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The sarcolemma of Aplysia smooth muscle in freeze-fracture preparations
TISSUE
& CELL
1976 8 (2) 241-258
Prlhli.slwd h.v Longmnn
Group l.td. Printed
in Great Britain
LISE PRESCOTT
and MILTON
THE SARCOLEMMA OF APLYSIA MUSCLE IN FREEZE-FRACTURE PREPARATIONS ABSTRACT.
W. BRIGHTMAN
SMOOTH
muscle cells in the sheath covering the visceral ganglion of with the techniques of freeze-fracture and conventional electron microscopy. The sarcolemma of these muscle cell invaginates to form myriad caveolae that have an intrinsic marker within their membrane. This intrinsic structure of the caveolar membrane is revealed by freeze-fracture and consists of rows of large particles in the outer half and matching grooves on the complementary inner half of the membrane. In thin plastic sections, parallel striations or shelves within the caveolar membrane appear to be the equivalent of the particles and grooves of the fractured membrane. Physical fixation of some specimens by rapid freezing in supercooled liquid nitrogen or in liquid helium suggests that in their natural state, the caveolar ostia are not uniform in size and that at any given moment a number of caveolae are flattened. When segments of the connective nerves which link the visceral ganglion to the cephalic ganglia are stretched in virro two to three times their in sifrr length, the caveolae lose their invaginated shape and are fully exposed to the extracellular space. The caveolar membrane, so stretched, is pulled into the line of fracture with the result that the large particles rather than the ostia appear on the cleaved surface. This flattening of the caveolae is reversible and suggests that they might serve as miniature stretch-receptors within the membrane of the smooth muscle cells. The caveolae are accessible to extracellular horseradish peroxidase but do not appear to pinocytose the protein. Aplwio
Smooth
culifomica
were examined
ceils has been noticed. These muscle cells are believed to account for the contractility of the sheath (Rosenbluth, 1963 ; Coggeshall, 1967). We have re-examined the sheath with the freeze-fracture technique in correlation with thin sectioning. Special attention was paid to the sarcolemmal features of these molluscan smooth muscle cells. They were compared with vertebrate smooth muscle cells in the toad (Byfo marinus) stomach, the mouse intestine and aorta, the turtle oviduct and vena cava, and found to have their own specific sarcolemmal characteristics. The carveolae of the Aplysia smooth muscle cells contain a natural marker which makes them a particularly suitable preparation for further combined chemical, physiological and ultrastructural studies of the sarcolemma belonging to the smooth muscle cell.
Introduction WHEN the visceral ganglion of Aplysia is stimulated mechanically or electrically, one can observe a slow retraction of the associated nerves (Strumwasser, 1961). The two long connectives which relate the visceral ganglion to the pleural ganglia in the cephalic region (Eales, 1921) take on a helical conformation. If the ganglion is left in artificial seawater for a while, the connectives will relax again. During previous ultrastructural examination of the sheath covering the ganglion and its nerves, the presence of smooth muscle
Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland. Received
I5 December
1975. 241
242
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Materials and Methods Aplysia californica,
weighing between 100 and 300 g were obtained from Pacific Bio-Marine Supply Co. (Venice, California) and kept from 2 to 15 days, at 15°C in a sea water aquarium through the whole year. The visceral ganglion and adjacent nerves were dissected out of the coelomic cavity and left for 5-60 min in physiological Aplysia saline without glucose: NaCl 460 mM, KC1 10 mM, CaCls 11 mM, McCls 50 mM and Tris-HCl 50 mM at a pH of 78 (Gainer and Wollberg, 1974). In a few Aplysia, the heart, the aorta, the crop and the stomach were also removed and prepared for conventional electron microscopy and freeze-fracture. Ultramicrotomy
The ganglion and viscera were fixed for 90 min in 2.5 % glutaraldehyde in a 0.1 M Na Cacodylate buffer and 0.8 M sucrose, at a pH of 7.6 (Henkart, 1975). Small pieces were then cut out of the viscera and from the sheath covering the nerves and body of the ganglion. After several rinses in buffer alone or buffer and sucrose, the tissues were osmicated for 3 hr, stained en bloc in 1% uranyl acetate (Heuser and Reese, 1973) dehydrated in methanol and embedded in Araldite (Ciba, Duxford, Cambridge, England). Thin sections were stained with 5 % uranyl acetate in 50 % methanol at 40°C for 20 min followed by lead citrate for 1 min (Venable and Coggeshall, 1965). A few specimens from the sheath were also fixed in 1.5% paraformaldehyde instead of glutaraldehyde. Samples from the toad (Bu~o marinas) stomach muscles, the turtle (Pseudemys elegans) vena cava and oviduct were fixed in 2.5 % glutaraldehyde by immersion buffered with 0.1 M sodium cacodylate at pH 7.4. Pieces from the intestine and aorta of mice, perfused with a mixture of aldehydes (Karnovsky, 1965), were also processed for electron microscopy. Freeze-fracture
Following fixation with glutaraldehyde, the specimens were rinsed briefly in buffer and left for 2 h in 20% glycerol. After glycerination, they were frozen in liquid Freon 22
AND
BRIGHTMAN
(monochlorodifluoromethane) at - 150°C and stored in liquid nitrogen. Some specimens were quenched directly in supercooled liquid nitrogen at -210°C (Moor, 1973) without prior fixation or glycerination. In one experiment, the specimens, kept for several hours at 4°C in Aplysia saline, unfixed and unglycerinated, were directly frozen in liquid helium at -270°C in a modified Van Harreveld apparatus (Heuser et al., 1975) and stored in liquid nitrogen. All frozen specimens were later fractured and replicated with platinum and carbon in a Balzer’s apparatus at - 110°C. Exposure to horseradish peroxidase
Shortly after removal from the coelomic cavity, a few connective nerves from Aplysia calijornica and one Aplysia dactylomela, were divided in segments 34 mm long and bathed for 30 min to 6 hr in a solution of Aplysia saline containing 2-5 mg/ml of horseradish peroxidase (Sigma Type VI, from Sigma Chem. Co., St Louis, MO.) at room temperature. The pieces were then fixed for 1 hr and incubated for l-3 hr at 4°C in 10 ml of 0.1 M Tris-HCl buffer to which 5 mg of diaminobenzidine and 0.1 ml of 1% Hz02 were added (Graham and Karnovsky, 1966). Following the incubation, the tissue was processed for electron microscopy. A few specimens were fixed for 2 hr prior to their exposure to horseradish peroxidase. Stretching experiment
After opening the coelomic cavity, each connective nerve was ligated at two points along its length. The distance between the two ligatures was then measured in situ. The ligated connectives were removed and remeasured when floating freely in the salt solution in order to establish their resting length. They were then pinned down, and while still submerged in saline, stretched to either two or three times their resting length, for 3, 10, 20, 35 and 55 min before being fixed with glutaraldehyde in the stretched position. Two nerves stretched twice their resting length, for 10 and 20 min were allowed to regain their resting state prior to fixation. All specimens were fixed for 1-2 hr, then prepared for freeze-fracture and thin sectioning.
SARCOLEMMA
OF SMOOTH
MUSCLE
Morphometry
A few replicas were systematically examined in the electron microscope and every membrane surface belonging to a muscle cell photographed. Measurements of membrane surface areas were made on enlarged micrographs, using a Hewlett-Packard calculator equipped with a digitizer and programmed to measure surface areas of any shape. Particle diameter and size of organelles were measured on micrographs with a calibrated IO x lens.
IN
APLYSlA
:a:
cellular space by means of an ostium. In many instances, several caveolae fuse to form a lobulated, tubular invagination of the sarcolemma (Figs. 3, 4). At high magnification, a tangential cut through the sarcolemma reveals a striated pattern in the caveolar membrane. The spacing of this pattern may vary between 80 and I50 A. When transversely sectioned, the striations appear as evenly spaced, short, pointed spicules emanating from the inner wall of the caveolae towards the caveolar lumen (Figs. 3, 9).
Observations In thin sections, the sheath surrounding the body of the visceral ganglion appears to be made of much collagen, numerous smooth muscle cells (SMC), some cell processes filled with dense granules and some glial elements (Fig. I ). In samples of the sheath taken around the nerves leading to or from the ganglion we have found mostly collagen and smooth muscle cells. These cells are not organized in bundles but isolated in the collagenous matrix, and establish very few contacts with their neighhours; gap junctions have been seen, but rarely. The SMC are long and thin with tiny lateral processes. The main axis of the SMC is roughly parallel to the long axis of the ganlion and the nerves. The full length of one cell is rarely seen in one section. The maximal length observed has been 8 pm. Their diameter in the region of the cell nucleus may vary between 0.8 and 5 pm, and, in the paranuclear region, between 0.2 and 6 /‘in. Embedded in the sarcoplasm are thick and thin filaments that are arranged parallel to the main axis of the cell but which do not form sarcomeres (Figs. I, 2, 12). In the cytoplasm, usually in the nuclear region, round bodies of variable electron density are observed (Fig. I). They resemble the pigmented granules seen in giant neurons of Aplysicr (Henkart, 1975). Hemidesmosomes which appear as focal mats of dense, amorphous. cytoplasmic material, lie subjacent to portions of the sarcolemma, opposite patches of extracellular fuzz (Fig. 12). The sarcolemma is frequently invaginated into the cytoplasm as simple flaskshaped caveolae or pits (Figs. I, 2, 3, 4). Each caveola communicates with the extra-
The plane of fracture follows a path inside the unit membrane and reveals two intramembranous faces: the cytoplasmic leaflet as seen from the extracellular space and the external leaflet viewed from the cytoplasm (Pinto da Silva and Branton. 1970). Extensive views of the sarcolemma may thus be obtained (Fig. 5). Of the two intramembranous faces of the sarcolemma, the cytoplasmic leaflet has four to five times more membrane particles. the ‘background’ particles, than the external leaflet. These particles, randomly distributed and 85 8, wide, are similar in size to the particles seen in tnost freeze-fractured biological membranes (Branton and Deamer. 1972). Two types of particle agglomerations are unique to the ApIysiu sarcolemma: ‘grills’ and ‘ridges’ (Figs. 5, 6, 15, 16, 18). The grills consist of particles, 95 A in diameter, which are uniformly spaced at 140 A intervals in parallel rows on the cytoplasmic half of the sarcolemmal membrane. Complementary images of the grills are rarely seen. The ridges are also situated on the cytoplasmic leaflet. and consist of linear arrays of particles which, in places, coalesce into an unbroken strand. On the external leaflet. matching grooves are easily recognized (Fig. 16). The most striking feature of the cleaved sarcolemma is the caveolar ostia whtcl; dominates the m/tire view. Fig. 5 represents a membrane area of 22 pm2 with an average of I4 ostia/pmZ. Behind each ostium there is a caveola invaginating the cytoplasm. The ostia are usually clustered in certain area\ of the sarcolemma. Wherever they are their density may vary from I to I 5/+mZ. However. areas of the sarcolemma as extensive as
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50 pm2 do not form ostia nor, therefore, caveolae. By tracing the perimeter of caveolae, as they appear in micrographs of replicas with the computer’s stylus, the amount of additional area contributed by the caveolae was estimated. The sarcolemma1 area, where the ostia are present was increased by as much as 23% by the caveolae. Since the caveolae invaginate into the cytoplasm, the internal structure of their membranes can only be visualized in crossfractured cells. A fracture along the caveolar membrane exposes two complementary faces (Fig. 4). One leaflet in continuity with the external leaflet of the sarcolemma has tightly clustered rows of particles about 125 8, in diameter, hence larger than the randomly dispersed particles (Fig. 4). The other leaflet in contact with the cytoplasm has matching grooves. The spacing of the rows and grooves, between 110 and 140 A, is comparable to the striated pattern seen on thin sections of caveolae. In comparison, caveolae in the vertebrate smooth muscles examined do not show any specific characteristics. In smooth muscles of the toad stomach, the turtle vena cava and oviduct, the mouse intestine and aorta,
Figs. 1, 2. N, nucleus; indicate caveolae. Fig. 1. Smooth
BRIGHTMAN
both faces of the fractured caveolar membranes are remarkably smooth (Fig. 14). Stretching effects
When the connective nerves are stretched to three times their resting length there is a striking difference in the appearance of the sarcolemma. Where there used to be clusters of ostia (Fig. 5), patches of agglomerated particles are present on the external leaflet (Fig. 7) and depressions with narrow, parallel grooves (Fig. 8) on the cytoplasmic leaflet. The caveolar membrane has been exteriorized or stretched into the same plane as the rest of the sarcolemma (Fig. 19). In thin sections many caveolae which used to be invaginations now look more like shallow undulations (Fig. 9). In some areas, the caveolae appear partially stretched and their ostia are much wider (Fig. 10). This appearance of the ostia is particularly common when the nerve is stretched only twice its resting length. The phenomenon is reversible within certain limits. A nerve stretched three times its resting length, for 1 hr, had several membranes that appeared to be ruptured when examined in replicas. Another nerve stretched three times its resting length, for 20 min,
F, filaments;
CL, collagen;
muscle cell cut transversely
Fig. 2. Longitudinal
AND
through
section of a muscle cell process.
D, pigmented the nuclear
bodies. Arrows region.
x 15,000.
x 22,000.
Fig. 3. Higher magnification of a cellular area containing indicates striations in a tangentially cut caveolar membrane. x 91,000.
several caveolae. Arrow 0, ostium of a caveola.
Fig. 4. Replica of a fracture across the cytoplasm. L, cytoplasmic leaflet of the sarcolemma. X and Y represent the two complementary faces of the plane of fracture inside the caveolar membrane. One leaflet, X, has rows of particles, the other leaflet, Y, has matching grooves. x 110,000. Fig. 5. Panoramic view of the cytoplasmic leaflet of the sarcclemma seen from the extracellular space. Arrow indicates a group of ostia. Out of the plane of fracture liut behind each ostium on this membrane there is a caveola invaginating into the cytoplasm. R, linear array of particles, called ‘ridge’. G, characteristic of this membrane, is an agglomeration of large particles that we have called ‘grill’. x 38,000. Fig. 6. Cytoplasmic leaflet of the sarcolemma. Compare the small ostia (arrows) with the larger ones on the left. G, grill. Crystallization of the extracellular space fluid in upper right corner. x 64,000.
PRESCOTT
took also 20 min to return to its original length. But nerves stretched two to three times their resting length for less than 10 min could regain their resting length almost immediately. At the ultrastructural level these replicated nerves showed no evidence of their previous stretched state. Freezing without prior jixatiotz and glycerination
In the specimens frozen directly in supercooled liquid nitrogen, the preservation of the tissue is rather poor. The cytoplasm and the extracellular space are full of replicated ice crystals. Nevertheless, patches of membranes carry interesting formations. The background particles of the sarcolemmal cytoplasmic leaflet are no longer separate or scattered as in the fixed tissue. Instead, they have conglomerated, forming many rows of particles, pairs or rows of paired particles (Fig. 15). Individual rows and doublets are not aligned with respect to each other. The external leaflet presents a matching pattern
Figs. 7, 8, 9, IO. Stretched
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of pits (Fig. 16). The appearance of the grills and ridges has not changed at all. Some caveolae appear flattened (Fig. 17) as they do in chemically fixed specimens. Freezing the specimens in liquid helium gives a much better preservation of the cytoplasm. Here, too, the background particles have agglomerated in the same way that they did in the specimens frozen in liquid nitrogen. The grills and ridges are always stable and some caveolae are flattened (Fig. 18). Immersion in a solution of’ HRP
Pieces of nerves bathed in a solution of HRP for less than 6 hr had only a very superficial layer of reaction product in their sheath. To obtain some information about the pinocytotic ability of the caveolae, the pieces of tissue had to be exposed to HRP for at least 6 hr. The reaction product then is seen in the extracellular space, trapped by the collagen, and inside the caveolae, coating their membrane (Fig. II). In the cytoplasm,
smooth
muscle cells. Compare
with Fig. 5.
Fig. 7. Replica of the external leaflet of the sarcolemma. In the stretched cell here, the ostia have been fully opened so that the caveolae are flattened out. Arrow indicates flattened caveola. x 86,000. Fig. 8. Replica of the cytoplasmic leaflet of the sarcolemma, showing the complementary face of stretched caveolae (arrow). 1, 2, ostia of caveolae unaffected by stretching. x 86,000. Fig. 9. In thin sections of stretched cells, the caveolae appear more often like undulations (arrows) rather than imaginations of the sarcolemma. 0, widened ostia. x I 15,000. Fig. 10. Cytoplasmic leaflet of the sarcolemma. Arrows indicate ostia of caveolae which have not been fully stretched. x 81,000.
several widened
Fig. 1I. Smooth muscle cell from Aplysia dacfylonwla, bathed in a solution of horseradish peroxidase for 6% hr. The peroxidase reaction product appears as a dense, armorphous material infiltrating the extracellular space E and coating (arrows) the interior of the caveolae. N, nucleus; 0, ostia. x 94,000. Fig. 12. Arrows indicate hemidesmosomes. Note dense extracellular material them. x 86,000. Insef: CL, collagen; S, subsurface cisterna. x 62,000. Fig. 13. Fracture across the cytoplasm lemma; S, subsurface cisterna. x 70,000.
(CT). L, cytoplasmic
facing
leaflet of the sarco-
Fig. 14. Turtle vena cava smooth muscle cell. View from the cytoplasm CT towards the external leaflet M. Arrows indicate the caveolar membrane bare of particles or grooves. x 92,000.
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caveolae whose ostia are out of the plane of section and which, therefore, appear as vesicles also contain the reaction product. Similar pictures are seen in the specimens which were fixed urior to their exoosure to HRP. Discussion Aplysia has an open circulation and, therefore, no capillaries or intermediary vessels (Mayeri et al., 1974). Metabolites must diffuse from the cells in the center of the viscera1 ganglion, through the sheath, towards the hemocoele fluid surrounding the ganglion. Some neurosecretory cell groups in the ganglion send their endings to the sheath (Coggeshall, 1967). It has been suggested that the sheath muscle cells may assist in the transport of neurosecretory material by means of their contraction (Rosenbluth, 1963). The slow peristaltic movement of the connective nerves observed during measurements of their resting length seems to support that idea. It is not known whether neighboring but isolated muscle cells contract synchronously in order to propel extracellular material in a given direction. That some of the cells operate in concert, however, is indicated by the contraction of the entire connective sheath. In some respects these muscle cells resemble, morphologically and functionally, the vertebrate smooth muscle cells found in capsules of the
Figs. 15, 16, 17. Cells quenched or glycerination.
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rat and human testicles (Gorgas and Bock. 1974; Langford and Heller, 1973), of the adrenals (Bressler, 1973) and of the spleen (Davies and Withrington, 1973). Sarcolemma
The sarcolemma of smooth muscle cells in Aplysia and in vertebrates is not an homogeneous surface. In Apfysia the caveolar ostia are clustered over certain areas of the cell, in particular those regions surrounding the nucleus and its immediate vicinity. They are also found in great number on some of the more peripheral attenuated extensions of the muscle cell. But it has been impossible to identify with certainty the regions of the cell from which they are absent. The caveolae contribute significantly to increasing the surface of the sarcolemma up to 23% in areas where they are present in Aplysia smooth muscle cells and probably more when the caveolae have a tubular conformation. Sarcolemmal invaginations in molluscan muscles have been compared to the T system in the striated muscle of vertebrates (Heyer and Kater, 1973). However, there is still no experimental evidence that the caveolae of molluscan or vertebrate smooth muscle cells are involved in the propagation of electrical or chemical excitation. In order to test the possibility that the caveolae are implicated in excitationcontraction coupling, it might be informative
in supercooled
Fig. 15. Cytoplasmic leaflet of the sarcolemma. field of agglomerated particles. x 148,000.
liquid nitrogen
without
prior fixation
G, grill; R, ridges. Arrow
indicates
Fig. 16. External leaflet of the sarcolemma. R, complementary groove of a ridge. Arrow indicates rows of pits matching agglomerated particles of the cytoplasmic leaflet. x 186,000. Fig. 17. External leaflet of the sarcolemma. Arrow indicates flattened caveola unstretched, quenched cell. 1, 2, 3, ostia ofinvaginated caveolae. x 66,000.
in an
Fig. 18. From a nerve directly frozen in liquid helium. Cytoplasmic leaflet of the sarcolemma. Arrow indicates flattened caveolae. 1, 2, partially flattened caveolae. R, ridges; 0, ostia. Note the pairing of the membrane particles. x 77,000.
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to stimulate selected spots on the cell surface. At this date, there has been no microelectrode exploration of the smooth muscle cell sarcolemma, analogous to the probing of the striated surface (Huxley and Taylor, 1958). Ideally, it should be done on large isolated smooth muscle cells, combining the identification of ultrastructural features of the membrane with ionophoretic stimulation. In Aplysiu, areas of the sarcolemma that are pitted by clusters of caveolae may be as large as 40 pm2 and areas where there are no caveolae just as large. Each areapitted and unpitted-seems to be wide enough to accommodate the tip of a microeltctrode. In resting connectives that had not been chemically fixed but, instead, directly frozen in liquid nitrogen or helium, the size of the caveolar ostia is not uniform at any given moment and a few caveolae always appear to be flattened (Figs. 17, 18). The fixation by liquid nitrogen or helium is more rapid than by chemical fixation (Heuser et al., 1975). The variability in size of the ostia after rapid freezing without fixation suggests that the caveolae have been arrested at different stages of opening to the extracellular space. Caveolar membrane
Striations of the caveolar membrane have been observed in smooth muscle cells of the aorta, the crop, the stomach, and in the pericardium as well as in the cardiac muscle of A&L+. Molluscan smooth muscle cells described so far have caveolae with no special membrane characteristics (Heyer and Kater, 1973). Somlyo et al. (1971) have reported striations of the caveolar membrane in thin sections of smooth muscle cells in the aorta, vena cava, and oviduct of the diamond-back turtle. That particular species being unavailable, we have examined, in thin sections and replicas, the smooth muscle cells of the vena cava and oviduct in the turtle Pseudemys elegans. There the caveolar membrane, like that in the mouse intestine and aorta and in the toad stomach, is smooth on both faces (Fig. 14). The spacing of the striations in the Aplysia smooth muscle is comparable to what has been found in the diamond-back turtle. In Aplysia, the striations are not shelves across the caveolae but represent, instead,
pREscoT-r.4~~
BRIGHTMAN
an intramembranous feature (Fig. 20). In thin sections, when the caveolae are cut transversely, one can see only spicules bulging into the caveolar space (Figs. 3, 9). This natural marker of the caveolar membrane in smooth muscle cells of Aplysia could be a useful tool to monitor membrane fractions, after homogenization, in order to study such problems as membrane synthesis, and enzymatic activity affecting ion fluxes that may occur at these portions of the plasmalemma. Entry of tracers in caveolae
In vertebrate smooth muscles, lanthanum and uranyl acetate have been used to show that the caveolae communicate with the extracellular space (Gabella, 1973). Horseradish peroxidase and ferritin also penetrate the caveolae of vertebrate smooth muscle cells but there is no evidence of pinocytosis (Devine et al., 1973). After more than 6 hr exposure to horseradish peroxidase, the caveolae of Aglysia muscle cells are coated with the enzyme smooth reaction product but there is no sign that the protein has been engulfed or is subjected to lysosomal activity (Simson and Spicer, 1973). The electron-dense pigmented bodies in the cytoplasm of the Aplysia muscle cells (Fig. I) could complicate the interpretation of the micrographs, but their appearance and number do not seem to be different after exposure of the cells to peroxidase. As yet, there is no evidence that pinocytosis is carried on by the caveolae in ApIysia smooth muscle. Grills and ridges
Other characteristics of the Ap/ysia sarcolemma include the grills, the ridges, and the background particles. The nature and function of the grills and ridges remain uncertain. They do not represent intercellular junctions, since the cells are separated by wide areas of extracellular space. In other systems, tight junctions and septate junctions have been recognized in replicas of freeze-fractured epithelia by their single or parallel rows of ridges or strands. Hemidesmosomes and desmosomes appear like clusters of big particles or ‘closely packed granules’ (McNutt and Weinstein, 1972; Kelly and Shienvold, 1973). Because the grills and ridges share some of the features of the
SARCOLEMMA
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MUSCLE
F
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G
H
Fig. 19. Diagram illustrating the plane of fracture L inside the cawolar membrane durinz stretchina: second row. EL. external leaflet: CL. cvtoolasmic leaflet. From A _ to D:-view from the cytoplasm towards the external leaflet EL. From E to H: view from the extracellular space towards the cytoplasmic leaflet CL. B, C. F. G, widened ostia during stretching; D, H, flattened caveolae, incorporated to the sarcolemmn.
junctions so far described, the possibility that they may represent sites of attachment of the sarcolemma to extracellular or intracellular elements is here posed but not yet demonstrated. In thin sections of vertebrate smooth muscle, hemidesmosomes are found easily, but grills and ridges have not been seen in replicas of these cells. The grills and ridges do not seem to bear any relation to the subsurface cisternae either. The sarcolemma facing the cisternae is unremarkable in structure (Figs. 12, inset, 13). The grills and ridges are very stable, being unaffected by diverse treatments like stretching, fixation with paraformaldehyde or glutaraldehyde, freezing without prior chemical fixation or glycerination in supercooled liquid nitrogen or in liquid helium. Buckgronnd
particles
The background particles are labile. They appear
on the other hand to be randomly
distributed in most replicas of specimens fixed in glutaraldehyde and glycerinated prior to freezing. Still, in some membranes fixed and glycerinated prior to freezing, the particles are paired (Fig. 6). The pairing or alignment of membrane particles is more frequent in specimens which had been fixed with paraformaldehyde or frozen directly in supercooled liquid nitrogen or in liquid helium (Figs. 15, 16, 17, 18). Crystallization of the extracellular and intracellular fluid is usually, but not always, detectable in replicas of membranes which have pairing of their particles. Pairing of the particles has been seen occasionally in well-fixed and wellfrozen specimens. What is the most representative picture of their natural state is. therefore, still uncertain. An inadequate cross-linking of membrane proteins (Elgsaeter and Branton, 1974), the fluid-gel state of the membrane lipids (Lee, 1975), and recrystallization, are among factors to consider in designing further experiments
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Fig. 20. Diagrammatic reconstruction of a triple caveola. The plane of fracture has revealed the inside of the membrane. S, sarcolemma in continuity with the caveola; 0, ostium; CP, caveolar particles; EL, external leaflet; OS, real outer surface facing the cytoplasm; IS, real inner surface in communication with the extracellular space; CL, cytoplasmic leaflet; CG, caveolar grooves.
to determine the causes of particle redistribution. Mechanosensitivity
of’ the sarcolemma
It is reasonable to assume that, in order for the individual separate muscle cells to be passively stretched, they must in some manner be anchored to the substance of the sheath. Many segments of the sacrolemma are specialized as hemidesmosomes which, in smooth muscle and other cells in many species, are supposed to be attachment devices between cell and extracellular material.
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The same assumption is made about the musculo-tendinous junctions, where hemidesmosomes occur on striated muscle near such junctions (Couteaux, 1959; Smith, 1972). In poorly fixed anterior byssus retractor muscle of Mytilus, Twarog et al. (1973) have seen the sarcolemma of muscle cells detached from the surrounding collagen except at the hemidesmosomes. Our observations lend indirect support to this supposition: even though the smooth muscle cells of the sheath are isolated they can be stretched when the entire sheath is elongated. The hemidesmosomes appear as the likely anchors although this observation does not rule out the less likely possibility that the rest of the sarcolemma might participate in attaching the cell. During stretching the caveolar membrane is exteriorized. This change appears to be reversible because when the stretching force is removed, the majority of caveolae are again fractured through their ostia rather than their delimiting membrane containing the large particles. A comparison between the specimens stretched three times and those stretched to twice their resting length reveals that the number of flattened caveolae may be proportional to the degree of stretching. In order to bolster this impression one would have to count the number of flattened caveolae in the sarcolemma of isolated cells frozen at increasing degrees of stretching. A number of observations have led to the recent formulation of a possible role of the caveolae in muscle activity. Thus, the absence of a T system and, in some vertebrate smooth muscle cells, the proximity of mitochondria and the cisternae of endoplasmic reticulum to caveolae has been taken to imply that caveolae may have a role in the coupling between excitation and contraction (Brading, 1975; Huddart, 1975; Devine et al., 1973). It has also been observed that in vertebrate smooth muscle sensitive to stretch, a minimum degree of stretch has to be attained before the sarcolemma depolarizes and the cell contracts (Marshall, 1974). These observations have led us to suggest that the caveolae might serve as stretchreceptors. Assuming that the caveolar membrane has special properties of ionic permeability, one can expect that a minimum number of caveolae have to be stretched or
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deformed to produce a depolarization of the membrane sufficient to cause a contraction of the muscle cell. Following physiological studies of stretch or tension receptors in vertebrate gastrointestinal tract, some investigators have concluded that these receptors are located in the smooth muscle layer and ‘appear to be in series with the smooth muscle cells’ (Leek, 1972). The caveolae, being a prominent feature of the sarcolemma in vertebrate smooth muscle ceils, may turn
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out to be the stretch-receptors so far eluded identification.
which
have
Acknowledgements We wish to thank Mrs J. Eskin, G. Goping, C. Mowry and T. Nicholson for their technical assistance and Dr T. S. Reese for his gracious help in handling the liquid helium freezing apparatus. Dr L. Prescott was a fellow of the Canadian Medical Research Council.
References BRADING,
A. F. 1975. Sodium/sodium
Physiol.
(Lond.),
exchange
in the smooth
muscle
of the guinea-pig
Tnwicr co/~. J.
251, 79-105.
BRANTON,
D. and DEAMER, D. W. 1972. Membrane structure. In Protop/usmnto/ogia, II/E,‘l. SpringerVerlag. BRESSLER,R. S. 1973. Myoid ceils in the capsule of the adrenal gland and in monolayers derived from cultured adrenal capsules. Anat. Rec., 177, 525-531. COCGESHALL, R. E. 1967. A light and electron microscope study of the abdominal ganglion of Apl,,sin calijbmica. J. Neurophysiol., 30, 1263-1287. COUTEAUX, R. 1959. Observations sur I’ultrastructure de la jonction musculo-tendineuse. c‘. Y. h&d. Skim. Acad. Sci., Paris, 249, 964-966. DAVIES.
B. N. and WITHRINGTON, P. G. 1973. The actions of drugs on the smooth muscle of the capsule and blood vessels of the spleen. Pharmac. Rev., 25, 373-413. Dr VINE, C. E,, SOMLYO, A. V. and SOMLYO, A. P. 1973. Sarcoplasmic reticulum and mitochondria as cation accumulation sites in smooth muscle. Phi/. Trans. R. Sot. Ser. B, 265, 17-23. EALES, N. B. 1921. Aplysia. Trans. Liverpool Biol. Sor., 35, 183-266. EL(;SAETER, A. and BRANTON, D. 1974. Intramembrane particle aggregation in erythrocyte ghosts. J. Cp// Biol., 63, 1018-1030.
C;ABLI,I.A.
G. 1973. Cellular structures
and electrophysiological
behaviour.
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GAINER, H. and WOLLBERG,Z. 1974. Specific protein metabolism J. Neurobiol.,
in identifiable
neurons ofAp/.vsio ca/ifixt,ir~rr.
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GOKc;As,
K. and B&K, P. 1974. Myofibroblasts in the rat testicular capsule. Cf>NTissur Res., 154. 533-541. GRAHAM, R. C. and KARNOVSKY, M. J. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem.
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HFNKART, M. 1975. Light-induced changes in the structure of pigmented granules in Aplysia neurons. Science, N. I’., 188, 155-157. HFIISEH, J. E. and REESE, 7. S. 1973. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol., 57, 3 15-344. HEIISEK, J. E., LANDIS, D. M. D. and REESE,T. S. 1975. Preservation of synaptic structure by rapid freezing. Cold Spring Harbor Symposium, ‘The Synapse’ (in press). HFY~R, C. B. and KATER, S. B. 1973. Neuromuscular systems in molluscs. Am. Zoo/., 13, 247-270, Hoooa~r, H. 1975. The Comparative Structure and Function qf Muscle. Pergamon Press. HL YLEY, A. F. and TAYLOR, R. E. 1958. Local activation of striated muscleJ. Physiol. (Land.). 144. 426-441. KAKNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27, 137A. Kbc LY, D. E. and SHIENVOLD, F. 1973. Freeze-fracture analysis of hemidesmosomes. J. Cell B;o/.. 59. 166A.
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258 LANGFORD, G. A. and HELLER, C. G. 1973. Fine structureofmusclecells of testicular contractions. Science, IV. Y., 179, 573-575. LEE, A. G. 1975. Functional properties of biological membranes: Biophys.,
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basis Prog.
29, 3-56.
LEEK, B. F. 1972. Abdominal visceral receptors. In Handbook qf’Sensory Physiology, III/I, ‘Enteroceptors’, pp. 144-l 60. MARSHALL, J. M. 1974. Vertebrate smooth muscle. In Medical Physiology (ed. Mountcastle, V. B.), pp. 121148. C. V. Mosby Co., St Louis. MAYERI, E., KOESTER, J. and LIEBESWAR,G. 1974. Functional organization of the neural control of circulation in Aplysia. Am. Zoo/., 14, 943-956. MCNUTT, N. S. and WEINSTEIN, R. S. 1972. Membrane ultrastructure at mammalian intercellular junctions. frog. Biophys., 26, 45-l 01. MOOR, H. 1973. In Freeze-etching, Techniques and Applications (ed. Benedetti, E. L. and Favard, P.), pp. 1l19. SocietC Franqaise de Microscopic Electronique, Paris. PINTO DA SILVA, P. and BRANTON, D. 1970. Membrane splitting in freeze-etching: covalently bound ferritin as a membrane marker. J. Cell Biol., 45, 598-605. ROSENBLUTH,J. 1963. Fine structure of epineural muscle cells in Aplysia californica. J. Cell Biol., 17, 455-460. SIMSON,J. V. and SPIC~R, S. S. 1973. Activities of specific cell constituents in phagocytosis (Endocytosis).
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SMITH, D. S. 1972. Muscle, pp. 55-57. Academic Press. SOMLYO, A. P., DEVINE, C. E., SOMLYO, A. V. and NORTH, S. R. 1971. Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium-free solutions. J. Cell Biol., 51, 722-741. STRUMWASSER,F. 1961. Neurally induced contraction of nerve trunks in Aplysia. The Physiologist, 4, 118. TWAROG, B. M., DEWEY, M. M. and HIDAKA, T. 1973. The structure of Mytilus smooth muscle and the electrical constants of the resting muscle. J. gen. Physiol., 61, 207-221. VENABLE, J. M. and COCGESHALL, R. 1965. Simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25, 407.
& CELL
1976 8 (2) 241-258
Prlhli.slwd h.v Longmnn
Group l.td. Printed
in Great Britain
LISE PRESCOTT
and MILTON
THE SARCOLEMMA OF APLYSIA MUSCLE IN FREEZE-FRACTURE PREPARATIONS ABSTRACT.
W. BRIGHTMAN
SMOOTH
muscle cells in the sheath covering the visceral ganglion of with the techniques of freeze-fracture and conventional electron microscopy. The sarcolemma of these muscle cell invaginates to form myriad caveolae that have an intrinsic marker within their membrane. This intrinsic structure of the caveolar membrane is revealed by freeze-fracture and consists of rows of large particles in the outer half and matching grooves on the complementary inner half of the membrane. In thin plastic sections, parallel striations or shelves within the caveolar membrane appear to be the equivalent of the particles and grooves of the fractured membrane. Physical fixation of some specimens by rapid freezing in supercooled liquid nitrogen or in liquid helium suggests that in their natural state, the caveolar ostia are not uniform in size and that at any given moment a number of caveolae are flattened. When segments of the connective nerves which link the visceral ganglion to the cephalic ganglia are stretched in virro two to three times their in sifrr length, the caveolae lose their invaginated shape and are fully exposed to the extracellular space. The caveolar membrane, so stretched, is pulled into the line of fracture with the result that the large particles rather than the ostia appear on the cleaved surface. This flattening of the caveolae is reversible and suggests that they might serve as miniature stretch-receptors within the membrane of the smooth muscle cells. The caveolae are accessible to extracellular horseradish peroxidase but do not appear to pinocytose the protein. Aplwio
Smooth
culifomica
were examined
ceils has been noticed. These muscle cells are believed to account for the contractility of the sheath (Rosenbluth, 1963 ; Coggeshall, 1967). We have re-examined the sheath with the freeze-fracture technique in correlation with thin sectioning. Special attention was paid to the sarcolemmal features of these molluscan smooth muscle cells. They were compared with vertebrate smooth muscle cells in the toad (Byfo marinus) stomach, the mouse intestine and aorta, the turtle oviduct and vena cava, and found to have their own specific sarcolemmal characteristics. The carveolae of the Aplysia smooth muscle cells contain a natural marker which makes them a particularly suitable preparation for further combined chemical, physiological and ultrastructural studies of the sarcolemma belonging to the smooth muscle cell.
Introduction WHEN the visceral ganglion of Aplysia is stimulated mechanically or electrically, one can observe a slow retraction of the associated nerves (Strumwasser, 1961). The two long connectives which relate the visceral ganglion to the pleural ganglia in the cephalic region (Eales, 1921) take on a helical conformation. If the ganglion is left in artificial seawater for a while, the connectives will relax again. During previous ultrastructural examination of the sheath covering the ganglion and its nerves, the presence of smooth muscle
Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland. Received
I5 December
1975. 241
242
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Materials and Methods Aplysia californica,
weighing between 100 and 300 g were obtained from Pacific Bio-Marine Supply Co. (Venice, California) and kept from 2 to 15 days, at 15°C in a sea water aquarium through the whole year. The visceral ganglion and adjacent nerves were dissected out of the coelomic cavity and left for 5-60 min in physiological Aplysia saline without glucose: NaCl 460 mM, KC1 10 mM, CaCls 11 mM, McCls 50 mM and Tris-HCl 50 mM at a pH of 78 (Gainer and Wollberg, 1974). In a few Aplysia, the heart, the aorta, the crop and the stomach were also removed and prepared for conventional electron microscopy and freeze-fracture. Ultramicrotomy
The ganglion and viscera were fixed for 90 min in 2.5 % glutaraldehyde in a 0.1 M Na Cacodylate buffer and 0.8 M sucrose, at a pH of 7.6 (Henkart, 1975). Small pieces were then cut out of the viscera and from the sheath covering the nerves and body of the ganglion. After several rinses in buffer alone or buffer and sucrose, the tissues were osmicated for 3 hr, stained en bloc in 1% uranyl acetate (Heuser and Reese, 1973) dehydrated in methanol and embedded in Araldite (Ciba, Duxford, Cambridge, England). Thin sections were stained with 5 % uranyl acetate in 50 % methanol at 40°C for 20 min followed by lead citrate for 1 min (Venable and Coggeshall, 1965). A few specimens from the sheath were also fixed in 1.5% paraformaldehyde instead of glutaraldehyde. Samples from the toad (Bu~o marinas) stomach muscles, the turtle (Pseudemys elegans) vena cava and oviduct were fixed in 2.5 % glutaraldehyde by immersion buffered with 0.1 M sodium cacodylate at pH 7.4. Pieces from the intestine and aorta of mice, perfused with a mixture of aldehydes (Karnovsky, 1965), were also processed for electron microscopy. Freeze-fracture
Following fixation with glutaraldehyde, the specimens were rinsed briefly in buffer and left for 2 h in 20% glycerol. After glycerination, they were frozen in liquid Freon 22
AND
BRIGHTMAN
(monochlorodifluoromethane) at - 150°C and stored in liquid nitrogen. Some specimens were quenched directly in supercooled liquid nitrogen at -210°C (Moor, 1973) without prior fixation or glycerination. In one experiment, the specimens, kept for several hours at 4°C in Aplysia saline, unfixed and unglycerinated, were directly frozen in liquid helium at -270°C in a modified Van Harreveld apparatus (Heuser et al., 1975) and stored in liquid nitrogen. All frozen specimens were later fractured and replicated with platinum and carbon in a Balzer’s apparatus at - 110°C. Exposure to horseradish peroxidase
Shortly after removal from the coelomic cavity, a few connective nerves from Aplysia calijornica and one Aplysia dactylomela, were divided in segments 34 mm long and bathed for 30 min to 6 hr in a solution of Aplysia saline containing 2-5 mg/ml of horseradish peroxidase (Sigma Type VI, from Sigma Chem. Co., St Louis, MO.) at room temperature. The pieces were then fixed for 1 hr and incubated for l-3 hr at 4°C in 10 ml of 0.1 M Tris-HCl buffer to which 5 mg of diaminobenzidine and 0.1 ml of 1% Hz02 were added (Graham and Karnovsky, 1966). Following the incubation, the tissue was processed for electron microscopy. A few specimens were fixed for 2 hr prior to their exposure to horseradish peroxidase. Stretching experiment
After opening the coelomic cavity, each connective nerve was ligated at two points along its length. The distance between the two ligatures was then measured in situ. The ligated connectives were removed and remeasured when floating freely in the salt solution in order to establish their resting length. They were then pinned down, and while still submerged in saline, stretched to either two or three times their resting length, for 3, 10, 20, 35 and 55 min before being fixed with glutaraldehyde in the stretched position. Two nerves stretched twice their resting length, for 10 and 20 min were allowed to regain their resting state prior to fixation. All specimens were fixed for 1-2 hr, then prepared for freeze-fracture and thin sectioning.
SARCOLEMMA
OF SMOOTH
MUSCLE
Morphometry
A few replicas were systematically examined in the electron microscope and every membrane surface belonging to a muscle cell photographed. Measurements of membrane surface areas were made on enlarged micrographs, using a Hewlett-Packard calculator equipped with a digitizer and programmed to measure surface areas of any shape. Particle diameter and size of organelles were measured on micrographs with a calibrated IO x lens.
IN
APLYSlA
:a:
cellular space by means of an ostium. In many instances, several caveolae fuse to form a lobulated, tubular invagination of the sarcolemma (Figs. 3, 4). At high magnification, a tangential cut through the sarcolemma reveals a striated pattern in the caveolar membrane. The spacing of this pattern may vary between 80 and I50 A. When transversely sectioned, the striations appear as evenly spaced, short, pointed spicules emanating from the inner wall of the caveolae towards the caveolar lumen (Figs. 3, 9).
Observations In thin sections, the sheath surrounding the body of the visceral ganglion appears to be made of much collagen, numerous smooth muscle cells (SMC), some cell processes filled with dense granules and some glial elements (Fig. I ). In samples of the sheath taken around the nerves leading to or from the ganglion we have found mostly collagen and smooth muscle cells. These cells are not organized in bundles but isolated in the collagenous matrix, and establish very few contacts with their neighhours; gap junctions have been seen, but rarely. The SMC are long and thin with tiny lateral processes. The main axis of the SMC is roughly parallel to the long axis of the ganlion and the nerves. The full length of one cell is rarely seen in one section. The maximal length observed has been 8 pm. Their diameter in the region of the cell nucleus may vary between 0.8 and 5 pm, and, in the paranuclear region, between 0.2 and 6 /‘in. Embedded in the sarcoplasm are thick and thin filaments that are arranged parallel to the main axis of the cell but which do not form sarcomeres (Figs. I, 2, 12). In the cytoplasm, usually in the nuclear region, round bodies of variable electron density are observed (Fig. I). They resemble the pigmented granules seen in giant neurons of Aplysicr (Henkart, 1975). Hemidesmosomes which appear as focal mats of dense, amorphous. cytoplasmic material, lie subjacent to portions of the sarcolemma, opposite patches of extracellular fuzz (Fig. 12). The sarcolemma is frequently invaginated into the cytoplasm as simple flaskshaped caveolae or pits (Figs. I, 2, 3, 4). Each caveola communicates with the extra-
The plane of fracture follows a path inside the unit membrane and reveals two intramembranous faces: the cytoplasmic leaflet as seen from the extracellular space and the external leaflet viewed from the cytoplasm (Pinto da Silva and Branton. 1970). Extensive views of the sarcolemma may thus be obtained (Fig. 5). Of the two intramembranous faces of the sarcolemma, the cytoplasmic leaflet has four to five times more membrane particles. the ‘background’ particles, than the external leaflet. These particles, randomly distributed and 85 8, wide, are similar in size to the particles seen in tnost freeze-fractured biological membranes (Branton and Deamer. 1972). Two types of particle agglomerations are unique to the ApIysiu sarcolemma: ‘grills’ and ‘ridges’ (Figs. 5, 6, 15, 16, 18). The grills consist of particles, 95 A in diameter, which are uniformly spaced at 140 A intervals in parallel rows on the cytoplasmic half of the sarcolemmal membrane. Complementary images of the grills are rarely seen. The ridges are also situated on the cytoplasmic leaflet. and consist of linear arrays of particles which, in places, coalesce into an unbroken strand. On the external leaflet. matching grooves are easily recognized (Fig. 16). The most striking feature of the cleaved sarcolemma is the caveolar ostia whtcl; dominates the m/tire view. Fig. 5 represents a membrane area of 22 pm2 with an average of I4 ostia/pmZ. Behind each ostium there is a caveola invaginating the cytoplasm. The ostia are usually clustered in certain area\ of the sarcolemma. Wherever they are their density may vary from I to I 5/+mZ. However. areas of the sarcolemma as extensive as
PRESCOTT
244
50 pm2 do not form ostia nor, therefore, caveolae. By tracing the perimeter of caveolae, as they appear in micrographs of replicas with the computer’s stylus, the amount of additional area contributed by the caveolae was estimated. The sarcolemma1 area, where the ostia are present was increased by as much as 23% by the caveolae. Since the caveolae invaginate into the cytoplasm, the internal structure of their membranes can only be visualized in crossfractured cells. A fracture along the caveolar membrane exposes two complementary faces (Fig. 4). One leaflet in continuity with the external leaflet of the sarcolemma has tightly clustered rows of particles about 125 8, in diameter, hence larger than the randomly dispersed particles (Fig. 4). The other leaflet in contact with the cytoplasm has matching grooves. The spacing of the rows and grooves, between 110 and 140 A, is comparable to the striated pattern seen on thin sections of caveolae. In comparison, caveolae in the vertebrate smooth muscles examined do not show any specific characteristics. In smooth muscles of the toad stomach, the turtle vena cava and oviduct, the mouse intestine and aorta,
Figs. 1, 2. N, nucleus; indicate caveolae. Fig. 1. Smooth
BRIGHTMAN
both faces of the fractured caveolar membranes are remarkably smooth (Fig. 14). Stretching effects
When the connective nerves are stretched to three times their resting length there is a striking difference in the appearance of the sarcolemma. Where there used to be clusters of ostia (Fig. 5), patches of agglomerated particles are present on the external leaflet (Fig. 7) and depressions with narrow, parallel grooves (Fig. 8) on the cytoplasmic leaflet. The caveolar membrane has been exteriorized or stretched into the same plane as the rest of the sarcolemma (Fig. 19). In thin sections many caveolae which used to be invaginations now look more like shallow undulations (Fig. 9). In some areas, the caveolae appear partially stretched and their ostia are much wider (Fig. 10). This appearance of the ostia is particularly common when the nerve is stretched only twice its resting length. The phenomenon is reversible within certain limits. A nerve stretched three times its resting length, for 1 hr, had several membranes that appeared to be ruptured when examined in replicas. Another nerve stretched three times its resting length, for 20 min,
F, filaments;
CL, collagen;
muscle cell cut transversely
Fig. 2. Longitudinal
AND
through
section of a muscle cell process.
D, pigmented the nuclear
bodies. Arrows region.
x 15,000.
x 22,000.
Fig. 3. Higher magnification of a cellular area containing indicates striations in a tangentially cut caveolar membrane. x 91,000.
several caveolae. Arrow 0, ostium of a caveola.
Fig. 4. Replica of a fracture across the cytoplasm. L, cytoplasmic leaflet of the sarcolemma. X and Y represent the two complementary faces of the plane of fracture inside the caveolar membrane. One leaflet, X, has rows of particles, the other leaflet, Y, has matching grooves. x 110,000. Fig. 5. Panoramic view of the cytoplasmic leaflet of the sarcclemma seen from the extracellular space. Arrow indicates a group of ostia. Out of the plane of fracture liut behind each ostium on this membrane there is a caveola invaginating into the cytoplasm. R, linear array of particles, called ‘ridge’. G, characteristic of this membrane, is an agglomeration of large particles that we have called ‘grill’. x 38,000. Fig. 6. Cytoplasmic leaflet of the sarcolemma. Compare the small ostia (arrows) with the larger ones on the left. G, grill. Crystallization of the extracellular space fluid in upper right corner. x 64,000.
PRESCOTT
took also 20 min to return to its original length. But nerves stretched two to three times their resting length for less than 10 min could regain their resting length almost immediately. At the ultrastructural level these replicated nerves showed no evidence of their previous stretched state. Freezing without prior jixatiotz and glycerination
In the specimens frozen directly in supercooled liquid nitrogen, the preservation of the tissue is rather poor. The cytoplasm and the extracellular space are full of replicated ice crystals. Nevertheless, patches of membranes carry interesting formations. The background particles of the sarcolemmal cytoplasmic leaflet are no longer separate or scattered as in the fixed tissue. Instead, they have conglomerated, forming many rows of particles, pairs or rows of paired particles (Fig. 15). Individual rows and doublets are not aligned with respect to each other. The external leaflet presents a matching pattern
Figs. 7, 8, 9, IO. Stretched
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BRIGHTMAN
of pits (Fig. 16). The appearance of the grills and ridges has not changed at all. Some caveolae appear flattened (Fig. 17) as they do in chemically fixed specimens. Freezing the specimens in liquid helium gives a much better preservation of the cytoplasm. Here, too, the background particles have agglomerated in the same way that they did in the specimens frozen in liquid nitrogen. The grills and ridges are always stable and some caveolae are flattened (Fig. 18). Immersion in a solution of’ HRP
Pieces of nerves bathed in a solution of HRP for less than 6 hr had only a very superficial layer of reaction product in their sheath. To obtain some information about the pinocytotic ability of the caveolae, the pieces of tissue had to be exposed to HRP for at least 6 hr. The reaction product then is seen in the extracellular space, trapped by the collagen, and inside the caveolae, coating their membrane (Fig. II). In the cytoplasm,
smooth
muscle cells. Compare
with Fig. 5.
Fig. 7. Replica of the external leaflet of the sarcolemma. In the stretched cell here, the ostia have been fully opened so that the caveolae are flattened out. Arrow indicates flattened caveola. x 86,000. Fig. 8. Replica of the cytoplasmic leaflet of the sarcolemma, showing the complementary face of stretched caveolae (arrow). 1, 2, ostia of caveolae unaffected by stretching. x 86,000. Fig. 9. In thin sections of stretched cells, the caveolae appear more often like undulations (arrows) rather than imaginations of the sarcolemma. 0, widened ostia. x I 15,000. Fig. 10. Cytoplasmic leaflet of the sarcolemma. Arrows indicate ostia of caveolae which have not been fully stretched. x 81,000.
several widened
Fig. 1I. Smooth muscle cell from Aplysia dacfylonwla, bathed in a solution of horseradish peroxidase for 6% hr. The peroxidase reaction product appears as a dense, armorphous material infiltrating the extracellular space E and coating (arrows) the interior of the caveolae. N, nucleus; 0, ostia. x 94,000. Fig. 12. Arrows indicate hemidesmosomes. Note dense extracellular material them. x 86,000. Insef: CL, collagen; S, subsurface cisterna. x 62,000. Fig. 13. Fracture across the cytoplasm lemma; S, subsurface cisterna. x 70,000.
(CT). L, cytoplasmic
facing
leaflet of the sarco-
Fig. 14. Turtle vena cava smooth muscle cell. View from the cytoplasm CT towards the external leaflet M. Arrows indicate the caveolar membrane bare of particles or grooves. x 92,000.
SARCOLEMMA
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SMOOTH
MUSCLE
IN
APL YSlA
249
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252
caveolae whose ostia are out of the plane of section and which, therefore, appear as vesicles also contain the reaction product. Similar pictures are seen in the specimens which were fixed urior to their exoosure to HRP. Discussion Aplysia has an open circulation and, therefore, no capillaries or intermediary vessels (Mayeri et al., 1974). Metabolites must diffuse from the cells in the center of the viscera1 ganglion, through the sheath, towards the hemocoele fluid surrounding the ganglion. Some neurosecretory cell groups in the ganglion send their endings to the sheath (Coggeshall, 1967). It has been suggested that the sheath muscle cells may assist in the transport of neurosecretory material by means of their contraction (Rosenbluth, 1963). The slow peristaltic movement of the connective nerves observed during measurements of their resting length seems to support that idea. It is not known whether neighboring but isolated muscle cells contract synchronously in order to propel extracellular material in a given direction. That some of the cells operate in concert, however, is indicated by the contraction of the entire connective sheath. In some respects these muscle cells resemble, morphologically and functionally, the vertebrate smooth muscle cells found in capsules of the
Figs. 15, 16, 17. Cells quenched or glycerination.
AND
BRIGHTMAN
rat and human testicles (Gorgas and Bock. 1974; Langford and Heller, 1973), of the adrenals (Bressler, 1973) and of the spleen (Davies and Withrington, 1973). Sarcolemma
The sarcolemma of smooth muscle cells in Aplysia and in vertebrates is not an homogeneous surface. In Apfysia the caveolar ostia are clustered over certain areas of the cell, in particular those regions surrounding the nucleus and its immediate vicinity. They are also found in great number on some of the more peripheral attenuated extensions of the muscle cell. But it has been impossible to identify with certainty the regions of the cell from which they are absent. The caveolae contribute significantly to increasing the surface of the sarcolemma up to 23% in areas where they are present in Aplysia smooth muscle cells and probably more when the caveolae have a tubular conformation. Sarcolemmal invaginations in molluscan muscles have been compared to the T system in the striated muscle of vertebrates (Heyer and Kater, 1973). However, there is still no experimental evidence that the caveolae of molluscan or vertebrate smooth muscle cells are involved in the propagation of electrical or chemical excitation. In order to test the possibility that the caveolae are implicated in excitationcontraction coupling, it might be informative
in supercooled
Fig. 15. Cytoplasmic leaflet of the sarcolemma. field of agglomerated particles. x 148,000.
liquid nitrogen
without
prior fixation
G, grill; R, ridges. Arrow
indicates
Fig. 16. External leaflet of the sarcolemma. R, complementary groove of a ridge. Arrow indicates rows of pits matching agglomerated particles of the cytoplasmic leaflet. x 186,000. Fig. 17. External leaflet of the sarcolemma. Arrow indicates flattened caveola unstretched, quenched cell. 1, 2, 3, ostia ofinvaginated caveolae. x 66,000.
in an
Fig. 18. From a nerve directly frozen in liquid helium. Cytoplasmic leaflet of the sarcolemma. Arrow indicates flattened caveolae. 1, 2, partially flattened caveolae. R, ridges; 0, ostia. Note the pairing of the membrane particles. x 77,000.
254
to stimulate selected spots on the cell surface. At this date, there has been no microelectrode exploration of the smooth muscle cell sarcolemma, analogous to the probing of the striated surface (Huxley and Taylor, 1958). Ideally, it should be done on large isolated smooth muscle cells, combining the identification of ultrastructural features of the membrane with ionophoretic stimulation. In Aplysiu, areas of the sarcolemma that are pitted by clusters of caveolae may be as large as 40 pm2 and areas where there are no caveolae just as large. Each areapitted and unpitted-seems to be wide enough to accommodate the tip of a microeltctrode. In resting connectives that had not been chemically fixed but, instead, directly frozen in liquid nitrogen or helium, the size of the caveolar ostia is not uniform at any given moment and a few caveolae always appear to be flattened (Figs. 17, 18). The fixation by liquid nitrogen or helium is more rapid than by chemical fixation (Heuser et al., 1975). The variability in size of the ostia after rapid freezing without fixation suggests that the caveolae have been arrested at different stages of opening to the extracellular space. Caveolar membrane
Striations of the caveolar membrane have been observed in smooth muscle cells of the aorta, the crop, the stomach, and in the pericardium as well as in the cardiac muscle of A&L+. Molluscan smooth muscle cells described so far have caveolae with no special membrane characteristics (Heyer and Kater, 1973). Somlyo et al. (1971) have reported striations of the caveolar membrane in thin sections of smooth muscle cells in the aorta, vena cava, and oviduct of the diamond-back turtle. That particular species being unavailable, we have examined, in thin sections and replicas, the smooth muscle cells of the vena cava and oviduct in the turtle Pseudemys elegans. There the caveolar membrane, like that in the mouse intestine and aorta and in the toad stomach, is smooth on both faces (Fig. 14). The spacing of the striations in the Aplysia smooth muscle is comparable to what has been found in the diamond-back turtle. In Aplysia, the striations are not shelves across the caveolae but represent, instead,
pREscoT-r.4~~
BRIGHTMAN
an intramembranous feature (Fig. 20). In thin sections, when the caveolae are cut transversely, one can see only spicules bulging into the caveolar space (Figs. 3, 9). This natural marker of the caveolar membrane in smooth muscle cells of Aplysia could be a useful tool to monitor membrane fractions, after homogenization, in order to study such problems as membrane synthesis, and enzymatic activity affecting ion fluxes that may occur at these portions of the plasmalemma. Entry of tracers in caveolae
In vertebrate smooth muscles, lanthanum and uranyl acetate have been used to show that the caveolae communicate with the extracellular space (Gabella, 1973). Horseradish peroxidase and ferritin also penetrate the caveolae of vertebrate smooth muscle cells but there is no evidence of pinocytosis (Devine et al., 1973). After more than 6 hr exposure to horseradish peroxidase, the caveolae of Aglysia muscle cells are coated with the enzyme smooth reaction product but there is no sign that the protein has been engulfed or is subjected to lysosomal activity (Simson and Spicer, 1973). The electron-dense pigmented bodies in the cytoplasm of the Aplysia muscle cells (Fig. I) could complicate the interpretation of the micrographs, but their appearance and number do not seem to be different after exposure of the cells to peroxidase. As yet, there is no evidence that pinocytosis is carried on by the caveolae in ApIysia smooth muscle. Grills and ridges
Other characteristics of the Ap/ysia sarcolemma include the grills, the ridges, and the background particles. The nature and function of the grills and ridges remain uncertain. They do not represent intercellular junctions, since the cells are separated by wide areas of extracellular space. In other systems, tight junctions and septate junctions have been recognized in replicas of freeze-fractured epithelia by their single or parallel rows of ridges or strands. Hemidesmosomes and desmosomes appear like clusters of big particles or ‘closely packed granules’ (McNutt and Weinstein, 1972; Kelly and Shienvold, 1973). Because the grills and ridges share some of the features of the
SARCOLEMMA
OF
SMOOTH
E
MUSCLE
F
IN
APLYSIA
G
H
Fig. 19. Diagram illustrating the plane of fracture L inside the cawolar membrane durinz stretchina: second row. EL. external leaflet: CL. cvtoolasmic leaflet. From A _ to D:-view from the cytoplasm towards the external leaflet EL. From E to H: view from the extracellular space towards the cytoplasmic leaflet CL. B, C. F. G, widened ostia during stretching; D, H, flattened caveolae, incorporated to the sarcolemmn.
junctions so far described, the possibility that they may represent sites of attachment of the sarcolemma to extracellular or intracellular elements is here posed but not yet demonstrated. In thin sections of vertebrate smooth muscle, hemidesmosomes are found easily, but grills and ridges have not been seen in replicas of these cells. The grills and ridges do not seem to bear any relation to the subsurface cisternae either. The sarcolemma facing the cisternae is unremarkable in structure (Figs. 12, inset, 13). The grills and ridges are very stable, being unaffected by diverse treatments like stretching, fixation with paraformaldehyde or glutaraldehyde, freezing without prior chemical fixation or glycerination in supercooled liquid nitrogen or in liquid helium. Buckgronnd
particles
The background particles are labile. They appear
on the other hand to be randomly
distributed in most replicas of specimens fixed in glutaraldehyde and glycerinated prior to freezing. Still, in some membranes fixed and glycerinated prior to freezing, the particles are paired (Fig. 6). The pairing or alignment of membrane particles is more frequent in specimens which had been fixed with paraformaldehyde or frozen directly in supercooled liquid nitrogen or in liquid helium (Figs. 15, 16, 17, 18). Crystallization of the extracellular and intracellular fluid is usually, but not always, detectable in replicas of membranes which have pairing of their particles. Pairing of the particles has been seen occasionally in well-fixed and wellfrozen specimens. What is the most representative picture of their natural state is. therefore, still uncertain. An inadequate cross-linking of membrane proteins (Elgsaeter and Branton, 1974), the fluid-gel state of the membrane lipids (Lee, 1975), and recrystallization, are among factors to consider in designing further experiments
PRESCOTT
256
Fig. 20. Diagrammatic reconstruction of a triple caveola. The plane of fracture has revealed the inside of the membrane. S, sarcolemma in continuity with the caveola; 0, ostium; CP, caveolar particles; EL, external leaflet; OS, real outer surface facing the cytoplasm; IS, real inner surface in communication with the extracellular space; CL, cytoplasmic leaflet; CG, caveolar grooves.
to determine the causes of particle redistribution. Mechanosensitivity
of’ the sarcolemma
It is reasonable to assume that, in order for the individual separate muscle cells to be passively stretched, they must in some manner be anchored to the substance of the sheath. Many segments of the sacrolemma are specialized as hemidesmosomes which, in smooth muscle and other cells in many species, are supposed to be attachment devices between cell and extracellular material.
AND
BRIGHTMAN
The same assumption is made about the musculo-tendinous junctions, where hemidesmosomes occur on striated muscle near such junctions (Couteaux, 1959; Smith, 1972). In poorly fixed anterior byssus retractor muscle of Mytilus, Twarog et al. (1973) have seen the sarcolemma of muscle cells detached from the surrounding collagen except at the hemidesmosomes. Our observations lend indirect support to this supposition: even though the smooth muscle cells of the sheath are isolated they can be stretched when the entire sheath is elongated. The hemidesmosomes appear as the likely anchors although this observation does not rule out the less likely possibility that the rest of the sarcolemma might participate in attaching the cell. During stretching the caveolar membrane is exteriorized. This change appears to be reversible because when the stretching force is removed, the majority of caveolae are again fractured through their ostia rather than their delimiting membrane containing the large particles. A comparison between the specimens stretched three times and those stretched to twice their resting length reveals that the number of flattened caveolae may be proportional to the degree of stretching. In order to bolster this impression one would have to count the number of flattened caveolae in the sarcolemma of isolated cells frozen at increasing degrees of stretching. A number of observations have led to the recent formulation of a possible role of the caveolae in muscle activity. Thus, the absence of a T system and, in some vertebrate smooth muscle cells, the proximity of mitochondria and the cisternae of endoplasmic reticulum to caveolae has been taken to imply that caveolae may have a role in the coupling between excitation and contraction (Brading, 1975; Huddart, 1975; Devine et al., 1973). It has also been observed that in vertebrate smooth muscle sensitive to stretch, a minimum degree of stretch has to be attained before the sarcolemma depolarizes and the cell contracts (Marshall, 1974). These observations have led us to suggest that the caveolae might serve as stretchreceptors. Assuming that the caveolar membrane has special properties of ionic permeability, one can expect that a minimum number of caveolae have to be stretched or
SARCOLEMMA
OF
SMOOTH
MUSCLE
IN APLYSIA
deformed to produce a depolarization of the membrane sufficient to cause a contraction of the muscle cell. Following physiological studies of stretch or tension receptors in vertebrate gastrointestinal tract, some investigators have concluded that these receptors are located in the smooth muscle layer and ‘appear to be in series with the smooth muscle cells’ (Leek, 1972). The caveolae, being a prominent feature of the sarcolemma in vertebrate smooth muscle ceils, may turn
157
out to be the stretch-receptors so far eluded identification.
which
have
Acknowledgements We wish to thank Mrs J. Eskin, G. Goping, C. Mowry and T. Nicholson for their technical assistance and Dr T. S. Reese for his gracious help in handling the liquid helium freezing apparatus. Dr L. Prescott was a fellow of the Canadian Medical Research Council.
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