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Alterations in the morphology of rabbit skeletal muscle plasma membrane during membrane isolation
JOURNAL OF ULTRASTRUCTURE RESEARCH 8 6 , 2 7 7 - - 2 9 3
(1984)
Alterations in the Morphology of Rabbit Skeletal Muscle Plasma Membrane during Membrane Isolation AKITSUGU SAITO, STEVEN SEILER, 1 AND SIDNEY FLEISCHER
Department of Molecular Biology, Station B, Vanderbilt University, Nashville, Tennessee 37235 Received December 14, 1983, and in revisedform March 14, 1984 This study describes changes in morphology of plasmalemma from fast skeletal muscle in the course of tissue disruption and isolation. We find that conditions used to solubilize muscle contractile elements, in the isolation of plasmalemma, including the use of 0.6 M KC1 or 0.4 M LiBr in the cold (0-4"C), lead to altered plasmalemma morphology. The intramembrane particles, as revealed by freeze-fracture electron microscopy, become aggregated, leaving large domains devoid of particles. The square arrays in the P face and the complementary "pits" in the E face also become aggregated, sometimes forming sizeable aggregates of square arrays. Thin-section electron microscopy using tannic acid enhancement reveals plasma membrane associated components, on both cytoplasmic and extracellular faces, are largely reduced by the salt treatment. Pyrophosphate and magnesium at lower concentrations, sometimes used instead of high salt, also resulted in particle aggregation, although less pronounced than with concentrated salt solutions. The plasma membrane-associated proteins on both plasma membrane surfaces were likewise decreased by this treatment. Pyrophosphate treatment also separated the basal lamina from the plasma membrane. Incubation of muscle in isoosmotic sucrose does not alter the morphology of the plasmalemma with regard to particle aggregation, diminution of membrane associated components,, or separation of the basal lamina. Our observations suggest that membrane-associated protein and/or cytoskeleton constrains the mobility of components in the plane of the membrane and that removal of this constraint leads to aggregation of intramembrane particles.
The isolation of plasmalemma from fast skeletal muscle is replete with difficulties. Muscle plasmalemma constitutes only a small percentage of the muscle membranes. Muscle fibers are entrapped in an extensive collagen network, making them resistant to disruption. Contractile filaments, which comprise the bulk of the muscle mass, tend to "glue" the membrane components together making separation of plasma membrane difficult and the yield low. Many of the current procedures for the isolation of plasmalemma from muscle use extraction with concentrated salt solutions or pyrophosphate in order to solubilize the contractile protein and enhance recovery of the sarcolemma. Such procedures give rise Present address: Department of Pharmacology, Indiana University of Medicine, Krannert Institute of Cardiology, 1001 West 10th Street, Indianapolis, Ind. 46202.
to enriched plasmalemma preparations with high specific activity of diagnostic enzymes and other plasma membrane markers, including (Na+,K+)-ATPase (Reddy et al., 1976), adenylate cyclase (Grefrath et al., 1978), [3H]dihydroalprenolol binding (Grefrath et al., 1978), [3H]saxitoxin binding (Weigele and Barchi, 1982), as well as ion transport (Seiler, a, 1982,b). This study compares the morphology of isolated plasma membrane with the plasmalemma, in situ, and is one in a series dealing with morphology of isolated muscle membranes. During the course of these studies, it was observed that conditions used to disaggregate muscle contractile elements resulted in profound changes in the morphology of the muscle plasmalemma. MATERIALS AND METHODS Glutaraldehyde (EM grade), tannic acid (EM grade), and osmium tetroxide (crystalline) were obtained from
277 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Polysciences, Inc. (Warrington, Pa). Imidazole (grade III) and cacodylate acid (sodium salt) were obtained from Sigma Chemical Company (St. Louis, Mo.). Hepes (ULTROL grade) was obtained from CalbiochemBehring Corporation (La Jolla, Calif.). Density gradient grade sucrose was obtained from EM laboratories (Elmsford, N.Y.), and pyrophosphate (tetra sodium salt) was obtained from Fisher Scientific Company (Pittsburgh, Pa.).
Treatment of Tissue Samples Female New Zealand White rabbits weighing between 1.0 and 3.5 kg were used. The rabbits were sacrificed by cervical dislocation and all subsequent steps were carried out at 0-4"C. A portion of predominantly fast muscle from the hind leg was cut with a razor blade into 0.2 to 0.5-mm cubes. The muscle cubes were then incubated for various lengths of time in one of several media. For "normal" muscle the cubes were immediately fixed in glutaraldehyde fixative containing 2% glutaraldehyde, 8% (w/v) sucrose, 100 m M Na2 cacodylate, pH 7.4. Some samples were fixed in tannic acid/glutaraldehyde (as described below) for thin section. Otherwise, the muscle cubes were incubated in the following solutions for various lengths of time before being fixed. The "KC1 medium" contained 0.6 M KC1, 4 mMimidazole, pH 7.4. The "pyrophosphate medium" contained 20 mMNa4P2Or, 20 mMNaH2PO4, 1 m M MgC12, pH 7.4, and the "sucrose medium" contained 0.3 M sucrose, 5 m M imidazole, pH 7.4. After incubation, the samples were fixed in glutaraldehyde as described above for 2 hr at 4°C (for freeze-fracture), or in tannic acid/glutaraldehyde for thin section (see below).
Tannic Acid/Glutaraldehyde Fixation Samples for thin section were fixed with tannic acid/ glutaraldehyde fixative, which contained 1% (w/v) tannic acid, 2% (v/v) glutaraldehyde, 8% (w/v) sucrose, 100 m M cacodylate (sodium salt), pH 7.2-7.3. The fixative solution was prepared by adding tannic acid powder to the 2% glutarldehyde solution and readjusting the pH to 7.2-7.3 with concentrated NaOH. This solution was made fresh and used immediately to minimize oxidation of the tannic acid. Samples for thin section were additionally fixed in 1% OsO4, 2.4 m M CaC12, 60 m M NaC1, 100 m M v e r onal-acetate, pH 7.2, for 2 hr at 40C and then blockstained with 0.5% uranyl acetate in 2.4 mMCaC12, 60 mMNaC1, 100 m M veronal-acetate, pH 6.0, for 2 hr at room temperature (Farquhar and Palade, 1965). The samples were then dehydrated in increasing concentrations of ethanol, then propylene oxide, prior to embedding in Epon 812 (Luft, 1961) and sectioned using an LKB Ultratome (LKB Instruments, Inc., Rockville, Md.). The sections were double-stained (first with 1% uranyl acetate in 50% ethanol for 10 min, and
then with lead citrate (Sato, 1968)). The samples were examined using JEOL JEM- 100S and Hitachi H U - 11B electron microscopes. Samples for freeze-fracture or negative staining were first fixed with 2% (v/v) glutaraldehyde, 8% (w/v) sucrose, 100 m M cacodylate (sodium salt), pH 7.2. For freeze-fracture, the fixed samples were immersed in 25% glycerol and incubated for at least 2 hr. Replicas were obtained using a Balzers BAF 300 apparatus (Balzers High Vacuum Corp., Santa Ana, Calif.). The specimens were fractured in vacuum of 6 x 10 7 at - 120"C, and shadowed within 2 sec of fracturing with 2 n m of platinum, then carbon, at an incident angle of approximately 45 ° (Moor, 1969). Some samples were rotaryshadowed to improve resolution of the structural detail (Margaritis et al., 1977). During the shadowing process, the stage, containing the sample, was rotated 360°/ sec for 3 sec (three revolutions). Two nanometers of platinum was deposited during this process. The platinum-carbon replicas were floated onto 10% Chlorox, incubated overnight, and washed with deionizedwater. Negative staining was carried out on membrane fractions which were fixed 2-18 hr in 2.5% glutaraldehyde by adding 1/10 vol of 25% glutaraldehyde, 200 m M cacodylate (sodium salt), pH 7.2, to the sample suspended in 0.3 M sucrose, 5 m M imidazole, pH 7.4. The fixed sample was diluted to approximately 1 mg/ ml with 0.3 Msucrose, 5 mMimidazole, pH 7.4. Two microliters of the sample were applied to a supporting membrane of a very thin carbon-coated Parlodion film over a 400-mesh grid. The support grid was covered with the sample and excess sample removed by touching the grid with the corner of a piece of filter paper. One drop of 1% sodium phosphotungstate, pH 7.2, was applied to the grid (the phosphotungstate solution was freshly filtered through a 0.22-~tm pore size Millipore filter attached to a syringe). After 1 rain incubation with the stain, the grid was washed with five drops of staining solution and allowed to incubate for 2 min more. The excess stain was removed by touching with filter paper and the grid was dried under vacuum in the electron microscope to minimize contact with the air.
Purified Plasmalemma Vesicles Plasma membrane vesicles were isolated from KC1treated muscle as described (Seller, 1982b) unless stated otherwise. Isolated subcellular fractions were fixed with glutaraldehyde or tannic acid/glutaraldehyde in suspension, and then pelleted in a 75 Ti rotor at 30 000 rpm for 30-45 min. RESULTS
"Normal" Muscle Studies "Normal" refers to the muscle fixed directly in glutaraldehyde upon removal from the animal. The skeletal muscle plasmalem-
MORPHOLOGICALCHANGESDURING MUSCLEPLASMALEMMAISOLATION ma has a distinctive appearance observed by freeze-fracture electron microscopy. The caveolae or invaginations of the plasma membrane can be seen at lower magnification (Fig. 1). As already described (Rash et al., 1974; Ellisman et al., 1976), the P face can be observed, at higher magnification, to contain numerous "orthogonal" or "square" arrays (Fig. 2). The square arrays consist of subunits (Figs. 2 and 4) with a center-to-center spacing of 5 nm in either direction. The square arrays appear on the P face of the plasmalemma, with complementary distinctive arrays of pits observed on the E face (Figs. 3 and 5). When rotary shadowing is used, the square arrays in the P face can be seen to be composed of subunits which are plainer and more irregular in shape than usually seen when shadowing is unidirectional (Figs. 4 and 6). Rotary shadowing of the E face also reveals arrays of pits but with significantly reduced contrast compared with unidirectional shadowing (not shown). The reduced contrast is indicative of flatter structures. In addition, large particles are observed which are devoid of shadowing material, appearing to have a hole in the center. These structures are reminiscent of those reported for the isolated (Na+,K+)-ATPase in membrane discs as visualized by the same rotary-shadowing technique (Maunsbach, 1979). Thus, the square arrays subunits appear to be morphologically distinct from the (Na+,K+)ATPase. Tannic acid enhancement reveals details of the cell surface in thin section not readily seen by glutaraldehyde fixation and/or osmium staining alone (Figs. 7 and 8). The collagen fibers (CF) and basal lamina (BL) are intensely stained with tannic acid enhancement. Membrane associated components on the cytoplasmic side of the membrane can be observed (Fig. 8). In some instances the basal lamina separates from the plasma membrane revealing extracellular projections approximately 12 nm high and 14 nm wide. These extracellular projections sometimes appear to coincide with
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components associated on the cytoplasmic face of the plasma membrane (Fig. 9). Salt- Treated M u s c l e
When muscle was treated with a solution containing 0.6 M KC1, the caveolae disappeared within 3 hr, and intramembrane particle aggregation was observed which increased with time of exposure (Figs. 10-12). Freeze-fracture electron micrographs of the plasmalemmal P face revealed intramembrane particle aggregation which included square arrays (Figs. 11 and 24). Both the square arrays (on the membrane P face), and the arrays of pits (on the membrane E face), aggregated in the plane of the membrane (Figs. 22 and 23). This provides evidence that the square arrays and pits reflect the same structure in complementary faces of the membrane. The square arrays sometimes seemed to aggregate radially around puckering in the plane of the membrane (Fig. 11). Puckering of the membrane may have been responsible for the radial arrangement of the square arrays; alternatively, the radial aggregation of the square arrays may have caused the bulging in the membrane. The altered morphology of the plasma membrane varied somewhat even within the sample being studied. The amount ofintramembrane particle aggregation depends on "penetration" of the incubation medium as well as exposure time of the salt treatment. Thin-section electron microscopy after tannic acid/glutaraldehyde fixation (Figs. 13-15) also revealed that the plasmalemma morphology was altered by salt treatment. There appeared to be fewer membrane-associated components on the cytoplasmic and extracellular faces of the plasma membrane after prolonged exposure to the KC1 medium. The decrease in such surface components seemed to correlate with particle aggregation, giving rise to areas devoid of intramembrane particles. The lack of visualization of cytoplasmic associated protein on the plasmalemma was due to extraction by salt solutions, rather than lack of penetration of the tannic acid,
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since the sarcoplasmic reticulum within the muscle fiber appeared highly asymmetric, typical o f tannic acid e n h a n c e m e n t (see, for example, Fig. 15) (Saito et al., 1978). T r e a t m e n t o f the muscle with 0.4 M LiBr also resulted in disappearance o f the caveolae and aggregation o f the i n t r a m e m b r a n e particles. The m o r p h o l o g y o f the plasma m e m b r a n e as observed in thin section and freeze-fracture resembled that o f the muscle treated with 0.6 M KC1 (data not shown).
It has been noted by others that perfusion o f cardiac muscle (Ashraf, 1979; Frank, 1982) or skeletal muscle (Eastwood et al., 1979) with Ca2+-free solutions or solutions containing excess E G T A caused the basal lamina to separate away from the plasma membrane. Divalent cations m a y bind the basal lamina to the plasma m e m b r a n e . Pyrophosphate treatment could sequester Ca/+, leading to separation o f the basal lamina.
Pyrophosphate- Treated Muscle
Sucrose- Treated Muscle
T r e a t m e n t o f the muscle in a m e d i u m containing m a g n e s i u m p y r o p h o s p h a t e also resulted in aggregation o f the m e m b r a n e particles. The aggregation, observed by freeze-fracture, increased with time o f exposure (Figs. 16-18). Thin section electron m i c r o s c o p y shows that there was also a progressive decrease in c o m p o n e n t s o f the cytoplasmic and extracellular surfaces o f the p l a s m a l e m m a as observed by tannic acid/ g l u t a r a l d e h y d e fixation (Figs. 1 9 - 2 1 ) . T r e a t m e n t o f the muscle with pyrophosphate did not result in as extensive or as rapid aggregation o f particldes as with 0.6 M KC1 or 0.4 M LiBr treatment, and less contractile protein appeared solubilized. However, p y r o p h o s p h a t e appears to be particularly effective in separating the basal lamina from the plasma m e m b r a n e (Figs. 20 and 21).
W h e n muscle was preincubated in "isoosmotic sucrose," the caveolae o f the plasma m e m b r a n e disappeared after 3-6 hr, however, incubation in this m e d i u m did not induce i n t r a m e m b r a n e particle aggregation (Fig. 25). Therefore, prolonged preincubation alone did not result in particle aggregation. The differences between KCl-treated and sucrose-treated muscle are m o r e readily seen when lower-magnification electron micrographs o f the freeze-fractured P face are c o m p a r e d (Figs. 24 and 25).
Isolated Plasma Membrane Freeze-fracture electron micrographs o f higly purified plasma m e m b r a n e vesicles isolated from KCl-treated muscle (Seiler, 1982b) had a lower density o f i n t r a m e m brane particles (Fig. 26) than plasma m e m brane observed in n o r m a l muscle (Fig. 1).
FIGS. 1-5. "Normal" muscle plasmalemma, in situ, observed by freeze-fracture electron microscopy. Figure 1 shows a low-magnificationview of the plasma membrane P face revealing caveolae, the invaginations of the plasmalemma (as pointed out by the double-headed arrow). The asterisk indicates the portion of the P face shown at higher magnification in Fig. 2. In Fig. 2 the arrows point to the square arrays. In this and other freezefracture micrographs, the large arrowhead in the top fight-hand comer points the direction of replica shadowing. Figure 3 is a freeze-fracture replica of the plasmalemmal E face. The arrows point to arrays of pits in the membrane. Figures 4 and 5 are higher magnifications showing square arrays. The large arrowhead points to a large particle which, under rotary shadowing, lacks electron-opaquematerial at its center (see also Fig. 6). Figure 4 the plasmalemma P face after rotary shadowing. Figure 5, pits in the plasmalemma E face (not rotary-shadowed). The subunit center-to-center distances for the square arrays (P face) and pits (E face) are the same for both structures (about 5 nm). The pits are not observed when rotary-shadowed. Fig. 1, x 50 000; Fig. 2, x 100 000; Fig. 3, x 160000; Figs. 4 and 5, x 400000. FIG. 6. Rotory-shadowed replica of the plasma membrane P face. The square arrays (arrows) appear to be composed of somewhat irregular subunits. Taller intramembrane particles in the P face (arrowheads) which lack electron-opaque material in their center can be observed, x 150 000.
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FIG. 7. Thin-section electron micrograph of the muscle cell surface fixed with glutaraldehyde, but without tannic acid. The plasmalemma (PL) and sarcoplasmic reticulum (SR) appear as symmetric trilaminar membranes (arrows). BL, basal lamina, x 200 000. FIGS. 8 AND 9. Thin-section electron micrographs of a portion of a muscle fiber fixed in glutaraldehyde with 1% tannic acid. FIG. 8. Note the increased contrast with tannic acid enhancement (compare with Fig. 7). Many structures stain with higher contrast including the collagen fibers (CF), the basal lamina (BL), and membrane-associated material on the cytoplasmic side of the plasma membrane (large arrowheads). There are also projections from the plasma membrane on the extracellular surface (small arrowheads). Note the symmetrical appearance of the plasmalemma (PL) and the characteristic asymmetry (broad outer band of the sarcoplasmic reticulum (SR) membrane (arrow). x 160 000. FIG. 9. This inset shows a case in which the basal lamina has separated from the muscle fiber surface revealing characteristic projections 12 nm high and 14 nm wide (arrowheads) at the extracellular face of the membrane. x 160000.
Intramembrane particles in such isolated vesicles, when found, were usually aggregated, and square arrays were seen rarely (Fig. 26). T h e c y t o p l a s m i c a n d e x t r a c e l l u l a r
surface components were also not observed (Figs. 3 0 - 3 2 ) . We repeated the isolation of sarcolemma according to the method of Kono and Co-
MORPHOLOGICAL CHANGES DURING MUSCLE PLASMALEMMA ISOLATION
lowick (1961), which made use of incubation in 0.4 M LiBr and then 0.6 M KC1 to extract the contractile protein. Freeze-fracture electron microscopy showed aggregation of intramembrane particles (Seiler, 1982a). Thin-section electron microscopy revealed that the basal lamina remained attached to the plasma membrane in this preparation; however, the cytoplasmic face
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of the membrane was devoid of membrane associated proteins (not shown).
Sucrose-Isolated Plasma Membrane Control studies (illustrated by Fig. 25) indicated that the particles, observed by freezefracture, including square arrays, were not aggregated when muscle was subject to an isoosmotic sucrose solution of low ionic
FIGS. 10-12. Freeze-fracture electron micrograph of the plasmalemma P face after treatment of muscle with 0.6 M KC1. FIG. 10. Treatment was for 3 hr. The caveolae have been lost, and the intramembrane particles, including square arrays (asterisks), aggregated with time of preincubation, x 120 000. FIG. 11. Treatment was for 6 hr. The intramembrane particles became more aggregated. Note the radial aggregation of the square arrays around a bulge in the membrane (asterisk). x 120 000. FIG. 12. Treatment was for 12 hr. The intramembrane particles are highly aggregated. Arrows point to square arrays within the particle aggregate, x 120 000. FIGS. 13-15. Thin-section electron micrograph showing plasma membrane after treatment of muscle with 0.6 M KC1. The samples were fixed using glutaraldehyde/tannic acid. FIG. 13. Treatment was for 3 hr. Electron densities on the cytoplasmic side of the membrane (arrowheads) are pronounced. The collagen fibers (CF) and basal lamina (BL) have remained attached while the contractile elements were solubilized, x 160000. FIG. 14. Treatment was for 6 hr. During the salt treatment the cytoplasmic surface material is removed and/ or aggregated leaving membrane devoid of associated material (between small arrowheads on opposite faces of the membrane). The arrowheads point to surface material remaining on the external surface. Patches of associated cytoplasmic material can still be observed in the same region on the inner face of the membrane. The bridge structures of the terminal cisternae (TC) of sarcoplasmic reticulum remain even though most of the contractile elements have been extracted by the procedure x 160 000. FIG. 15. Treatment was for 12 hr. Practically all the cytoplasm-associated material has been extracted; the arrowhead and asterisk refer to residual remaining membrane and cytoplasmic material, respectively. The absence of tannic acid staining material on the cytoplasmic side of the plasma membrane is not the result of inadequate tannic acid penetration, since the characteristic asymmetry of sarcoplasmic reticulum (SR) using tannic acid enhancement (Saito et al., 1978) can be observed (arrow). The basal lamina appear largely intact, x 160 000. FIGS. 16-18. Freeze-fracture electron micrographs of the plasmalemma P face after the muscle cube was treated with pyrophosphate solution. In Fig. 16 treatment was for 3 hr. The pyrophosphate solution contained 20 m M pyrophosphate, 1 mMMgC12, 20 mMphosphate, pH 7.4. As for treatment with 0.6 MKC1, the caveolae disappeared in less than 3 hr, and the intramembrane particles aggregated with time (Fig. 10). The arrows point to square arrays. In Fig. 17 treatent was for 6 hr. There is more aggregation of the intramembrane particles. Treatment was for 12 hr in Fig. 18. The intramembrane particles are highly aggregated. The arrow points to a square array within intramembrane particle aggregates, x 160 000. FIGS. 19-21. Thin section of muscle with emphasis on the surface after treatment with pyrophosphate solution. Tannic acid enhancement was used in these figures. FIG. 19. Treatment was for 3 hr. The basal lamina (BL) is still associated with the plasmalemma. The asymmetry of the SR membrane can be observed (arrow). x 160 000. FIG. 20. Treatment with pyrophosphate for 6 hr caused separation of the basal lamina (no longer visualized) from the plasma membrane, revealing extracellular projections from the plasma membrane (small arrows). Membrane-associated components can also be seen (arrowheads) on the cytoplasmic side. x 160000. FIG. 21. Treatent was for 12 hr. The proteins on both sides of the membrane can be observed to have been extracted or aggregated, leaving patches of membrane devoid of membrane-associatedprotein (see region between the small arrowheads on opposite faces of the membrane). Some small amounts of cytoplasmic and extracellular associated material remain (in region between arrow and large arrowhead on opposite faces of the membrane), although much less than after shorter incubation times, x 160 000.
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strength in the cold. We, therefore, attempt to obtain plasma m e m b r a n e vesicles from muscle exposed only to buffered sucrose solutions. U n d e r these conditions plasma m e m b r a n e vesicles were located in denser regions o f a sucrose step gradient using isopycnic centrifugation, i.e., in a fraction o f the gradient enriched in sarcoplasmic reticulum vesicles (cf. legend, Fig. 27). The plasm a m e m b r a n e vesicles constituted less than 5% o f the total vesicle population, but the m o r p h o l o g y o f the plasma m e m b r a n e was preserved (Figs. 27-29) even though the basal lamina had separated away (Figs. 33 and 34). In thin-section electron micrographs, intracellular c o m p o n e n t s (presumably contractile a n d / o r cytoskeletal protein) were observed within the vesicle (Figs. 33 and 34), and the morphological characteristics o f muscle plasma m e m b r a n e , in situ, were retained. That is, membrane-associated c o m p o n e n t s were observed and rand o m l y distributed in thin section and by negative staining, on both faces o f the m e m brane, similar to that in situ (Figs. 35 and 36). DISCUSSION The p l a s m a l e m m a o f skeletal muscle in situ has a characteristic morphology. As observed by freeze-fracture electron microscopy, it reveals an appreciable density o f particles, more or less r a n d o m l y distributed, with a higher density o f particles on the
P face than E face. Square arrays are observed on the p l a s m a l e m m a P face, with c o m p l e m e n t a r y pits in the E face (see also Rash et aL, 1974; Ellisman, 1976). Characteristic caveolae are observed. In this study, thin-section electron m i c r o s c o p y using tannic acid e n h a n c e m e n t reveals m e m brane-associated structures at the cytoplasmic face that have not been previously reported for skeletal muscle. Some surface structures are also seen on the external face, especially under conditions which induce separation o f the basal lamina. These surface structures appear to be involved in the association o f the m e m b r a n e with the basal lamina. This study shows that conditions frequently used for subcellular fractionation o f muscle m e m b r a n e s result in extraction o f m e m b r a n e c o m p o n e n t s and aggregation o f c o m p o n e n t s in skeletal muscle plasmalemma. The rearrangement is induced by high salt and other reagents which are c o m m o n l y used to solubilize muscle contractile protein in the isolation o f the p l a s m a l e m m a (Kono and Colowick, 1961; K o n o et aL, 1964; Koketsu et aL, 1964; A b o o d et al., 1966; Sulakhe et al., 1971, 1973a,b; Severson et aL, 1972; Drabikowski and Zubrzycka, 1976; Boegman et al., 1970; A n d r e w and Appel, 1973; F e s t o f f a n d Engel, 1974; R e d d y et aL, 1976; A n d r e w et aL, 1974; Schapira et aL, 1974; Agapito and Cabezas, 1977; Smith et al., 1977; Barchi et al., 1979; Seiler, 1982b).
FIGS. 22--24. Freeze-fractureelectron micrographs of skeletal muscle after treatment with 0.6 M KC1 for 6 hr. The arrowheads in the upper right-hand corners of Figs. 22-24 show the direction of shadowing. FIG. 22. Aggregatedsquare arrays (arrows) in the plasmalemma P face. x 160 000. FIG. 23. Aggregatedpits (arrows) in the plasmalemma E face. Figures 22 and 23 also contain selected linear aggregatesof square arrays, 0.37 ~m in length (within verticalbars) on the plasma membrane P face and aggregated pit arrays on the E face. These micrographs illustrate one of several aggregationpatterns that both square arrays and pits can undergo and provide further evidence that square arrays and pits are from complementary halves of the membrane, x 160 000. FIG. 24. Lowermagnification of the plasma membrane P face shows the particle aggregation (asterisk) and regions of the plasma membrane relatively depleted of intramembrane particles, x 90 000. FiG. 25. Electron micrograph of the muscle plasmalemmal P face after treatment of the muscle in an isoosmoticsucrose solution for 12 hr. The caveolaehave been lost, but the square arrays and other intramembrane particles remain randomly distributed. Comparison of Figs. 24 and 25 reveals the extent of intramembrane particle aggregation by treatment in 0.6 M KC1 for 6 hr. x 90 000.
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FIGS. 26-29. Freeze-fracture micrographs of purified plasma membrane vesicles. Figure 26, purified plasmalemma vesicles were prepared as previously descibed from 0.6 M KCl-treated muscle (Seiler, 1982b). The intramembrane particle density of the vesicles is low compared with muscle plasmalemma in situ (Fig. 1). On occasion square arrays could be observed on the plasma membrane vesicles (small arrowhead). Figures 27-29 are from muscle homogenized in 0.3 M sucrose. These plasmalemma vesicles were prepared from muscle not treated with conditions used to solubilize contractile elements. Ground muscle was homogenized in cold 0.3 M
MORPHOLOGICAL CHANGES DURING MUSCLE PLASMALEMMAISOLATION These isolated p l a s m a l e m m a preparations have not been well characterized with respect to in situ morphology. This study suggests that the m o r p h o l o g y o f these preparations m a y be considerably different from that in situ. For the best characterized plasm a l e m m a preparation (Seiler, 1982a,b), the particle density is greatly decreased; the square arrays are rarely observed, and the caveolae are missing. The surface structures are likewise absent. The consequence o f such salt extraction is that the purified plasmal e m m a vesicles have a lower isopycnic density since they contain only a fraction o f the i n t r a m e m b r a n e particle density as compared with in situ. By homogenizing and sedimenting skeletal muscle in a sucrose m e d i u m devoid o f salt, the p l a s m a l e m m a vesicles have a higher isopycnic density and are morphologically m o r e comparable to plasma m e m b r a n e observed in situ. Such vesicles have a higher particles density and retain the characteristic square arrays. This study suggests that high-salt conditions extract m e m b r a n e - a s s o c i a t e d proteins, including the cytoskeleton, so that t r a n s m e m b r a n e c o m p o n e n t s are no longer anchored and tend to aggregate in the plane o f the m e m b r a n e . In this regard, the skeletal muscle p l a s m a l e m m a behave similar to the well-studied red blood cell, in that the cytoskeleton constrains lateral mobility o f t r a n s m e m b r a n e proteins and helps to maintain the cell structure (Elgsaeter and Branton, 1974; Elgsaeter et al., 1976; Nicolson and Painter, 1973; Nigg and Cherry, 1980; Yu and Branton, 1976; Shotton et al., 1978;
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Jacobson et al., 1982; Bennett and Stenbuck, 1979; Fowler and Bennett, 1978). Red blood cells can be hemolyzed with retention o f the cytoskeleton so that the ghost remains in the right-side-out configuration. When the c y t o s k e l e t o n is e x t r a c t e d , albeit u n d e r somewhat different conditions, the red cell m e m b r a n e gives rise to vesicles o f insideout orientation. Skeletal muscle plasmalemm a vesicles obtained in the absence o f salt are right-side out (Figs. 28, 29, and 33-35) in contrast with the inside-out vesicles which are derived when salt conditions are used (Seller, 1982b). The cytoskeletal system in the red cell has been extensively studied (Steck, 1974; Bennett and Stenbuck, 1979; Branton et aL, 1981). It consists o f spectrin and actin which are linked via ankyrin to band 3, the t r a n s m e m b r a n e anion channel. Cytoskeleton c o m p o n e n t s resemble constituents o f muscle filaments. Spectrin-like proteins have also been observed in nonerythroid cells, including e m b r y o n i c chicken myocytes, rat h e p a t o m a cells, and mouse fibroblasts ( G o o d m a n et al., 1981), as well as skeletal and heart muscle associated with the p l a s m a l e m m a (Nelson and Lazirides, 1983). By analogy, a similar cytoskeletal system appears to be associated with skeletal muscle p l a s m a l e m m a which m a y be rem o v e d by high ionic strength solutions used in the process o f p l a s m a l e m m a preparation. Square arrays are a characteristic o f skeletal muscle p l a s m a l e m m a and because o f their unique appearance allow easy visualization o f their aggregation a m o n g other aggregated particles. They have been observed
sucrose-5 re_Mrimidazole, pH 7.4, or 0.3 M sucrose-0.5 mM EDTA, pH 7.2, using a Waring blender (maximum speed for 1 min). Microsomes were obtained by differential centrifugation. In some cases microsomes were also obtained by rehomogenization of a low-speed sediment (8000 rpm for 10 min in a JA 10 rotor of a J2-21 Beckman centrifuge). The microsomes were then suspended on 0.3 M sucrose and layered onto a discontinuous sucrose gradient using a swing-bucket rotor (Beckman SW 27 rotor). The fraction, which was isopycnic at the 38-45% (w/w) sucrose interface, contained mostly terminal cisternae of sarcoplasmic reticulum and some plasmalemma vesicles (somewhat less than 5%). Figure 27 shows the E and P faces are replete with particles. At high magnification, these "heavy" plasmalemma vesicles can be observed to contain numerous square arrays in the P face (arrows) (Fig. 29) and pits in the E face (arrowheads) (Fig. 28). Note, the nonaggregated intact square arrays, yet, there are patches of membrane which are relatively devoid of particles. Fig. 26, x 160 000; Fig. 27, x 100 000; Figs. 28 and 29, x 200 000.
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~r
~q 1I~
O. Ll.lm
FIG. 30. Thin section of (purified) plasma membrane vesicles from KCl-t1"eated muscle, using tannic acid enhancement (Seller, 1982b). Practically no cytoplasmic or extracellular membrane associated proteins are observed, x 260 000. FIGS. 31 AND 32. Negative staining with 1% sodium phosphotungstate of purified plasmalemma vesicles from 0.6 M KCl-treated muscle (Seiler, 1982b).
MORPHOLOGICAL CHANGES DURING MUSCLE PLASMALEMMA ISOLATION in fast skeletal muscle (Rash et aL, 1974) a n d cardiac muscle (McNutt, 1975), b u t not in slow muscle (Ellisman et aL, 1976, 1978) and in adult, but not in fetal muscle (Schmalbruch, 1979). T h e y are r a n d o m l y oriented with respect to s a r c o m e r e - r e p e a t distances (Smith et al., 1975). T h e density o f square arrays varies with the position along the muscle fiber. R a s h and Ellisman (1974) found that square arrays are absent f r o m the postsynaptic region o f the neurom u s c u l a r junction. T h e y are at their highest density (approximately 5 0 / # m 2) at 0.5 m m f r o m the postsynaptic region and b e c o m e less concentrated further away f r o m the postsynaptic region (Rash a n d Ellisman, 1974). Square arrays h a v e also been found in the p l a s m a m e m b r a n e s o f other tissues (see R o b e n e k and G r e v e n , 1980, for a list) such as astrocytes (Landis and Reese, 1974), intestinal epithelia (Staehelin, 1972), a n d hepatocytes (Kreutziger, 1968). T h e i r function is not known, however, we h a v e shown, using rotary shadowing, that the subunits o f the square arrays are morphologically different f r o m the (Na+,K+)-ATPase (Maunsb a c h et al., 1979). T h e y do not a p p e a r to be gap junctions as originally thought (Staehelin, 1972), since the cells are separated b y a thick basal l a m i n a a n d no other cell is in direct contract with the muscle cell at that point (Rash and Ellisman, 1974). Aggregation o f i n t r a m e m b r a n e particles
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has been o b s e r v e d in heart (Ashraf and H a l verson, 1977) a n d kidney ( C o l e m a n a n d Duggan, 1976) when the organs r e m a i n ischemic at b o d y t e m p e r a t u r e for m o r e t h a n 30 min. Reperfusion o f the heart led to even m o r e aggregation (Ashraf and H a l v e r s o n , 1977) rather than to reverse aggregation. W h e n cardiac muscle is perfused with Ca 2+free media, the basal l a m i n a separates away f r o m the p l a s m a l e m m a ; the m e m b r a n e becomes m o r e permeable (Ashraf, 1979; F r a n k et al., 1982), and slight aggregation o f the i n t r a m e m b r a n e particles is observed. Neither separation o f the basal l a m i n a n o r aggregation o f the i n t r a m e m b r a n e particles was o b s e r v e d when perfusion was carried out at lower t e m p e r a t u r e s (Frank et aL, 1982). M o s t o f the m e m b r a n e proteins (in the muscle p l a s m a l e m m a which h a v e been studied, such as the acetylcholine receptor (Axelrod et aL, 1976, 1978; Prives et aL, 1982), the concanavalin A receptor ( T a n k et aL, 1982), and N a + channel (Almers et al., 1983; S t u h m e r et al., 1982), are constrained in the plane o f the m e m b r a n e probably b y interacting with m e m b r a n e - a s s o c i ated proteins or cytoskeleton. M o s t o f the p l a s m a m e m b r a n e preparation procedures utilize high ionic strength (typically 0.4 M LiBr, 0.6 M KCI, or both) to solubilize the contractile protein, enhancing the recovery o f the sarcolemma. We n o w find that such t r e a t m e n t has a p r o f o u n d
FIG. 31. These vesicles are practically devoid of membrane-associated protein. The asterisk denotes the area illustrated under higher magnification in Fig. 32. × 60 000. FIG. 32. Note the relative absence of membrane-associated material on the surface of the vesicle, x 260 000. FIGS.33 AND34. Thin sections of plasma membrane vesicles obtained from muscle homogenized in sucrose. The vesicles were prepared as described in legend to Fig. 27. FIG. 33. Tannic acid enhancement reveals both intracellular (arrowheads) and extraeellular membrane associated material (arrows). × 260 000. FIG. 34. Thin section of plasmalemma vesicle at lower magnification. In rare cases caveolae are retained (arrow). Filaments can be seen entrapped within the vesicle, x 100 000. FIGS. 35 AND36. Negative staining with 1% sodium phosphotungstate of a plasmalemma vesicle prepared from muscle homogenized in sucrose. FIG. 35. The extravesicular surface reveals components (arrows) which are similar in shape and size (12 x 14 nm) to those revealed using tannic acid enhancement (Fig, 14). These structures appear randomly spaced over the surface of the plasma membrane, x 260 000. FIG. 36. Lower magnification ofplasmalemma vesicle. Membrane-associated protein can readily be observed at the outer surface of the vesicle, x 100 000.
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deleterious effect on muscle plasma membrane morphology. Conditions used to isolate plasma membranes lead both to loss of membrane-associated proteins and to aggregation of intrinsic proteins within the plasma membrane. Many intrinsic membrane proteins remain with the plasma membrane, including (Na+,K+)-ATPase (Reddy et al., 1976), adenylate cyclase (Grefrath et al., 1978), dihydroalprenolol binding site (Grefrath et aL, 1978), saxitoxin binding site (Weigele and Barchi, 1982), as well as Na+-K + pump and Na+/Ca 2+ exchange transporter (Seiler, 1982a). This study shows that the plasma membrane vesicles, isolated using such conditions are not morphologically characteristic of the plasmalemma, in situ, and suggests a basis for such alteration. New methodology, which avoids the use of high salt or pyrophosphate, is clearly necessary to yield more representative plasmalemma fractions. The experiments presented here (Figs. 33-36) describe a lead in this direction. These studies were supported in part by grants from the National Institutes of Health (AM 14632) and the
Muscular Dystrophy Association. REFERENCES ABOOD, L. G., KURAHASI,K., BRUNNGRABER,E., AND KOKETSU, K. (1966) Biochim. Biophys. Acta 112, 330-339. AGAPITO, M. T., AND CABEZAS,J. A. (1977) Int. J. Biochem. 8, 811-817. ALMERS,W., STAr,~-IELD,P. R., ANDSTUHMER,W. (1983) J. Physiol. (London) 336, 261-284. ANDREW, C. G., AND APPEL, A., (1973) J. Biol. Chem. 248, 5156-5163. ANDREWS, C. G., ALMON, R. R., AND APPEL, S. H. (1974) J. Biol. Chem. 249, 6163-6165. ASHRAE,M., AND HALVERSON,C. (1977) Amer. J. Pathol. 88, 583-594. ASHRAE, M. (1979)Amer. J. Pathol. 97, 411-432. AXELROD, D., RAVDIN, P., KOPPEL, D. E., SCHLESSINGER, J., WEBB, W. W., ELSON, E. L., AND PODLESKI, T. R. (1976) Proe. Natl. Acad. Sci. USA 73, 45944598. AXELROD,D., RAVDIN,P., AND PODLESgJ,T. R. (1978) Biochim. Biophys. Acta 511, 23-38. BARCHI, R. L., WEIGELE, J. B., CHALIKIAN, D. M., AND MURPHY, L. E. (1979) Biochim. Biophys. Acta 550, 59-76.
BENNETT,V., ANDSTENBUCK,P. J. (1979) J. Biol. Chem. 254, 2533-2541. BOEGMAN,R. J., MANERY,J. F., ANDPrNTERIC,L. (1970) Biochim. Biophys. Acta 203, 506-530. BRANTON, D., COHEN, C. M., AND TYLER, J. (1981) Cell 24, 24-32. COLEMAN, S. E., AND DUGGAN, R. L. (1976) Lab. 1nvest. 35, 63-70. DP,ABIKOWSrd, W., AND ZUBRZYCKA,E. (1976) Recent Adv. Stud. Card. Struct. Metab. 9, 133-147. EASTWOOD, A. B., WOOD, D. S., BOCK, K. L., AND SORENSON, M. M. (1979) Tissue Cell 11, 553-566. ELGSAETER, A., AND BRANTON, D. (1974) J. Cell Biol. 63, 1018-1030. ELGSAETER, A., SHOTTON, D. M., AND BRANTON, D. (1976) Biochim. Biophys. Acta 426, 101-122. ELLISMAN, M. H., BROOKE, M. H., STAEHELIN,L. A., AND PORTER, K. R. (1976) J. CellBioL 68, 752-774. ELLISMAN,M. H., BROOKE,M. H., KAISER,K. K., AND RASH, J. E. (1978) Exp. Neurol. 58, 59-67. FARQUHAR, M. G., AND PALADE, G. E. (1965) J. Cell Biol. 26, 263-291. FESTOFF, B. W., AND ENGEL, W. K. (1974) Proc. NatL Acad. Sci. USA 71, 2435-2439. FLEISCHER, S., FLEISCHER, B., AND STOCKENIUS, W. (1967) J. Cell Biol. 31, 193-208. FOWLER, V., AND BENNETT, V. (1978) J. Supramol. Struct. 8, 215-221. FRANK, J. S., BEYDLER, S., KREMAN, M., AND RAU, E. (1980) Circ. Res. 47, 131-143. FRANK, J. S., RICH, T. L., BEYDLER, S., AND KREMAN, M. (1982) Circ. Res. 51, 117-130. GOODMAN, S. R., ZAGON, I. S., AND KULIKOWSKI,R. R. (1981) Proc. Natl. Acad. Sci. USA 78, 7570-7574. GREFRaTH, S. P., SMITH,P. B., ANDAPPEL, S. H. (1978) Arch. Biochem. Biophys. 188, 328-337. JACOBSON, K., ELSON, E., KOPPEL, D. E., AND WEBB, W. W. (1982) Nature (London) 295, 283-284. KOKETSU, K., KITAMURA, R., AND TANAKA, R. (1964) Amer. J. Physiol. 207, 509-5t2. KONO, T., AND COLOWlCK, S. P. (1961) Arch. Biochem. Biophys. 93, 520-533. KONO, T., KAKUMA,F., HOMMA, M., AND FUKUDA,S. (1964) Biochim. Biophys. Acta 88, 155-176. KREUTZlGER,G. O. (1968) in ARCENEOUX,C. J. (Ed.), Proc. of 26th Meeting of the Electron Microscopy Society, pp. 234-235, Claitors, Baton Rouge, La. LANDIS,D. M. D., AND REESE,T. S. (1974) J. CellBiol. 60, 316-320. LOFT, J. H. (1961) J. Biophys. Biochem. Cytol. 9, 409414. MARGARITIS,L. H., ELGSAETER,A., AND BRANTON, D. (1977) J. CellBiol. 72, 47-56. MAUNSBACH,A. B., SKRIVER, E., AND JORGENSEN, P. L. (1979) in SKou, J. C., AND NORBY, J. G. (Eds.), Na, K-ATPase: Structure and Kinetics, pp. 3-13, Academic Press, London.
MORPHOLOGICAL CHANGES D U R I N G MUSCLE PLASMALEMMA ISOLATION MOOR, H. (1969) Int. Rev. CytoL 25, 391-412. McNuTT, N. S. (1975) Circ. Res. 37, 1-13. NELSON, W. J., AND L~ZARIDES,E. (1983) Proc. Natl. Acad. Sci. USA 80, 363-367. NICOLSON, G. L., AND PAINTER, R. G. (1973) J. Cell Biol. 59, 395-406. NIGG, E. A., AND CHERRY, R. J. (1980) Proc. Natl. Acad. Sci. USA 77, 4702-4706. PRIVES, J., FULTON, A. B., PENMAN, S., DANIELS, M. P., AND CHRISTIAN, C. N. (1982) J. Cell Biol. 92, 231-236. RASH, J. E., AND ELLISMAN,M. H. (1974) J. Cell Biol. 63, 567-586. RASH, J. E., STAEHELIN,L. A., AND ELLISMAN,M. H. (1974) Exp. CellRes. 86, 187-190. REDDY, N. B., ENGEL, W. K., AND FESTOFF, B. W. (1976) Biochim. Biophys. Acta 433, 343-359. ROnENEK, H., AND GREVEN, H. (1980) J. Ultrastruct. Res. 72, 119-122. SAITO, A., WANG, C.-T., AND FLEISCHER,S. (1978) J. Cell Biol. 49, 601-616. SATO, T. (1968) J. Electron Microsc. 17, 158-159. SCHAPIRA,G., DOBOCZ,I., PIAU, J. P., AND DELAIN,E. (1974) Biochim. Biophys. Acta 345, 348-358. SCHMALBRUCH,H. (1979) Nature (London) 281, 145146. SMITH, P. B., AND APPEL, S. H. (1977) Biochim. Biophys. Acta 466, 109-122.
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SEILER, S. (1982a) Ph.D. Dissertation, Vanderbilt University. SELLER, S., AND FLEISCHER, S. (1982b) J. Biol. Chem. 257, 13862-13871. SEVERSON,D. t., DRUMMOND,G. I., AND SULAKHE,P. V. (1972) J. Biol. Chem. 247, 2949-2958. SHOTTON,D., THOMPSON,K., WozsY, L., ANDBRANTON, D. (1978)J. CellBiol. 76, 512-531. SMITH,D. S., BAERWALD,R. J., ANDHART, M. A. (1975) Tissue Cell 7, 369-382. STAEHELIN, L. A. (1972) Proc. Natl. Acad. Sci. USA 69, 1318-1321. STECg, T. L. (1974) J. CellBiol. 62, 1-19. STUHMER,W., STANFIELD,P. R., ANDALMERS,W. (1982) Biophys. J. 37, 171a. SULAYd-IE,P. V., FEDELESOVA,M., MCNAMARA,D. B., AND DHALLA,N. S. (1971) Biochem. Biophys. Res. Comm. 42, 793-800. SULAKHE, P. V., DRUMMOND, G. I., AND NG, D. C. (1973a) J. Biol. Chem. 248, 4150-4157. SULAKHE, P. V., DRUMMOND, G. I., AND NG, D. C. (1973a) J. Biol. Chem. 248, 4158-4163. TANK, D. W., WU, E. S., AND WEBB, W. W. (1982) J. Cell Biol. 92, 207-212. WEIGELE, J. B., AND BARCIJI, R. L. (1982) Proc. Natl. Acad. Sci. USA 79, 3651-3655. Yu, J., AND BRANTON,D. (1976) Proe. Natl. Acad. Sci. USA 73, 3891-3895.
(1984)
Alterations in the Morphology of Rabbit Skeletal Muscle Plasma Membrane during Membrane Isolation AKITSUGU SAITO, STEVEN SEILER, 1 AND SIDNEY FLEISCHER
Department of Molecular Biology, Station B, Vanderbilt University, Nashville, Tennessee 37235 Received December 14, 1983, and in revisedform March 14, 1984 This study describes changes in morphology of plasmalemma from fast skeletal muscle in the course of tissue disruption and isolation. We find that conditions used to solubilize muscle contractile elements, in the isolation of plasmalemma, including the use of 0.6 M KC1 or 0.4 M LiBr in the cold (0-4"C), lead to altered plasmalemma morphology. The intramembrane particles, as revealed by freeze-fracture electron microscopy, become aggregated, leaving large domains devoid of particles. The square arrays in the P face and the complementary "pits" in the E face also become aggregated, sometimes forming sizeable aggregates of square arrays. Thin-section electron microscopy using tannic acid enhancement reveals plasma membrane associated components, on both cytoplasmic and extracellular faces, are largely reduced by the salt treatment. Pyrophosphate and magnesium at lower concentrations, sometimes used instead of high salt, also resulted in particle aggregation, although less pronounced than with concentrated salt solutions. The plasma membrane-associated proteins on both plasma membrane surfaces were likewise decreased by this treatment. Pyrophosphate treatment also separated the basal lamina from the plasma membrane. Incubation of muscle in isoosmotic sucrose does not alter the morphology of the plasmalemma with regard to particle aggregation, diminution of membrane associated components,, or separation of the basal lamina. Our observations suggest that membrane-associated protein and/or cytoskeleton constrains the mobility of components in the plane of the membrane and that removal of this constraint leads to aggregation of intramembrane particles.
The isolation of plasmalemma from fast skeletal muscle is replete with difficulties. Muscle plasmalemma constitutes only a small percentage of the muscle membranes. Muscle fibers are entrapped in an extensive collagen network, making them resistant to disruption. Contractile filaments, which comprise the bulk of the muscle mass, tend to "glue" the membrane components together making separation of plasma membrane difficult and the yield low. Many of the current procedures for the isolation of plasmalemma from muscle use extraction with concentrated salt solutions or pyrophosphate in order to solubilize the contractile protein and enhance recovery of the sarcolemma. Such procedures give rise Present address: Department of Pharmacology, Indiana University of Medicine, Krannert Institute of Cardiology, 1001 West 10th Street, Indianapolis, Ind. 46202.
to enriched plasmalemma preparations with high specific activity of diagnostic enzymes and other plasma membrane markers, including (Na+,K+)-ATPase (Reddy et al., 1976), adenylate cyclase (Grefrath et al., 1978), [3H]dihydroalprenolol binding (Grefrath et al., 1978), [3H]saxitoxin binding (Weigele and Barchi, 1982), as well as ion transport (Seiler, a, 1982,b). This study compares the morphology of isolated plasma membrane with the plasmalemma, in situ, and is one in a series dealing with morphology of isolated muscle membranes. During the course of these studies, it was observed that conditions used to disaggregate muscle contractile elements resulted in profound changes in the morphology of the muscle plasmalemma. MATERIALS AND METHODS Glutaraldehyde (EM grade), tannic acid (EM grade), and osmium tetroxide (crystalline) were obtained from
277 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Polysciences, Inc. (Warrington, Pa). Imidazole (grade III) and cacodylate acid (sodium salt) were obtained from Sigma Chemical Company (St. Louis, Mo.). Hepes (ULTROL grade) was obtained from CalbiochemBehring Corporation (La Jolla, Calif.). Density gradient grade sucrose was obtained from EM laboratories (Elmsford, N.Y.), and pyrophosphate (tetra sodium salt) was obtained from Fisher Scientific Company (Pittsburgh, Pa.).
Treatment of Tissue Samples Female New Zealand White rabbits weighing between 1.0 and 3.5 kg were used. The rabbits were sacrificed by cervical dislocation and all subsequent steps were carried out at 0-4"C. A portion of predominantly fast muscle from the hind leg was cut with a razor blade into 0.2 to 0.5-mm cubes. The muscle cubes were then incubated for various lengths of time in one of several media. For "normal" muscle the cubes were immediately fixed in glutaraldehyde fixative containing 2% glutaraldehyde, 8% (w/v) sucrose, 100 m M Na2 cacodylate, pH 7.4. Some samples were fixed in tannic acid/glutaraldehyde (as described below) for thin section. Otherwise, the muscle cubes were incubated in the following solutions for various lengths of time before being fixed. The "KC1 medium" contained 0.6 M KC1, 4 mMimidazole, pH 7.4. The "pyrophosphate medium" contained 20 mMNa4P2Or, 20 mMNaH2PO4, 1 m M MgC12, pH 7.4, and the "sucrose medium" contained 0.3 M sucrose, 5 m M imidazole, pH 7.4. After incubation, the samples were fixed in glutaraldehyde as described above for 2 hr at 4°C (for freeze-fracture), or in tannic acid/glutaraldehyde for thin section (see below).
Tannic Acid/Glutaraldehyde Fixation Samples for thin section were fixed with tannic acid/ glutaraldehyde fixative, which contained 1% (w/v) tannic acid, 2% (v/v) glutaraldehyde, 8% (w/v) sucrose, 100 m M cacodylate (sodium salt), pH 7.2-7.3. The fixative solution was prepared by adding tannic acid powder to the 2% glutarldehyde solution and readjusting the pH to 7.2-7.3 with concentrated NaOH. This solution was made fresh and used immediately to minimize oxidation of the tannic acid. Samples for thin section were additionally fixed in 1% OsO4, 2.4 m M CaC12, 60 m M NaC1, 100 m M v e r onal-acetate, pH 7.2, for 2 hr at 40C and then blockstained with 0.5% uranyl acetate in 2.4 mMCaC12, 60 mMNaC1, 100 m M veronal-acetate, pH 6.0, for 2 hr at room temperature (Farquhar and Palade, 1965). The samples were then dehydrated in increasing concentrations of ethanol, then propylene oxide, prior to embedding in Epon 812 (Luft, 1961) and sectioned using an LKB Ultratome (LKB Instruments, Inc., Rockville, Md.). The sections were double-stained (first with 1% uranyl acetate in 50% ethanol for 10 min, and
then with lead citrate (Sato, 1968)). The samples were examined using JEOL JEM- 100S and Hitachi H U - 11B electron microscopes. Samples for freeze-fracture or negative staining were first fixed with 2% (v/v) glutaraldehyde, 8% (w/v) sucrose, 100 m M cacodylate (sodium salt), pH 7.2. For freeze-fracture, the fixed samples were immersed in 25% glycerol and incubated for at least 2 hr. Replicas were obtained using a Balzers BAF 300 apparatus (Balzers High Vacuum Corp., Santa Ana, Calif.). The specimens were fractured in vacuum of 6 x 10 7 at - 120"C, and shadowed within 2 sec of fracturing with 2 n m of platinum, then carbon, at an incident angle of approximately 45 ° (Moor, 1969). Some samples were rotaryshadowed to improve resolution of the structural detail (Margaritis et al., 1977). During the shadowing process, the stage, containing the sample, was rotated 360°/ sec for 3 sec (three revolutions). Two nanometers of platinum was deposited during this process. The platinum-carbon replicas were floated onto 10% Chlorox, incubated overnight, and washed with deionizedwater. Negative staining was carried out on membrane fractions which were fixed 2-18 hr in 2.5% glutaraldehyde by adding 1/10 vol of 25% glutaraldehyde, 200 m M cacodylate (sodium salt), pH 7.2, to the sample suspended in 0.3 M sucrose, 5 m M imidazole, pH 7.4. The fixed sample was diluted to approximately 1 mg/ ml with 0.3 Msucrose, 5 mMimidazole, pH 7.4. Two microliters of the sample were applied to a supporting membrane of a very thin carbon-coated Parlodion film over a 400-mesh grid. The support grid was covered with the sample and excess sample removed by touching the grid with the corner of a piece of filter paper. One drop of 1% sodium phosphotungstate, pH 7.2, was applied to the grid (the phosphotungstate solution was freshly filtered through a 0.22-~tm pore size Millipore filter attached to a syringe). After 1 rain incubation with the stain, the grid was washed with five drops of staining solution and allowed to incubate for 2 min more. The excess stain was removed by touching with filter paper and the grid was dried under vacuum in the electron microscope to minimize contact with the air.
Purified Plasmalemma Vesicles Plasma membrane vesicles were isolated from KC1treated muscle as described (Seller, 1982b) unless stated otherwise. Isolated subcellular fractions were fixed with glutaraldehyde or tannic acid/glutaraldehyde in suspension, and then pelleted in a 75 Ti rotor at 30 000 rpm for 30-45 min. RESULTS
"Normal" Muscle Studies "Normal" refers to the muscle fixed directly in glutaraldehyde upon removal from the animal. The skeletal muscle plasmalem-
MORPHOLOGICALCHANGESDURING MUSCLEPLASMALEMMAISOLATION ma has a distinctive appearance observed by freeze-fracture electron microscopy. The caveolae or invaginations of the plasma membrane can be seen at lower magnification (Fig. 1). As already described (Rash et al., 1974; Ellisman et al., 1976), the P face can be observed, at higher magnification, to contain numerous "orthogonal" or "square" arrays (Fig. 2). The square arrays consist of subunits (Figs. 2 and 4) with a center-to-center spacing of 5 nm in either direction. The square arrays appear on the P face of the plasmalemma, with complementary distinctive arrays of pits observed on the E face (Figs. 3 and 5). When rotary shadowing is used, the square arrays in the P face can be seen to be composed of subunits which are plainer and more irregular in shape than usually seen when shadowing is unidirectional (Figs. 4 and 6). Rotary shadowing of the E face also reveals arrays of pits but with significantly reduced contrast compared with unidirectional shadowing (not shown). The reduced contrast is indicative of flatter structures. In addition, large particles are observed which are devoid of shadowing material, appearing to have a hole in the center. These structures are reminiscent of those reported for the isolated (Na+,K+)-ATPase in membrane discs as visualized by the same rotary-shadowing technique (Maunsbach, 1979). Thus, the square arrays subunits appear to be morphologically distinct from the (Na+,K+)ATPase. Tannic acid enhancement reveals details of the cell surface in thin section not readily seen by glutaraldehyde fixation and/or osmium staining alone (Figs. 7 and 8). The collagen fibers (CF) and basal lamina (BL) are intensely stained with tannic acid enhancement. Membrane associated components on the cytoplasmic side of the membrane can be observed (Fig. 8). In some instances the basal lamina separates from the plasma membrane revealing extracellular projections approximately 12 nm high and 14 nm wide. These extracellular projections sometimes appear to coincide with
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components associated on the cytoplasmic face of the plasma membrane (Fig. 9). Salt- Treated M u s c l e
When muscle was treated with a solution containing 0.6 M KC1, the caveolae disappeared within 3 hr, and intramembrane particle aggregation was observed which increased with time of exposure (Figs. 10-12). Freeze-fracture electron micrographs of the plasmalemmal P face revealed intramembrane particle aggregation which included square arrays (Figs. 11 and 24). Both the square arrays (on the membrane P face), and the arrays of pits (on the membrane E face), aggregated in the plane of the membrane (Figs. 22 and 23). This provides evidence that the square arrays and pits reflect the same structure in complementary faces of the membrane. The square arrays sometimes seemed to aggregate radially around puckering in the plane of the membrane (Fig. 11). Puckering of the membrane may have been responsible for the radial arrangement of the square arrays; alternatively, the radial aggregation of the square arrays may have caused the bulging in the membrane. The altered morphology of the plasma membrane varied somewhat even within the sample being studied. The amount ofintramembrane particle aggregation depends on "penetration" of the incubation medium as well as exposure time of the salt treatment. Thin-section electron microscopy after tannic acid/glutaraldehyde fixation (Figs. 13-15) also revealed that the plasmalemma morphology was altered by salt treatment. There appeared to be fewer membrane-associated components on the cytoplasmic and extracellular faces of the plasma membrane after prolonged exposure to the KC1 medium. The decrease in such surface components seemed to correlate with particle aggregation, giving rise to areas devoid of intramembrane particles. The lack of visualization of cytoplasmic associated protein on the plasmalemma was due to extraction by salt solutions, rather than lack of penetration of the tannic acid,
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since the sarcoplasmic reticulum within the muscle fiber appeared highly asymmetric, typical o f tannic acid e n h a n c e m e n t (see, for example, Fig. 15) (Saito et al., 1978). T r e a t m e n t o f the muscle with 0.4 M LiBr also resulted in disappearance o f the caveolae and aggregation o f the i n t r a m e m b r a n e particles. The m o r p h o l o g y o f the plasma m e m b r a n e as observed in thin section and freeze-fracture resembled that o f the muscle treated with 0.6 M KC1 (data not shown).
It has been noted by others that perfusion o f cardiac muscle (Ashraf, 1979; Frank, 1982) or skeletal muscle (Eastwood et al., 1979) with Ca2+-free solutions or solutions containing excess E G T A caused the basal lamina to separate away from the plasma membrane. Divalent cations m a y bind the basal lamina to the plasma m e m b r a n e . Pyrophosphate treatment could sequester Ca/+, leading to separation o f the basal lamina.
Pyrophosphate- Treated Muscle
Sucrose- Treated Muscle
T r e a t m e n t o f the muscle in a m e d i u m containing m a g n e s i u m p y r o p h o s p h a t e also resulted in aggregation o f the m e m b r a n e particles. The aggregation, observed by freeze-fracture, increased with time o f exposure (Figs. 16-18). Thin section electron m i c r o s c o p y shows that there was also a progressive decrease in c o m p o n e n t s o f the cytoplasmic and extracellular surfaces o f the p l a s m a l e m m a as observed by tannic acid/ g l u t a r a l d e h y d e fixation (Figs. 1 9 - 2 1 ) . T r e a t m e n t o f the muscle with pyrophosphate did not result in as extensive or as rapid aggregation o f particldes as with 0.6 M KC1 or 0.4 M LiBr treatment, and less contractile protein appeared solubilized. However, p y r o p h o s p h a t e appears to be particularly effective in separating the basal lamina from the plasma m e m b r a n e (Figs. 20 and 21).
W h e n muscle was preincubated in "isoosmotic sucrose," the caveolae o f the plasma m e m b r a n e disappeared after 3-6 hr, however, incubation in this m e d i u m did not induce i n t r a m e m b r a n e particle aggregation (Fig. 25). Therefore, prolonged preincubation alone did not result in particle aggregation. The differences between KCl-treated and sucrose-treated muscle are m o r e readily seen when lower-magnification electron micrographs o f the freeze-fractured P face are c o m p a r e d (Figs. 24 and 25).
Isolated Plasma Membrane Freeze-fracture electron micrographs o f higly purified plasma m e m b r a n e vesicles isolated from KCl-treated muscle (Seiler, 1982b) had a lower density o f i n t r a m e m brane particles (Fig. 26) than plasma m e m brane observed in n o r m a l muscle (Fig. 1).
FIGS. 1-5. "Normal" muscle plasmalemma, in situ, observed by freeze-fracture electron microscopy. Figure 1 shows a low-magnificationview of the plasma membrane P face revealing caveolae, the invaginations of the plasmalemma (as pointed out by the double-headed arrow). The asterisk indicates the portion of the P face shown at higher magnification in Fig. 2. In Fig. 2 the arrows point to the square arrays. In this and other freezefracture micrographs, the large arrowhead in the top fight-hand comer points the direction of replica shadowing. Figure 3 is a freeze-fracture replica of the plasmalemmal E face. The arrows point to arrays of pits in the membrane. Figures 4 and 5 are higher magnifications showing square arrays. The large arrowhead points to a large particle which, under rotary shadowing, lacks electron-opaquematerial at its center (see also Fig. 6). Figure 4 the plasmalemma P face after rotary shadowing. Figure 5, pits in the plasmalemma E face (not rotary-shadowed). The subunit center-to-center distances for the square arrays (P face) and pits (E face) are the same for both structures (about 5 nm). The pits are not observed when rotary-shadowed. Fig. 1, x 50 000; Fig. 2, x 100 000; Fig. 3, x 160000; Figs. 4 and 5, x 400000. FIG. 6. Rotory-shadowed replica of the plasma membrane P face. The square arrays (arrows) appear to be composed of somewhat irregular subunits. Taller intramembrane particles in the P face (arrowheads) which lack electron-opaque material in their center can be observed, x 150 000.
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FIG. 7. Thin-section electron micrograph of the muscle cell surface fixed with glutaraldehyde, but without tannic acid. The plasmalemma (PL) and sarcoplasmic reticulum (SR) appear as symmetric trilaminar membranes (arrows). BL, basal lamina, x 200 000. FIGS. 8 AND 9. Thin-section electron micrographs of a portion of a muscle fiber fixed in glutaraldehyde with 1% tannic acid. FIG. 8. Note the increased contrast with tannic acid enhancement (compare with Fig. 7). Many structures stain with higher contrast including the collagen fibers (CF), the basal lamina (BL), and membrane-associated material on the cytoplasmic side of the plasma membrane (large arrowheads). There are also projections from the plasma membrane on the extracellular surface (small arrowheads). Note the symmetrical appearance of the plasmalemma (PL) and the characteristic asymmetry (broad outer band of the sarcoplasmic reticulum (SR) membrane (arrow). x 160 000. FIG. 9. This inset shows a case in which the basal lamina has separated from the muscle fiber surface revealing characteristic projections 12 nm high and 14 nm wide (arrowheads) at the extracellular face of the membrane. x 160000.
Intramembrane particles in such isolated vesicles, when found, were usually aggregated, and square arrays were seen rarely (Fig. 26). T h e c y t o p l a s m i c a n d e x t r a c e l l u l a r
surface components were also not observed (Figs. 3 0 - 3 2 ) . We repeated the isolation of sarcolemma according to the method of Kono and Co-
MORPHOLOGICAL CHANGES DURING MUSCLE PLASMALEMMA ISOLATION
lowick (1961), which made use of incubation in 0.4 M LiBr and then 0.6 M KC1 to extract the contractile protein. Freeze-fracture electron microscopy showed aggregation of intramembrane particles (Seiler, 1982a). Thin-section electron microscopy revealed that the basal lamina remained attached to the plasma membrane in this preparation; however, the cytoplasmic face
283
of the membrane was devoid of membrane associated proteins (not shown).
Sucrose-Isolated Plasma Membrane Control studies (illustrated by Fig. 25) indicated that the particles, observed by freezefracture, including square arrays, were not aggregated when muscle was subject to an isoosmotic sucrose solution of low ionic
FIGS. 10-12. Freeze-fracture electron micrograph of the plasmalemma P face after treatment of muscle with 0.6 M KC1. FIG. 10. Treatment was for 3 hr. The caveolae have been lost, and the intramembrane particles, including square arrays (asterisks), aggregated with time of preincubation, x 120 000. FIG. 11. Treatment was for 6 hr. The intramembrane particles became more aggregated. Note the radial aggregation of the square arrays around a bulge in the membrane (asterisk). x 120 000. FIG. 12. Treatment was for 12 hr. The intramembrane particles are highly aggregated. Arrows point to square arrays within the particle aggregate, x 120 000. FIGS. 13-15. Thin-section electron micrograph showing plasma membrane after treatment of muscle with 0.6 M KC1. The samples were fixed using glutaraldehyde/tannic acid. FIG. 13. Treatment was for 3 hr. Electron densities on the cytoplasmic side of the membrane (arrowheads) are pronounced. The collagen fibers (CF) and basal lamina (BL) have remained attached while the contractile elements were solubilized, x 160000. FIG. 14. Treatment was for 6 hr. During the salt treatment the cytoplasmic surface material is removed and/ or aggregated leaving membrane devoid of associated material (between small arrowheads on opposite faces of the membrane). The arrowheads point to surface material remaining on the external surface. Patches of associated cytoplasmic material can still be observed in the same region on the inner face of the membrane. The bridge structures of the terminal cisternae (TC) of sarcoplasmic reticulum remain even though most of the contractile elements have been extracted by the procedure x 160 000. FIG. 15. Treatment was for 12 hr. Practically all the cytoplasm-associated material has been extracted; the arrowhead and asterisk refer to residual remaining membrane and cytoplasmic material, respectively. The absence of tannic acid staining material on the cytoplasmic side of the plasma membrane is not the result of inadequate tannic acid penetration, since the characteristic asymmetry of sarcoplasmic reticulum (SR) using tannic acid enhancement (Saito et al., 1978) can be observed (arrow). The basal lamina appear largely intact, x 160 000. FIGS. 16-18. Freeze-fracture electron micrographs of the plasmalemma P face after the muscle cube was treated with pyrophosphate solution. In Fig. 16 treatment was for 3 hr. The pyrophosphate solution contained 20 m M pyrophosphate, 1 mMMgC12, 20 mMphosphate, pH 7.4. As for treatment with 0.6 MKC1, the caveolae disappeared in less than 3 hr, and the intramembrane particles aggregated with time (Fig. 10). The arrows point to square arrays. In Fig. 17 treatent was for 6 hr. There is more aggregation of the intramembrane particles. Treatment was for 12 hr in Fig. 18. The intramembrane particles are highly aggregated. The arrow points to a square array within intramembrane particle aggregates, x 160 000. FIGS. 19-21. Thin section of muscle with emphasis on the surface after treatment with pyrophosphate solution. Tannic acid enhancement was used in these figures. FIG. 19. Treatment was for 3 hr. The basal lamina (BL) is still associated with the plasmalemma. The asymmetry of the SR membrane can be observed (arrow). x 160 000. FIG. 20. Treatment with pyrophosphate for 6 hr caused separation of the basal lamina (no longer visualized) from the plasma membrane, revealing extracellular projections from the plasma membrane (small arrows). Membrane-associated components can also be seen (arrowheads) on the cytoplasmic side. x 160000. FIG. 21. Treatent was for 12 hr. The proteins on both sides of the membrane can be observed to have been extracted or aggregated, leaving patches of membrane devoid of membrane-associatedprotein (see region between the small arrowheads on opposite faces of the membrane). Some small amounts of cytoplasmic and extracellular associated material remain (in region between arrow and large arrowhead on opposite faces of the membrane), although much less than after shorter incubation times, x 160 000.
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strength in the cold. We, therefore, attempt to obtain plasma m e m b r a n e vesicles from muscle exposed only to buffered sucrose solutions. U n d e r these conditions plasma m e m b r a n e vesicles were located in denser regions o f a sucrose step gradient using isopycnic centrifugation, i.e., in a fraction o f the gradient enriched in sarcoplasmic reticulum vesicles (cf. legend, Fig. 27). The plasm a m e m b r a n e vesicles constituted less than 5% o f the total vesicle population, but the m o r p h o l o g y o f the plasma m e m b r a n e was preserved (Figs. 27-29) even though the basal lamina had separated away (Figs. 33 and 34). In thin-section electron micrographs, intracellular c o m p o n e n t s (presumably contractile a n d / o r cytoskeletal protein) were observed within the vesicle (Figs. 33 and 34), and the morphological characteristics o f muscle plasma m e m b r a n e , in situ, were retained. That is, membrane-associated c o m p o n e n t s were observed and rand o m l y distributed in thin section and by negative staining, on both faces o f the m e m brane, similar to that in situ (Figs. 35 and 36). DISCUSSION The p l a s m a l e m m a o f skeletal muscle in situ has a characteristic morphology. As observed by freeze-fracture electron microscopy, it reveals an appreciable density o f particles, more or less r a n d o m l y distributed, with a higher density o f particles on the
P face than E face. Square arrays are observed on the p l a s m a l e m m a P face, with c o m p l e m e n t a r y pits in the E face (see also Rash et aL, 1974; Ellisman, 1976). Characteristic caveolae are observed. In this study, thin-section electron m i c r o s c o p y using tannic acid e n h a n c e m e n t reveals m e m brane-associated structures at the cytoplasmic face that have not been previously reported for skeletal muscle. Some surface structures are also seen on the external face, especially under conditions which induce separation o f the basal lamina. These surface structures appear to be involved in the association o f the m e m b r a n e with the basal lamina. This study shows that conditions frequently used for subcellular fractionation o f muscle m e m b r a n e s result in extraction o f m e m b r a n e c o m p o n e n t s and aggregation o f c o m p o n e n t s in skeletal muscle plasmalemma. The rearrangement is induced by high salt and other reagents which are c o m m o n l y used to solubilize muscle contractile protein in the isolation o f the p l a s m a l e m m a (Kono and Colowick, 1961; K o n o et aL, 1964; Koketsu et aL, 1964; A b o o d et al., 1966; Sulakhe et al., 1971, 1973a,b; Severson et aL, 1972; Drabikowski and Zubrzycka, 1976; Boegman et al., 1970; A n d r e w and Appel, 1973; F e s t o f f a n d Engel, 1974; R e d d y et aL, 1976; A n d r e w et aL, 1974; Schapira et aL, 1974; Agapito and Cabezas, 1977; Smith et al., 1977; Barchi et al., 1979; Seiler, 1982b).
FIGS. 22--24. Freeze-fractureelectron micrographs of skeletal muscle after treatment with 0.6 M KC1 for 6 hr. The arrowheads in the upper right-hand corners of Figs. 22-24 show the direction of shadowing. FIG. 22. Aggregatedsquare arrays (arrows) in the plasmalemma P face. x 160 000. FIG. 23. Aggregatedpits (arrows) in the plasmalemma E face. Figures 22 and 23 also contain selected linear aggregatesof square arrays, 0.37 ~m in length (within verticalbars) on the plasma membrane P face and aggregated pit arrays on the E face. These micrographs illustrate one of several aggregationpatterns that both square arrays and pits can undergo and provide further evidence that square arrays and pits are from complementary halves of the membrane, x 160 000. FIG. 24. Lowermagnification of the plasma membrane P face shows the particle aggregation (asterisk) and regions of the plasma membrane relatively depleted of intramembrane particles, x 90 000. FiG. 25. Electron micrograph of the muscle plasmalemmal P face after treatment of the muscle in an isoosmoticsucrose solution for 12 hr. The caveolaehave been lost, but the square arrays and other intramembrane particles remain randomly distributed. Comparison of Figs. 24 and 25 reveals the extent of intramembrane particle aggregation by treatment in 0.6 M KC1 for 6 hr. x 90 000.
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FIGS. 26-29. Freeze-fracture micrographs of purified plasma membrane vesicles. Figure 26, purified plasmalemma vesicles were prepared as previously descibed from 0.6 M KCl-treated muscle (Seiler, 1982b). The intramembrane particle density of the vesicles is low compared with muscle plasmalemma in situ (Fig. 1). On occasion square arrays could be observed on the plasma membrane vesicles (small arrowhead). Figures 27-29 are from muscle homogenized in 0.3 M sucrose. These plasmalemma vesicles were prepared from muscle not treated with conditions used to solubilize contractile elements. Ground muscle was homogenized in cold 0.3 M
MORPHOLOGICAL CHANGES DURING MUSCLE PLASMALEMMAISOLATION These isolated p l a s m a l e m m a preparations have not been well characterized with respect to in situ morphology. This study suggests that the m o r p h o l o g y o f these preparations m a y be considerably different from that in situ. For the best characterized plasm a l e m m a preparation (Seiler, 1982a,b), the particle density is greatly decreased; the square arrays are rarely observed, and the caveolae are missing. The surface structures are likewise absent. The consequence o f such salt extraction is that the purified plasmal e m m a vesicles have a lower isopycnic density since they contain only a fraction o f the i n t r a m e m b r a n e particle density as compared with in situ. By homogenizing and sedimenting skeletal muscle in a sucrose m e d i u m devoid o f salt, the p l a s m a l e m m a vesicles have a higher isopycnic density and are morphologically m o r e comparable to plasma m e m b r a n e observed in situ. Such vesicles have a higher particles density and retain the characteristic square arrays. This study suggests that high-salt conditions extract m e m b r a n e - a s s o c i a t e d proteins, including the cytoskeleton, so that t r a n s m e m b r a n e c o m p o n e n t s are no longer anchored and tend to aggregate in the plane o f the m e m b r a n e . In this regard, the skeletal muscle p l a s m a l e m m a behave similar to the well-studied red blood cell, in that the cytoskeleton constrains lateral mobility o f t r a n s m e m b r a n e proteins and helps to maintain the cell structure (Elgsaeter and Branton, 1974; Elgsaeter et al., 1976; Nicolson and Painter, 1973; Nigg and Cherry, 1980; Yu and Branton, 1976; Shotton et al., 1978;
289
Jacobson et al., 1982; Bennett and Stenbuck, 1979; Fowler and Bennett, 1978). Red blood cells can be hemolyzed with retention o f the cytoskeleton so that the ghost remains in the right-side-out configuration. When the c y t o s k e l e t o n is e x t r a c t e d , albeit u n d e r somewhat different conditions, the red cell m e m b r a n e gives rise to vesicles o f insideout orientation. Skeletal muscle plasmalemm a vesicles obtained in the absence o f salt are right-side out (Figs. 28, 29, and 33-35) in contrast with the inside-out vesicles which are derived when salt conditions are used (Seller, 1982b). The cytoskeletal system in the red cell has been extensively studied (Steck, 1974; Bennett and Stenbuck, 1979; Branton et aL, 1981). It consists o f spectrin and actin which are linked via ankyrin to band 3, the t r a n s m e m b r a n e anion channel. Cytoskeleton c o m p o n e n t s resemble constituents o f muscle filaments. Spectrin-like proteins have also been observed in nonerythroid cells, including e m b r y o n i c chicken myocytes, rat h e p a t o m a cells, and mouse fibroblasts ( G o o d m a n et al., 1981), as well as skeletal and heart muscle associated with the p l a s m a l e m m a (Nelson and Lazirides, 1983). By analogy, a similar cytoskeletal system appears to be associated with skeletal muscle p l a s m a l e m m a which m a y be rem o v e d by high ionic strength solutions used in the process o f p l a s m a l e m m a preparation. Square arrays are a characteristic o f skeletal muscle p l a s m a l e m m a and because o f their unique appearance allow easy visualization o f their aggregation a m o n g other aggregated particles. They have been observed
sucrose-5 re_Mrimidazole, pH 7.4, or 0.3 M sucrose-0.5 mM EDTA, pH 7.2, using a Waring blender (maximum speed for 1 min). Microsomes were obtained by differential centrifugation. In some cases microsomes were also obtained by rehomogenization of a low-speed sediment (8000 rpm for 10 min in a JA 10 rotor of a J2-21 Beckman centrifuge). The microsomes were then suspended on 0.3 M sucrose and layered onto a discontinuous sucrose gradient using a swing-bucket rotor (Beckman SW 27 rotor). The fraction, which was isopycnic at the 38-45% (w/w) sucrose interface, contained mostly terminal cisternae of sarcoplasmic reticulum and some plasmalemma vesicles (somewhat less than 5%). Figure 27 shows the E and P faces are replete with particles. At high magnification, these "heavy" plasmalemma vesicles can be observed to contain numerous square arrays in the P face (arrows) (Fig. 29) and pits in the E face (arrowheads) (Fig. 28). Note, the nonaggregated intact square arrays, yet, there are patches of membrane which are relatively devoid of particles. Fig. 26, x 160 000; Fig. 27, x 100 000; Figs. 28 and 29, x 200 000.
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SAITO, SEILER, AND FLEISCHER
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FIG. 30. Thin section of (purified) plasma membrane vesicles from KCl-t1"eated muscle, using tannic acid enhancement (Seller, 1982b). Practically no cytoplasmic or extracellular membrane associated proteins are observed, x 260 000. FIGS. 31 AND 32. Negative staining with 1% sodium phosphotungstate of purified plasmalemma vesicles from 0.6 M KCl-treated muscle (Seiler, 1982b).
MORPHOLOGICAL CHANGES DURING MUSCLE PLASMALEMMA ISOLATION in fast skeletal muscle (Rash et aL, 1974) a n d cardiac muscle (McNutt, 1975), b u t not in slow muscle (Ellisman et aL, 1976, 1978) and in adult, but not in fetal muscle (Schmalbruch, 1979). T h e y are r a n d o m l y oriented with respect to s a r c o m e r e - r e p e a t distances (Smith et al., 1975). T h e density o f square arrays varies with the position along the muscle fiber. R a s h and Ellisman (1974) found that square arrays are absent f r o m the postsynaptic region o f the neurom u s c u l a r junction. T h e y are at their highest density (approximately 5 0 / # m 2) at 0.5 m m f r o m the postsynaptic region and b e c o m e less concentrated further away f r o m the postsynaptic region (Rash a n d Ellisman, 1974). Square arrays h a v e also been found in the p l a s m a m e m b r a n e s o f other tissues (see R o b e n e k and G r e v e n , 1980, for a list) such as astrocytes (Landis and Reese, 1974), intestinal epithelia (Staehelin, 1972), a n d hepatocytes (Kreutziger, 1968). T h e i r function is not known, however, we h a v e shown, using rotary shadowing, that the subunits o f the square arrays are morphologically different f r o m the (Na+,K+)-ATPase (Maunsb a c h et al., 1979). T h e y do not a p p e a r to be gap junctions as originally thought (Staehelin, 1972), since the cells are separated b y a thick basal l a m i n a a n d no other cell is in direct contract with the muscle cell at that point (Rash and Ellisman, 1974). Aggregation o f i n t r a m e m b r a n e particles
291
has been o b s e r v e d in heart (Ashraf and H a l verson, 1977) a n d kidney ( C o l e m a n a n d Duggan, 1976) when the organs r e m a i n ischemic at b o d y t e m p e r a t u r e for m o r e t h a n 30 min. Reperfusion o f the heart led to even m o r e aggregation (Ashraf and H a l v e r s o n , 1977) rather than to reverse aggregation. W h e n cardiac muscle is perfused with Ca 2+free media, the basal l a m i n a separates away f r o m the p l a s m a l e m m a ; the m e m b r a n e becomes m o r e permeable (Ashraf, 1979; F r a n k et al., 1982), and slight aggregation o f the i n t r a m e m b r a n e particles is observed. Neither separation o f the basal l a m i n a n o r aggregation o f the i n t r a m e m b r a n e particles was o b s e r v e d when perfusion was carried out at lower t e m p e r a t u r e s (Frank et aL, 1982). M o s t o f the m e m b r a n e proteins (in the muscle p l a s m a l e m m a which h a v e been studied, such as the acetylcholine receptor (Axelrod et aL, 1976, 1978; Prives et aL, 1982), the concanavalin A receptor ( T a n k et aL, 1982), and N a + channel (Almers et al., 1983; S t u h m e r et al., 1982), are constrained in the plane o f the m e m b r a n e probably b y interacting with m e m b r a n e - a s s o c i ated proteins or cytoskeleton. M o s t o f the p l a s m a m e m b r a n e preparation procedures utilize high ionic strength (typically 0.4 M LiBr, 0.6 M KCI, or both) to solubilize the contractile protein, enhancing the recovery o f the sarcolemma. We n o w find that such t r e a t m e n t has a p r o f o u n d
FIG. 31. These vesicles are practically devoid of membrane-associated protein. The asterisk denotes the area illustrated under higher magnification in Fig. 32. × 60 000. FIG. 32. Note the relative absence of membrane-associated material on the surface of the vesicle, x 260 000. FIGS.33 AND34. Thin sections of plasma membrane vesicles obtained from muscle homogenized in sucrose. The vesicles were prepared as described in legend to Fig. 27. FIG. 33. Tannic acid enhancement reveals both intracellular (arrowheads) and extraeellular membrane associated material (arrows). × 260 000. FIG. 34. Thin section of plasmalemma vesicle at lower magnification. In rare cases caveolae are retained (arrow). Filaments can be seen entrapped within the vesicle, x 100 000. FIGS. 35 AND36. Negative staining with 1% sodium phosphotungstate of a plasmalemma vesicle prepared from muscle homogenized in sucrose. FIG. 35. The extravesicular surface reveals components (arrows) which are similar in shape and size (12 x 14 nm) to those revealed using tannic acid enhancement (Fig, 14). These structures appear randomly spaced over the surface of the plasma membrane, x 260 000. FIG. 36. Lower magnification ofplasmalemma vesicle. Membrane-associated protein can readily be observed at the outer surface of the vesicle, x 100 000.
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deleterious effect on muscle plasma membrane morphology. Conditions used to isolate plasma membranes lead both to loss of membrane-associated proteins and to aggregation of intrinsic proteins within the plasma membrane. Many intrinsic membrane proteins remain with the plasma membrane, including (Na+,K+)-ATPase (Reddy et al., 1976), adenylate cyclase (Grefrath et al., 1978), dihydroalprenolol binding site (Grefrath et aL, 1978), saxitoxin binding site (Weigele and Barchi, 1982), as well as Na+-K + pump and Na+/Ca 2+ exchange transporter (Seiler, 1982a). This study shows that the plasma membrane vesicles, isolated using such conditions are not morphologically characteristic of the plasmalemma, in situ, and suggests a basis for such alteration. New methodology, which avoids the use of high salt or pyrophosphate, is clearly necessary to yield more representative plasmalemma fractions. The experiments presented here (Figs. 33-36) describe a lead in this direction. These studies were supported in part by grants from the National Institutes of Health (AM 14632) and the
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