Structure and Function of Cell Membranes1,2 Marvin H

Home , Fluid mosaic model

STRUCTURE AND FUNCTION OF CELL MEMBRANES1,2 MARVIN H. STROMER AND WILLIAM J. REVILLE Iowa State University3, Ames, Iowa 50010

INTRODUCTION

The purpose of this presentation is to provide some backgrmnd information regarding the organization and function of cell membranes, in general, and then to focus attention on the membranes of the skeletal muscle cell. The interrelationships of the specialized membranes of the skeletal muscle cell will be discussed in order to develop a framework into which the succeeding three papers can be integrated.

In attempting to ascribe a purpose or function to any biological membrane in a cell, one is rapidly forced to conclude that membranes sequester or segregate components of the cell or the cell itself from its environment and regulate what external stimuli or components may enter the cell. This is the situation for a wide variety of cells, including muscle. Mernbranes inside a cell may maintain favorable microenvironments within closed tubular or vesicular systems and prmide a structural matrix on which enzymes may be favorably oriented to interact with substrates and on which certain essential features such as ribosomes may be located to maximize their efficiency. Processes such as protein synthesized for export may be regulated or modulated by interactions with membranes.

MEMBRANE MODELS a. Unit membrane model

Research on membranes from a variety of sources is a very active and diverse area of investigation. The two models that attempt to explain membrane structure and components are the unit membrane or bimolecular leaflet model of Danielli-Davson-Robertson (6, 2) and the fluid mosaic model of Singer and Nicolson (28). Danielli and Davson published their original unit membrane model in 1936, and Robertson (26) has, since the 1950's, been engaged in research which supports this

Journal Paper No. 5-7963 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Projects No. 1795, 1796, 2025. This review supported in part by grants from the American Heart Association (No. 71-679), and the Muscular Dystrophy Associations of America. * Presented at the 27th Annual Reciprocal Meat Conference of the American Meat Science Association, 1974. 3 Muscle Biology Group, Departments of Food Technology and Animal Science cooperating 299 model. The unit membrane consists of a core of phospholipid molecules with their fatty acid tails directed inward and the polar and ionic heads directed toward the outer margins of the membrane (Fig. 1). This phospholipid core is covered on each side by a monolayer of nonlipid material, thought by many to be protein or protein-like. The work of Robertson and associates has suggested that the two covering monolayers are chemically different from each Dtner (Fig. 4) and may thereby confer an inner and an outer orientation to membranes. The unit membrane is typically observed in the electron microscope as a structure about 75 x thick and composed of two dense lines each 20 8 in thickness, which are separated by a 35 8 space of decreased electron opacity as shown in the left schematic of Fig. 2. These same dimensions are obtained from electron microscope images and X-ray diffraction patterns of membranes. Robertson and others have obtained such images fram conventional intact membranes and from myelin and have obtained corroborating evidence from lipid model systems. Of the three possible arrangements shown in Fig. 2a-c for the components of the unit membrane (namely, polar lipid heads outward with nonlipid outer covering as in a, polar lipid heads directed inward toward the core of the membrane with a nonlipid outer covering as in b, and polar heads directed inward toward a nonlipid or protein core as in c, only the first satisfies the image seen in the electron microscope. The polar ends of the lipid molecdes and the covering layer produce the dark staining zones while the nonpolar carbon chains are responsible for the lighter zone. If either of the other two pmsibilities were correct, a membrane would consist of three dense lines and two light zones as in b or a single dense band as in c rather than the tripartite structure usually seen. Multiple layers of this tripartite membrane structure would generate myelin sheaths (Fig. 3) as described by Finean and Burge (10).

Permeability of unit membranes to small ions has been described as being accomplished by a carrier molecule binding to the ion and transporting it to the other side. Robertson, however, believes that, if an ion was bound by the covering protein coat at a particular site, an allosteric change in the protein molecule could occur and the ion could be transported across the protein layer and reside, temporarily, adjacent to the lipid core. If several ions accumulated next to the polar groups, the potential across the bilayer could increase to -400 mv. Experiments with model membrane systems indicate that such a potential is sufficient to break the lipid bilayer. This would afford a passage- way for ions to cross the remaining portion of the membrane.

Three pohts regarding the present concept of the unit membrane model as shown in Fig. 4 must be stressed. First, structural features of the model are based primarily on electron microscope and X-ray diffraction analysis of intact red blood cell and myelin membranes and lipid model systems. Second, the model clearly is based on a continuous lipid bilayer covered by continuous coats of nonlipid material, presumably protein. Third, certain specialized membranes in very rare instances present structural images that can not be interpreted as typical unit membranes (16). Nevertheless, many membranes in biological systems do present this appearance. Exterior -- ..

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interior Fig. 1. The Danielli-Davson membrane model consists of a lipid bilayer of unspecified thickness and two monolayers of protein. Note that the polar heads of the lipid molecules are directed outward and that the membrane is symmetrical. From Danielli and Davson, 1935, by permission of the author and the Wistar Press.

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Fig. 2. The typical appearance of a unit membrane and the dhensions observedI in the electron microscope are shown on the lef't side of this diagram. Two dark 20 8 lines separated by a 35 8 space give a total membrane thickness of 75 8. Three possible arrangements of the molecular components in the unit membrane model are shown in a, b and c. Reaction of electron-dense stains with membrane components dictates that the only arrangement possible is that sham in a. From J.D. Robertson, Arch. Int. Med., Feb. 1972, Vol 129, Copyright 1972, American Medical Assn. 3 01

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Fig. 3. Finean's model for the repeating unit in the myelin sheath was based on X-ray and electron microscope evidence and consists of two unit membranes which, although apparently symmetrical, give different reactions at their two surfaces to fixation and staining. From Finean, 1955 by permission of the author.

Flg. 4. Schematic diagram of Robertson's concept of the unit membrane. Comparison with Fig. 1 reveals that, although polar lipid heads still point outward, the two covering layers of non-lipid are chemically different and cause the membrane to be asymmetrical. From J. D. Robertson, Arch, Int. Med., Feb. 1972, Vol. 129, Copyright 1972, American Medical Assn. b. Fluid mosaic model

The second membrane model is called the fluid mosaic model (Fig. 5). Globular protein molecules are randomly dispersed in a lipid bilayer matrix. Orientatim of the protein molecules is such that the more polar portions of the molecule are directed outward toward the water interface while the less ionic portions of the molecules are found in the center of the lipid core (Fig. 6). This model is clearly based on a discontinuous distribution of protein in the membrane. Direct evidence for such a distribution comes from antibody localization and from freeze-etching experiments. Certain of the proteins known to be located in membranes were isolated and purified and used as antigens. Localization of antibodies to these antigens at the electron microscope level by Nicolson and Associates (19, 20, 21) clearly showed that the antigen was distributed randomly in the plane of the membrane. The appearance of membranes observed after the freeze etching technique (3O), parCicularly if the fracture plane occurs down the center of the lipid bilayer, is that of a fairly smooth matrjx containing a large number of usually randomly distributed particles. This is the characteristic appearance of membranes from nuclei, plasmalemma, mitochondria, bacteria and vacuoles (2, 3, 29). The size of the particle seems to be particularly characteristic of the membrane in which the particle resides. For example, in erythrory-te membranes these particles seem to be about 85 W in diameter. There also seems to be two classes of membrane proteins as reported by Singer's laboratory (27) and by Green's laboratory (24). The peripheral proteins are removed by mild treatments, dissociate free of lipids, and once dissociated, are relatively soluble in neutral aqueous buffers. The integral proteins require very strong treatments to free them from membranes, often remain associated with lipids after isolation, and become very insoluble if totally freed from lipid. In the fluid mosaic model, the peripheral proteins would be those only partly embedded in the lipid bilayer; the integral proteins are those more completely embedded in the lipid rnatrix and may even extend completely across the bilayer.

Thermodynamic considerations of membranes and their components were the main impetus for the developnent of the fluid mosaic model of cell membranes. These considerations are centered on the hydrophobic and hydrophylic interactions known to occur either in a membrane or at its surface. To have a favorable thermodynamic state, the nonpolar portions of the protein molecules and the fatty acid chains of the phospholipids should be as far removed as possible fromthe water in contact with the outer surface of the membrane and, if possible, should be buried in the interior of the lipid bilayer core. If the proteins formed a continuous coat as proposed in the unit membrane model, certain nonpolar regions of the protein molecules would have to be in fairly close contact with the water-rich environment. The thermodynamic consequences of this arrangement are described by Singer and Nicolson (28). Other evidence 303

Fig. 5. The fluid mosaic membrane model proposed by Singer and Nicolson consists of a phospholipid bilayer matrix with ionic and polar heads directed outward and globular protein molecules embedded in this matrix. These protein molecules may extend across the bilayer and are free to move in the plane of the membrane. Note that both the phospholipid and proteins are arranged in discontinuous fashion. From Singer and Nicolson, 1972. Copyright 1972 By the American Association for the Advancement of Science. c-

Fig. 6. A schematic drawing of the cross-section of a membrane as described by the fluid mosaic model. The lipid bilayer is arranged as described in Fig. 5. Two globular proteFns represented by heavy, wavy lines extend either partially or fully across the bilayer. The folded protein chains are arranged so that the more highly charged, ionic residues are protruding from the surface of the membrane while the nonpolar portions are embedded in the lipid bilayer. The arrow indicates a frequently encountered cleavage plane when the freeze etching technique is used. Ram Singer and Nicolson, 1972. Copyright 1972 by the American Association for the Advancement of Science. 304 from X-ray diffraction studies (g), from spin-labeling experiment, and from differential calorimetry indicates that, under physiological conditions, cell membranes contain a fluid lipid matrix which permits lateral movement of proteins. Thus far, myelin seems to be an exception to this observation. The fluid mosaic model is a much mwe dynamic structure than the more rigid unit membrane and would require lower energies of activation to move a protein in the plane of the membrane.

Although it is not possible to finally choose between the two models for membrane structure since neither has been conclusively proven or disproven, it is clear that the fluid mosaic model provides attractive alternatives to consider in the interpretation of recently published data. Because much of the supporting data for the unit membrane model were obtained from studies on red blood cell membranes and myelin sheaths, two highly specialized membrane systems, it seems reasonable to question whether information fr9m these membranes is directly and totally applicable to the wide diversity of membranes found in the cell.

CHBMICAL COIJlPOSITION

Coupled to the uncertainty as to which of these models best describes membrane structure is the variability in chemical composition of membranes. Red blood cell ghosts, a widely studied sc)urce of membranes, reportedly contain about 6@ protein and 4do lipid by weight. The lipid portion is about 7W$ phospholipid and nearly 304b cholesterol. In frog muscle sarcolemme (l), protein was about 6fi0, and lipids about 16$ (table 1) with cholester51 composing about 3@ of the lipid fraction. Rabbit muscle sarcolemma (16) contains 634 protein and l7.5$ lipid (table 1). The cholesterol content of membranes varies widely with the lowest levels found in mitochondrial membranes and the highest found in membranes prepared from brain or myelin sheaths.

MUSCLE CELL MEMBRANES By their unique overall arrangement, many of the membranes in the skeletal muscle cell attest to their residence in a highly differentiated and specialized cell. For reviews or additional information, the reader is referred to Peachey (22) and Franzini-Armstrong and Porter (11, 12). Because a skeletal muscle cell exports little, if any, protein for utilization outside the cell, the golgi apparatus and rough endoplasmic reticulum are rarely seen. Mitochondria are always present with numbers being dependent on the type of muscle cell in question. Red muscle fibers or cells normally contain a greater number of mitochondria than do white fibers from the same animal. Nuclei and their surrounding membranes have no particularly unique attributes in the skeletal muscle cell except for their location just under the cell membrane. c\! 0 +It- 0

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The cell membrane of a muscle cell is called the sarcolem. Unique to the skeletal muscle cell sarcolemma is a highly ordered array of deep invaginations that occur at each I band of the underlying myofibril. The continuity between the sarcolemma and the transverse or T-tubule membrane invagination has been demonstrated by Franzini-Armstrong and Porter (11) who observed the morphological connection in the electron microscope and by both Peachey and Schild (23) and Huxley (14) who followed the penetration of ferritin into the muscle cell. Ferritin penetrates only into the T-tubule and not into other membranes if the sarcolermna is intact. These observations regarding the continuity between T-tubules and sarcolemma have been confirmed both in freeze- etch preparatjms and by scanning electron microscopy. The T-tubule is not simply an open channel to the cell's environment. Electron micro- graphs of suitably fixed muscle preparations often show an unstructured matrix in the tubule. Hypotonic fixatives will usually cause distension of the tubules and hypertonic fixatives will cause collapse of the lumen. A paradoxical situation occurs if sucrose is included in the fixing solution. The more sucrose included in the fixative, the greater will be the T-tubule distension. This observation has led to the proposal that some form of regulatory gel exists in these tubules. Experiments with microelectrodes have shown that the T-tubules also function in conducting nerve impulses into the cell. Regular spacing of tubules at each I band obviously facilitates the coordinated shortening of all myofibrils in the cell. It should be stressed that this sys&em of regularly spaced T-tubules, which are oriented at right angles to the long axis of the cell, is properly considered to be formed by invaginations from the sarcolemma. Although they are in close proximity to the sarcoplasmic reticulum, the tubules of the sarcoplasmic reticulum are intracellular in origin and are analogous to the endoplasmic reticulum of other cells. b . Sarcoplasmic reticulum The sarcoplasmic reticular membranes are also called the L-system because they are oriented predominately parallel to the long &is of the cell. The L-system tubules have bulbous endings called the lateral or terminal cisternae, which are located adjacent to the T-tubule. A typical longitudinal section of vertebrate skeletal muscle will show a T-tubule cut in cross section and two of the bulbous lateral cisternae on opposite sides of the T-tubule. This tripartrite structure is called a triad. Its location within the I band varies slightly depending on the class of animal from which the muscle came. L-system tubules anastromose around the center of the sarcomere to form the fenestrated collar. The structures are shown in Fig. 7.

From a functional standpoint, the lateral cisternae are of great interest. Constantin, Franzini-Armstrong and Podolsky in 1965 (5) clearly localized calcium accumulation in the muscle cell in these cisternae. Previous studies had demonstrated that isolated sarcoplasmic reticular vesicles would accumulate calcium, but the unique calcium L

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Sarcotubu les 1) Fig. 7. A three-dimensional drawing of a portion of a skeletal muscle cell. Note that one transverse tubule and triad are located at the level of each Z-line in this example which is typical of frog muscle. In mammalian muscle, two transverse tubules and triads would be located bilaterally with respect to each Z-line. The anastrxnosing bracelet formed by the sarcoplasmic reticulum at the center of the sarcomere is called the fenestrated collar. From Bloom and Fawcett, A Textbook of Histolo& ninth edition, W. B. Saunders Co ., Publisher.

Fig. 8. Inesi's concept of the sarcoplasmic reticulum includes an overall thickness of 80 8 and an outer granular layer (shown here as a shaded layer) which is observed in the electron microscope in negatively stained membr e preparations. The 60 a spheres which are spaced approximately 1002 apart center-to-center depict the arrangement of the ATPase enzyme in the membrane. The in situ spacing of the ATPase may not be this uniform. Reproduced, with permission, from "Active Transport of Calcium Ion in Sarcoplasmic Membranes, " Annual Review of Biophysics and Bioengineering, Vol. 1, page 205. Copyright 1972 by Annual Reviews Inc. All right reserved. 3@3 accwrmlating ability of these cisternae was not appreciated fro these biochemical experiments . The finding by Winegrad (31, 32) thatm45Ca could be used to monitor the movement of calcium during contraction and during recovery from tetanus was direct evidence that calcium did move with respect to the sarcoplasmic reticulum. The experimentation by Greaser, Hasselbach, MacLennan, Inesi, Martonosi, Fleischer and others on isolated sarcoplasmic reticulum has significantly advanced our under- standing of the properties of the system. Worthy of special note are the localization of functionally vital sulfnydryl groups in the sarcoplasmic reticulum membranes with a heavy metal label as reported by Hasselbach and colleagues (13) and the report of the ability to dissociate and reconstitute functional sarcoplasmic reticulum vesicles published by Meissner and Fleischer (18). Inesi's concept of the SR membrane (14) includes a protein-rich outer layer shown here as shaded and ATPase enzyme molecules embedded in a lipid matrix (Fig. 8 ) .

SUMMARY

With this abbreviated overview of muscle cell membranes, one might ask what does this mean from the standpoint of the final end product; i.e., a desirable consumable portion of meat. In my opinion, some of the more direct implications are these. If any exogenous substance, be it enzymic, curing ingredients, etc., is going to be introduced into the muscle cell, it will most certainly encounter membrane barriers that must be crossed if interaction with the myofibrillar proteins is to occur. On the other hand, the pstmortem decline in ATP concentration and pH would be expected to decrease the ability of the sarcoplasmic reticular membranes to hold calcium. This release of calcium probably triggers postmrtem contraction and, in this way, influences tenderness of the muscle postmortem. This intracellular release of calcium would also raise the free calcium concentration sufficiently to activate the endogeneous calcium-activated muscle protease, which is specific for Z-line removal (4,7,8). It seem clear that muscles or aninrals having higher intramuscular concentrations of this protease should experience greater postmmtem tenderization. The corollary is that, if any limiting factor (e .g., calcium concentration, pH, etc .) required for optimum activity of the enzyme could be supplied to the cell, a rapid and hexpensive tenderization might be feasible.

ACKNOWLEDGEMENTS

We are gratefhl to Mary Arthur for skilled assistance in preparation in figures and to Joan Andersen and Barbara Hallman for typing the manuscript. 309 BIBLIOGRAPHY

1. Abood, L. G., K. Kurshasi, E. Brunngraber and K. Koketsu. 1966. Biochemical analysis of isolated bullfrog sarcolemma. Biochem. Biophys . Acta 112 :330. 2. Bayer, M. E. and C . C . Remsen. 1970. Structure of Escherichia -coli after freeze-etching. J, Bacteriol. lOl:3&. 3. Branton, D. 1966. Fracture faces of frozen membranes. Proc. Natl. Acad . Sci. 55 :1048. 4. Busch W. A., M. H. Stromer, D. E. Go11 and A. Suzuki. 1972. Ca2'-specific removal of Z lines from rabbit skeletal muscle. J. Cell Biol. 52:367. Constantin, L. L., C. Franzini-Armstrong and R. J. Podolsky. 5. Localization of calcium-accumulating structures in 1965. striated muscle fibers . Science 147:158. 6. Danielli, J. F. and H . A. Davson . 1936. A contribution to the theory of permeability of thin films. J. Cell. Comp. Physiol. 9:89.

Dayton, W. R., D. E. Goll, W. J. Reville, M. G. Zeece, M. H. Stromer, and R. M. Robsm. 1974. Purification and some properties of a muscle enzyme that degrades myofibrils. Fed. Proc. 33:lSO.

8. Dayton, W. R., 11. J. Reville, D. E. Goll, M. H. Stromer and R. M. Robson. 1973. Properties of a Ca2+-activated proteolytic enzyme from porcine muscle. J. him. Sci. 37:259. EngeIman, D. M. X-ray diffraction studies of phase 9. 1970. transitions in the membrane of Mycoplasma laidlawii. J. Mol. Biol. 47:ll5.

10. Finean, J. B. and R. E. Burge. 1962. The determination of the fourier transformation of the myelin layer from a study of swelling phenomena. J . Mol. Biol . 7:672.

11. Franzini-Armstrong, C. and K. R. Porter. 1964. Sarcolemmal invaginations constituting the T-system in fish muscle fibers. J . Cell Biol. 22:675. 12. Franzini-Armstrong, C. and K. R. Porter. 1965. The sarcoplasmic reticulum. Sci. Am. 2U:72.

Hasselbach, and L. G. Elfvin. 13 W . 1967. Structural and chemical asymmetry of the calcium-transporting membranes of the sarcotubular system as revealed by electron microscopy. J. Ultrastruct . Res. 17:598 14. Huxley, H. E. 1964. Evidence for continuity between the central elements of the triads and extracellular space in frog sartorius muscle. Nature 202 :lO67.

15. Inesi, G. 1972. Active transport of calcium ion in sarcoplasmic membranes. Biophysics and Bioengineering 1:191. 16. Madeira, V.M.C. and M. C. Antunesaadeira. 1973. Chemical composition of sarcolemma isolated from rabbit skeletal muscle. Biochem. Biophys . Acta. 298:230. 17. McNutt, N . S . and R. S . Weinstein. 1970. The ultrastructure of the nexus. A correlated thin-section and freeze-cleave study. J. Cell Biol. 47:666. 18. Meissner, G. and S . Fleischer . 1974. Dissociation arid reconstitution of f'unctional sarcoplasmic reticulum vesicles. J. Biol. Clem. 249:302.

19. Nicolson, G. L., R. Hyman and S. J. Singer. 1971. The two- dimensional topographic distribution of H-2 histocompatibility alloantigens on mouse red blood cell membranes. J. Cell. Biol. 50: 905.

20. Hicolson, G. L., S. P. Masouredis and S. J. Singer. 1971. Quantitative two-dimensional ultrastructural distribution of Rh*(D) antigenic sites on human erythrocyte membranes. Proc . Nat . Acad . Sci . 68 ~416. 21. Nicolson, G. L. and S. J. Singer. 1971. Ferritin-conjugated plant agglutinins as specific saccharide stains for electron microscope: Application to saccharides bound to cell membranes. Proc. Nat. Acad. Sci. 68:942.

22, Peachey, L. D. 1970. Form of the sarcoplasmic reticulum and T- system of striated muscle, pg. 273. In The Physiology and Biochemistry of Muscle as a Food 2. E. J. Briskey, R. Go Cassens and B. B. Marsh (eds .) . Univ . Wisconsin Press, Madison. 23. Peachey, L. D. and R. F. Schild. 1968. The distribution of the T-system along the sarcomeres of frog and toad sartorius muscles. J. Physiol. 194:249.

24. Richardson, S. H., H. 0. Hultin and D. E. Green. 1963. Structural proteins of membrane systems. Proc . Hat. Acad. Sci. 50:821.

25. Robertson, J. D . 1969. Molecular structure of biological membranes, pg . 1403. In Handbook of Molecular Cytology. A. Lima-de-Faria ( ed .) . North-Holland Publishing Co ., London. 26. Robertson, J. D. 1972. The structure of biological membranes. Arch. Int. Med. E9:2O2. 27. Singer, S . J. 1971. The molecular organization of biological membranes, pg. 145. In Structure and Function of Biological Membranes. L. I. RotEield (ed .) . Academic Press, New York. 28. Singer, S. J. and G.-L. Nicolson. 1972. The fluid mosaic model of the structure of cell membranes. Science 175:720.

29. Staehelin, L. A. 1968. The interpretation of freeze-etched artificial and biological membranes. J. Ultrastruct . Res. 22 :326. 30. Steere, R. L. 1969. Freeze-etching simplified. Cryobiology 5:306. 31. Winegrad, S . 1968. Intracellular calcium movements of frog skeletal muscle during recovery from tetanus. J . Gen. Physiol. 51:65.

32. Winegrad, S. 1970. The intracellular site of calcium activation of contraction in frog skeletal muscle. J. Gen. Physiol. 55:77. C. E. Allen: When we think about muscle contracting and relaxing, I've never quite pictured what happens t:, the fenestrated cDllar as we go from, say 3 1/2 microns to 1 1/2. Does it just fold, or how would you describe what is happening?

Marv Stromer: I don't know of any definitive studies in this area myself, but I can tell you what my ideas are. The situation here is that I see this fenestrated collar on the one hand as being a very elaborate interconnection between membranes from adjacent 1-bands; but on the other hand, I see it also as a very flexible component. It is pretty well accepted that, as the muscle cell contracts, its volume remains fairly constant, so as the length decreases, there must be a comensurate increase in diameter.. This fenestrated collar is arranged in such a way that it can, in fact, accommodate this lateral increase in dimension of the cell. The work of Winegrad indicates rather clearly that the calcium-45 used in his experiments did not rebind directly into the lateral cisternae, but was probably rebound somewhere near the fenestrated collar region. This may, in fact, be a very important region which functions in some way to bind back some of the calcium in the relaxation part of the contraction cycle. Those are my ideas and are strictly hypo- theses; I don't know of definitive studies in the area. M. D . Judge: Would pu comment on the location of the triad in mammals? Are there twice as many triads by virtue of their being at the A-1 junction, as compared to the system you showed? And are there any comments you could make on how such a development would take place, evolution-w ise? M. H. Stromer: I guess this discussion period could be called "Speculate with Stromer," because I'm not sure it is possible to come up with any definitive ideas here either. It's true that in the amphibian there tends to be one triad located right at the Z line, and in mammalian muscle there tends to be one located at each A-1 junction. The only thing I could hypothesize in that case is that we have moved a little bit further up the order, so to speak; perhaps there has been a need to differentiate and specialize beyond what the fish or amphibian needs for survival.

B . B . Marsh: Thank you very much, Marv, for a fine address . Our second paper is concerned more specifically with the sarcolemma. "Properties of the Sarcolemma," to be given by Dr. Cedric Matsushima of California State Polytechnic University -Pomona .