid molecules and by specifying the location of less firmly bound proteins. The two models differ with respect to the mode of interaction between the protein molecules and the lipid bilayer. The firstmentioned model leaves a larger part of the globular protein molecules in contact with an aqueous medium than the second model. In the latter model, so large parts of the protein molecules are exposed to a nonpolar, liquid environment that it seems possible that this environment could greatly affect the conformation of the protein molecules. This effect of the nonpolar environment would be less pronounced if the pro-
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tein molecules located in the lipid bilayer were associated in the form of multimolecular complexes. It is obvious that these models depict features of the membrane that are far beyond the actual observations. We therefore must distinguish between what interpretations are based on observations and what are reasonable deductions from these observations. The asymmetric arrangement of proteins and lipids in the membrane belongs to interpretations based on observations, while the various possible modes of interaction between proteins and lipids represent reasonable deductions. This illustrates the limitations of the freeze-fracturing technique and the great need of developing improved preparatory procedures that can further extend the range of specimen resolution toward molecular dimensions.
The Development of Outer Segment Disks The structural arrangement of the outer segment plasma membrane as deduced here has certain similarities to that of the disk membranes. In both cases it was concluded that the membrane was a layered structure. However, the size distribution of isodiametric particles differed in the two types of membrane. The cytoplasmic surface of both membranes exposed large particles, but these particles were larger and were considerably more numerous in the case of the plasma membrane. Areas with a fine particulate structure alternating with smooth areas were not observed in the case of the disk membranes except after extensive cross-linking. The outer segment disks develop as invaginations extending from the outer segment plasma membrane (SjSstrand 1959; Nilsson, 1964). It is then of interest to compare the two membranes in order to find some clues to the mechanism of disk formation. Both the plasma membrane and the disk membrane face the cytoplasm with a surface which is smooth in comparison to the surface facing away from the cytoplasm.
This means that during the process of formation the material in the two membranes that gives rise to smooth fracture faces is continuous. The fact that the plasma membrane differs structurally from the disk membrane in other respects shows that the disk membrane is not a simple invagination of the plasma membrane formed in connection with a general growth of the plasma membrane. Instead, it shows that a membrane that is structurally different, and structurally simpler than the plasma membrane, is formed at the invagination. This is an example where continuity does not necessarily mean that the structures involved are identical. The crista membranes and the innermost surface membrane of mitochondria represent a second example of such a relationship (SjSstrand, 1977). There are now several features that characterize the formation of the disks. First, the disk develops from a narrow zone of the plasma membrane which is extending perpendicular to the long axis of the outer segment. The length of this zone is not known, but from the fact that the invagination is easily observed in thin sections excludes that it is a tubular invagination. Second, the disk develops in one plane without any wrinkling of the newly formed membrane. Third, the growth of the disk stops when it has reached a certain dimension and the control of the size is executed with a high degree of precision. Fourth, the disk edge has a different structure when compared with that of the disk membranes. The fact that the disk grows in one plane without any random wrinkling of the newly formed membrane is here considered to require a particular organization of the components responsible for the assembly of the new membrane. The narrow zone of plasma membrane, from which the invagination develops, must during the first stage of disk formation be the growth zone where the new membrane is assembled.
O U T E R S E G M E N T PLASMA M E M B R A N E
It would be simple to explain the directionality of the membrane growth if the membrane assembly were restricted to this narrow zone and if it became part of the edge of the invagination. It will then be translocated further and further in the interior of the outer segment when the invagination increases in size. If we furthermore assume that the growth zone splits up into two zones in the guinea pig outer segment disks, it is possible to explain the formation of the incision that gives the disk its characteristic shape. The growth of the invagination involves the assemblage of lipids and proteins, and the assemblage of the edge structure which appears to be a predominantly protein structure. The proteins are synthesized in the inner segment and are transported through the cytoplasm to the invagination. The structure of the disk membrane that was proposed by SjSstrand and Kreman (1978) means that the membrane proteins are located at the side of the membrane facing away from the cytoplasm and that a lipid layer is interposed between them and the cytoplasm. This structure makes it likely that the assemblage of the membrane involves a more or less simultaneous incorporation of lipids and proteins which is possible at the edge of the invagination. This way the photopigment molecules would not have to penetrate into and pass through a lipid layer to arrive at their destination. While the site of synthesis of membrane proteins is known from the distribution of ribosomes in the receptor cell, the site of synthesis of the membrane lipids is not known. It is appealing to speculate that the lipids are synthesized in the edge structure of the disk and that this synthesis is one feature characterizing the growth zone. We then need only one mechanism for trapping the molecules in the growth zone which is one for trapping the proteins. There is one advantage of restricting the membrane assembly to the edge of the invagination. It will automatically favor a
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growth in one plane. Through protein-protein interaction between the two disk membranes, the disk would furthermore tend to form a planar compact structure with no tendency for the development of a vesicular disk. The size of the disk could possibly be controlled by the time the phospholipid synthesis can proceed before the enzyme systems involved become spontaneously inactivated.
The Absence of Complementary Fracture Faces It was characteristic for the examined material that no fracture faces were observed that could have been complementary to the two types of fracture faces described here. Every convex fracture face showed the dense particulate structure, and every concave face showed a relatively smooth surface. This means that there must be certain definite restrictions involved with respect to how the fracture proceeds at the plasma membrane when fracturing by sectioning. We will try to furnish an explanation for these observations by analyzing in a simple way how the fracturing of the material proceeds. We start out from the conclusion
7
Fro. 12. Fracturing by sectioning means that pieces of the frozen material are chipped off. The two concentric rings symbolize the plasma membrane of, for instance, an outer segment.
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A
S
C
\ \
Fro. 13. (A) The three stages characterizingthe chipping off of a piece of the frozenmaterial. 1, A crack is formed; 2, the two surfaces at the crack becomeseparated; and 3, a piece is lifted off the newlyformedsurface through the developmentof a vertical crack. (B-D) Consecutive stages in the chipping off of a piece of the frozen material. t h a t sectioning makes pieces of material become lifted off the newly formed surface in the form of small chips. Most of the crack through the material initiated by the moving knife edge proceeds in a plane located below the plane in which the knife edge moves. Th e pieces of material that are being chipped off therefore must be lifted from the level of the crack to the front face of the knife (Fig. 12). It seems reasonable to distinguish between three phases in this chipping off of the material. T h e first phase involves the formation of a crack through the material without much separation of the two surfaces at the crack. T he second phase involves a separation of the two surfaces at the crack which leaves a wedgeshaped space between these surfaces, with
the edge of the wedge facing in the direction the crack proceeds. T h e third stage involves the separation of a piece of material through a more or less vertical crack through the material and the translocation of this piece of material to the front face of the knife (Fig. 13A). During this process the material located above the fracture plane has been exposed to considerable deformation. T he piece of the material t hat eventually is chipped off must have been exposed to a horizontal force "compression" as the wedge-shaped space between the two surfaces of the crack is developing, before a vertical crack introduces a release in the stress to which the material is exposed. (We will not include other horizontal forces in this discussion.)
OUTER SEGMENT PLASMA MEMBRANE As a consequence, any structures that are present in the pieces that are chipped off must have been exposed to stress. The deformation caused by stress is likely to be only partially reversible {elastic deformation). Because the material that is being chipped off is lifted up from the new surface formed by the crack, it is clear that the material in this surface must be exposed to a force with a large vertical component. If the crack reaches the convex outer surface of the plasma membrane the orientation of this vertical component relative to the membrane surface will vary for different parts of the surface. Where the surface is oriented parallel to the direction in which the cleavage is initiated, this vertical force will tend to pull material out of the surface as the cleavage proceeds. The effect of such a pulling force on the exposed surface structure will obviously depend on the structure of the exposed material. Since the force can be considered to be acting uniformly over fairly large areas of the surface, it will have little effect if the material at the exposed surfaces is homogeneous, while it can be expected that it can have considerable influence on the structure of the surface in the case in which the material at the crack is nonhomogeneous, for instance, when the material has a particulate structure. The action of a force with a vertical orientation explains the observation made by SjSstrand and Kreman (1978) that particles in the exposed particulate surface of the outer segment disk membranes protruded further than what corresponded to the total thickness of the particulate layer. For those parts of the plasma membrane surface, which are oriented at an angle relative to the direction in which the cleavage is initiated, the vertical force acting at the fracturing will have one component that is oriented tangential to the surface. This means that the material in these parts is exposed to a sheering force. This sheering force will be greater the more the orientation of the surface of the plasma membrane
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A
C FIG. 14. The permitted (arrows) and the excluded (crossed over) fracture faces associated with the plasma membrane. approaches an orientation perpendicular to the direction in which the material is being cleaved. The sheering effect means that the material at the surface will be exposed to a force acting parallel to the surface, and oriented perpendicular to the edge of the area of exposed membrane. It offers an explanation for the appearance of a pattern located at this edge which consists of paraUel ridges. This pattern is shown in Figs. 2 and 3. Also in this case there should be little deformation introduced by this force in the case in which the material is homogeneous since the force is uniformly distributed over rather large areas of the surface. No deformation that could be linked to this force was observed at smooth membrane surfaces. The observations made so far have shown that the extent to which the material is deformed due to plastic flow varies considerably in different specimens. One factor that will have considerable influence on the extent of deformation is the temperature at which the fracturing is performed. The lower this temperature, the less deformation due to plastic flow is to be expected. It therefore appears essential to pursue the
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This is illustrated in Figs. 13 and 15. As the crack reached the membrane surface this stress must have involved parts of the membrane located just above the level to which the crack had progressed. This stress followed by deformation is likely to favor the crack cutting across the membrane over the crack following the first natural cleavage plane that it reaches. The cleavage across the membrane is facilitated by the arrangement of the molecules in the membrane, with lipid molecules predominantly oriFIG. 15. Deformationof a membranedue to plastic ented perpendicular to the plane of the and elastic deformationof the frozenmaterial as the membrane and with protein molecules with two surfacesof a crack becomeseparated. a globular shape. It is obvious that the same conditions as fracturing at the lowest possible tempera- discussed for B apply to C in Fig. 14. ture. From this we can conclude that only In Fig. 14 are shown the planes along double replicas could furnish fracture faces which the plasma membranes are fractured complementary to the two types of faces and the excluded fracture planes. The fig- observed here. However, since the fracture ure shows the two possibilities, a crack ex- planes are located between materials with panding from the outside of the outer seg- different properties, it is likely that the ment, and a crack that has cut through the complementary faces differ considerably plasma membrane on one side and ap- from what would be expected from a real proaches the plasma membrane from its complementarity, that is, that elevations on cytoplasmic side on the opposite side of the one surface correspond to depressions on outer segment. There is a simple rule with the complementary face. Considerable derespect to permissible and excluded frac- viation from complementarity can be preture paths. If the angle between the plane dicted particularly for the peripheral of the cleavage and the first surface of the plasma membrane surface because the parmembrane reached by the crack is larger ticulate structure has been enhanced by than 90 ° the fracture follows the first sur- plastic deformation, while the extracellular face reached by the crack as shown at A medium is little affected by plastic deforand D in Fig. 14. If this angle is less than mation as shown by the smooth fracture 90 ° the crack cuts across the membrane faces it exposes. and follows the more distant surface of the This analysis explains the fact that only membrane. This is illustrated at B and C in two types of fracture faces were observed, Fig. 14. In A and D, the preference for the and never any that could have been comlocation of the fracture plane can be ex- plementary faces to these two faces. The plained by these locations representing the deformation to which the membrane is exfirst natural cleavage plane reached by the posed as the crack proceeds in B and C in crack. Fig. 14, makes it highly unlikely that the In B and C another explanation must be crack would continue through the middle found. Considering case B it becomes clear of the membrane. If the fracturing still that the membrane must have been ex- would have exposed inner membrane faces, posed to considerable stress and finally to the lack of complementarity of the two breakage as the crack proceeded and ma- faces would then necessitate the conclusion terial was being chipped off the surface. that the membrane consists of two layers
OUTER SEGMENT PLASMA MEMBRANE with very different properties. T h e s e layers would then, however, be located in a way opposite to t h a t described here, with the s m o o t h material at the peripheral surface and the particulate material at the cytoplasmic surface. It would then be difficult to explain the presence of large particles extending high above the surface of the concave face and the thickness of the mem~ brane would t h e n be a b o u t 200/~. We wish to express our gratitude to Dr. J. Frank, Department of Physiology,for her generosity in allow-
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ing us to use her freeze-fracture equipment, and to Mrs. S. Beydler for her excellent technical assistance. This research was supported by NSF Grant 74-20390 and by a Biomedical Research Support Grant, University of California. REFERENCES NILSSON,S.-E. (1964) J. Ultrastruct. Res. 11, 581. SINGER, S. J., AND NICHOLSON,G. L. {1972) Science 175, 720. SJOSTRAND, F. S. (1959) Rev. Mod. Phys. 31, 301. SJOSTRAND,F. S. (1977) J. Ultrastruct. Res. 59, 292. SJOSTRAND, F. S., ANDKREMAN,M. (1978) J. Ultrastruct. Res. 65, 195.
